Functional subcellular distribution of β1- and β2-adrenergic receptors in rat ventricular cardiac myocytes

PubMed Central

Cros, Caroline; Brette, Fabien

2013-01-01

β-adrenergic stimulation is a key regulator of cardiac function. The localization of major cardiac adrenergic receptors (β1 and β2) has been investigated using biochemical and biophysical approaches and has led to contradictory results. This study investigates the functional subcellular localization of β1- and β2-adrenergic receptors in rat ventricular myocytes using a physiological approach. Ventricular myocytes were isolated from the hearts of rat and detubulated using formamide. Physiological cardiac function was measured as Ca2+ transient using Fura-2-AM and cell shortening. Selective activation of β1- and β2-adrenergic receptors was induced with isoproterenol (0.1 μmol/L) and ICI-118,551 (0.1 μmol/L); and with salbutamol (10 μmol/L) and atenolol (1 μmol/L), respectively. β1- and β2-adrenergic stimulations induced a significant increase in Ca2+ transient amplitude and cell shortening in intact rat ventricular myocytes (i.e., surface sarcolemma and t-tubules) and in detubulated cells (depleted from t-tubules, surface sarcolemma only). Both β1- and β2-adrenergic receptors stimulation caused a greater effect on Ca2+ transient and cell shortening in detubulated myocytes than in control myocytes. Quantitative analysis indicates that β1-adrenergic stimulation is ∼3 times more effective at surface sarcolemma compared to t-tubules, whereas β2- adrenergic stimulation occurs almost exclusively at surface sarcolemma (∼100 times more effective). These physiological data demonstrate that in rat ventricular myocytes, β1-adrenergic receptors are functionally present at surface sarcolemma and t-tubules, while β2-adrenergic receptors stimulation occurs only at surface sarcolemma of cardiac cells. PMID:24303124

Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes.

PubMed

Bryant, Simon; Kimura, Tomomi E; Kong, Cherrie H T; Watson, Judy J; Chase, Anabelle; Suleiman, M Saadeh; James, Andrew F; Orchard, Clive H

2014-03-01

L-type Ca channels (LTCC), which play a key role in cardiac excitation-contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

α4-Integrin Mediates Neutrophil-Induced Free Radical Injury to Cardiac Myocytes

PubMed Central

Poon, Betty Y.; Ward, Christopher A.; Cooper, Conan B.; Giles, Wayne R.; Burns, Alan R.; Kubes, Paul

2001-01-01

Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via β2-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the α4-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) β2-integrins (CD18) still signal oxidant production, or if this process is now coupled to the α4-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte–PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30–50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both α4-integrin antibody (Ab)-treated PMNs and NADPH oxidase–deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti–α4-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the α4-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed α4-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the α4-integrin–coupled NADPH

Pharmacological targeting of CDK9 in cardiac hypertrophy.

PubMed

Krystof, Vladimír; Chamrád, Ivo; Jorda, Radek; Kohoutek, Jirí

2010-07-01

Cardiac hypertrophy allows the heart to adapt to workload, but persistent or unphysiological stimulus can result in pump failure. Cardiac hypertrophy is characterized by an increase in the size of differentiated cardiac myocytes. At the molecular level, growth of cells is linked to intensive transcription and translation. Several cyclin-dependent kinases (CDKs) have been identified as principal regulators of transcription, and among these CDK9 is directly associated with cardiac hypertrophy. CDK9 phosphorylates the C-terminal domain of RNA polymerase II and thus stimulates the elongation phase of transcription. Chronic activation of CDK9 causes not only cardiac myocyte enlargement but also confers predisposition to heart failure. Due to the long interest of molecular oncologists and medicinal chemists in CDKs as potential targets of anticancer drugs, a portfolio of small-molecule inhibitors of CDK9 is available. Recent determination of CDK9's crystal structure now allows the development of selective inhibitors and their further optimization in terms of biochemical potency and selectivity. CDK9 may therefore constitute a novel target for drugs against cardiac hypertrophy.

Cardiac myocyte-protective effect of microRNA-22 during ischemia and reperfusion through disrupting the caveolin-3/eNOS signaling

PubMed Central

Chen, Zhenfei; Qi, Yinliang; Gao, Chao

2015-01-01

MicroRNA-22 (miR-22) was previously reported to elicit cardiac myocyte hypertrophy and had an anti-apoptotic effect on neurons. However, its effects on cardiac myocyte apoptosis and cardiac function during ischemia and reperfusion (I/R) are not clear. In the present study, we demonstrate that pre-administration of miR-22 mimic reduced I/R-induced cardiac dysfunction significantly in a rat model. We found that miR-22 overexpression inhibited cardiac myocyte apoptosis, and reduced cardiac remodeling during I/R. Significant cardiac myocyte apoptosis was also observed in a cardiac myocyte model after hypoxia/reoxygenation (H/R), a representative process of I/R. Further experiments showed that eNOS activity and the following NO production were significantly decreased during I/R and H/R, while such decrease was inhibited by overexpression of miR-22. Mechanistically, overexpression of miR-22 had little effect on the total protein level of eNOS, but restored the level of p-eNOS (Ser1177) which was down-regulated during H/R. Further RT-PCR results demonstrated that Caveolin 3 (Cav3), an upstream negative regulator of eNOS, was upregulated during H/R, resulting in a decrease of p-eNOS. However, such upregulation of Cav3 transcript level was inhibited directly by miR-22 during H/R, leading to a restored p-eNOS level and followed NO production in cardiac myocytes. Together, the present study revealed that miR-22 down-regulated Cav3, leading to restored eNOS activity and NO production, which further inhibited cardiac myocyte apoptosis and promoted cardiac function after I/R. Of clinical interest, the present study may highlight miR-22 as a potential therapeutic agent for reducing I/R induced cardiac injury. PMID:26191152

Protection of adult rat cardiac myocytes from ischemic cell death: role of caveolar microdomains and delta-opioid receptors.

PubMed

Patel, Hemal H; Head, Brian P; Petersen, Heidi N; Niesman, Ingrid R; Huang, Diane; Gross, Garrett J; Insel, Paul A; Roth, David M

2006-07-01

The role of caveolae, membrane microenvironments enriched in signaling molecules, in myocardial ischemia is poorly defined. In the current study, we used cardiac myocytes prepared from adult rats to test the hypothesis that opioid receptors (OR), which are capable of producing cardiac protection in vivo, promote cardiac protection in cardiac myocytes in a caveolae-dependent manner. We determined protein expression and localization of delta-OR (DOR) using coimmunohistochemistry, caveolar fractionation, and immunoprecipitations. DOR colocalized in fractions with caveolin-3 (Cav-3), a structural component of caveolae in muscle cells, and could be immunoprecipitated by a Cav-3 antibody. Immunohistochemistry confirmed plasma membrane colocalization of DOR with Cav-3. Cardiac myocytes were subjected to simulated ischemia (2 h) or an ischemic preconditioning (IPC) protocol (10 min ischemia, 30 min recovery, 2 h ischemia) in the presence and absence of methyl-beta-cyclodextrin (MbetaCD, 2 mM), which binds cholesterol and disrupts caveolae. We also assessed the cardiac protective effects of SNC-121 (SNC), a selective DOR agonist, on cardiac myocytes with or without MbetaCD and MbetaCD preloaded with cholesterol. Ischemia, simulated by mineral oil layering to inhibit gas exchange, promoted cardiac myocyte cell death (trypan blue staining), a response blunted by SNC (37 +/- 3 vs. 59 +/- 3% dead cells in the presence and absence of 1 muM SNC, respectively, P < 0.01) or by use of the IPC protocol (35 +/- 4 vs. 62 +/- 3% dead cells, P < 0.01). MbetaCD treatment, which disrupted caveolae (as detected by electron microscopy), fully attenuated the protective effects of IPC or SNC, resulting in cell death comparable to that of the ischemic group. By contrast, SNC-induced protection was not abrogated in cells incubated with cholesterol-saturated MbetaCD, which maintained caveolae structure and function. These findings suggest a key role for caveolae, perhaps through enrichment of

The Interplay of Rogue and Clustered Ryanodine Receptors Regulates Ca2+ Waves in Cardiac Myocytes.

PubMed

Chen, Xudong; Feng, Yundi; Huo, Yunlong; Tan, Wenchang

2018-01-01

Ca 2+ waves in cardiac myocytes can lead to arrhythmias owing to delayed after-depolarisations. Based on Ca 2+ regulation from the junctional sarcoplasmic reticulum (JSR), a mathematical model was developed to investigate the interplay of clustered and rogue RyRs on Ca 2+ waves. The model successfully reproduces Ca 2+ waves in cardiac myocytes, which are in agreement with experimental results. A new wave propagation mode of "spark-diffusion-quark-spark" is put forward. It is found that rogue RyRs greatly increase the initiation of Ca 2+ sparks, further contribute to the formation and propagation of Ca 2+ waves when the free Ca 2+ concentration in JSR lumen ([Ca 2+ ] lumen ) is higher than a threshold value of 0.7 mM. Computational results show an exponential increase in the velocity of Ca 2+ waves with [Ca 2+ ] lumen . In addition, more CRUs of rogue RyRs and Ca 2+ release from rogue RyRs result in higher velocity and amplitude of Ca 2+ waves. Distance between CRUs significantly affects the velocity of Ca 2+ waves, but not the amplitude. This work could improve understanding the mechanism of Ca 2+ waves in cardiac myocytes.

The Interplay of Rogue and Clustered Ryanodine Receptors Regulates Ca2+ Waves in Cardiac Myocytes

PubMed Central

Chen, Xudong; Feng, Yundi; Huo, Yunlong; Tan, Wenchang

2018-01-01

Ca2+ waves in cardiac myocytes can lead to arrhythmias owing to delayed after-depolarisations. Based on Ca2+ regulation from the junctional sarcoplasmic reticulum (JSR), a mathematical model was developed to investigate the interplay of clustered and rogue RyRs on Ca2+ waves. The model successfully reproduces Ca2+ waves in cardiac myocytes, which are in agreement with experimental results. A new wave propagation mode of “spark-diffusion-quark-spark” is put forward. It is found that rogue RyRs greatly increase the initiation of Ca2+ sparks, further contribute to the formation and propagation of Ca2+ waves when the free Ca2+ concentration in JSR lumen ([Ca2+]lumen) is higher than a threshold value of 0.7 mM. Computational results show an exponential increase in the velocity of Ca2+ waves with [Ca2+]lumen. In addition, more CRUs of rogue RyRs and Ca2+ release from rogue RyRs result in higher velocity and amplitude of Ca2+ waves. Distance between CRUs significantly affects the velocity of Ca2+ waves, but not the amplitude. This work could improve understanding the mechanism of Ca2+ waves in cardiac myocytes. PMID:29755362

The Electrophysiological Effects of Qiliqiangxin on Cardiac Ventricular Myocytes of Rats

PubMed Central

Wei, Yidong; Liu, Xiaoyu; Wei, Haidong; Hou, Lei; Che, Wenliang; The, Erlinda; Li, Gang; Jhummon, Muktanand Vikash; Wei, Wanlin

2013-01-01

Qiliqiangxin, a Chinese herb, represents the affection in Ca channel function of cardiac myocytes. It is unknown whether Qiliqiangxin has an effect on Na current and K current because the pharmacological actions of this herb's compound are very complex. We investigated the rational usage of Qiliqiangxin on cardiac ventricular myocytes of rats. Ventricular myocytes were exposed acutely to 1, 10, and 50 mg/L Qiliqiangxin, and whole cell patch-clamp technique was used to study the acute effects of Qiliqiangxin on Sodium current (I Na), outward currents delayed rectifier outward K+ current (I K), slowly activating delayed rectifier outward K+ current (I Ks), transient outward K+ current (I to), and inward rectifier K+ current (I K1). Qiliqiangxin can decrease I Na by 28.53% ± 5.98%, and its IC50 was 9.2 mg/L. 10 and 50 mg/L Qiliqiangxin decreased by 37.2% ± 6.4% and 55.9% ± 5.5% summit current density of I to. 10 and 50 mg/L Qiliqiangxin decreased I Ks by 15.51% ± 4.03% and 21.6% ± 5.6%. Qiliqiangxin represented a multifaceted pharmacological profile. The effects of Qiliqiangxin on Na and K currents of ventricular myocytes were more profitable in antiarrhythmic therapy in the clinic. We concluded that the relative efficacy of Qiliqiangxin was another choice for the existing antiarrhythmic therapy. PMID:24250713

PGC-1{alpha} accelerates cytosolic Ca{sup 2+} clearance without disturbing Ca{sup 2+} homeostasis in cardiac myocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Min, E-mail: chenminyx@gmail.com; Yunnan Centers for Diseases Prevention and Control, Kunming 650022; Wang, Yanru

2010-06-11

Energy metabolism and Ca{sup 2+} handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1{alpha}) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1{alpha} in cardiac Ca{sup 2+} signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1{alpha} via adenoviral transduction. Our data shows that overexpressing PGC-1{alpha} improved myocyte contractility without increasing the amplitude of Ca{sup 2+}more » transients, suggesting that myofilament sensitivity to Ca{sup 2+} increased. Interestingly, the decay kinetics of global Ca{sup 2+} transients and Ca{sup 2+} waves accelerated in PGC-1{alpha}-expressing cells, but the decay rate of caffeine-elicited Ca{sup 2+} transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca{sup 2+}-ATPase (SERCA2a), but not Na{sup +}/Ca{sup 2+} exchange (NCX) contribute to PGC-1{alpha}-induced cytosolic Ca{sup 2+} clearance. Furthermore, PGC-1{alpha} induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1{alpha} did not disturb cardiac Ca{sup 2+} homeostasis, because SR Ca{sup 2+} load and the propensity for Ca{sup 2+} waves remained unchanged. These data suggest that PGC-1{alpha} can ameliorate cardiac Ca{sup 2+} cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1{alpha}-calcium handing pathway sheds new light on the role of PGC-1{alpha} in the therapy of cardiac diseases.« less

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy

PubMed Central

Zhou, Jibin; Ahmad, Firdos; Parikh, Shan; Hoffman, Nichole E.; Rajan, Sudarsan; Verma, Vipin K.; Song, Jianliang; Yuan, Ancai; Shanmughapriya, Santhanam; Guo, Yuanjun; Gao, Erhe; Koch, Walter; Woodgett, James R.; Muniswamy, Madesh; Kishore, Raj; Lal, Hind; Force, Thomas

2016-01-01

Rationale Cardiac myocyte-specific deletion of either Glycogen Synthase Kinase (GSK)3A or GSK3B leads to cardiac protection following myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration due to the fact that all GSK-3–targeted drugs including the drugs already in clinical trial target both isoforms of GSK-3 and none are isoform specific. Objective To identify the consequences of combined deletion of cardiac myocyte GSK3A and GSK3B in heart function. Methods and Results We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout, DKO). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, DKO hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from DKO implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. DKO cardiac myocytes showed cell cycle progression resulting in increased DNA content and multi-nucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. Conclusion Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis and its loss is incompatible with life due to cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy. PMID:26976650

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy.

PubMed

Zhou, Jibin; Ahmad, Firdos; Parikh, Shan; Hoffman, Nichole E; Rajan, Sudarsan; Verma, Vipin K; Song, Jianliang; Yuan, Ancai; Shanmughapriya, Santhanam; Guo, Yuanjun; Gao, Erhe; Koch, Walter; Woodgett, James R; Madesh, Muniswamy; Kishore, Raj; Lal, Hind; Force, Thomas

2016-04-15

Cardiac myocyte-specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3β leads to cardiac protection after myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration because of the fact that all GSK-3-targeted drugs, including the drugs already in clinical trial target both isoforms of GSK-3, and none are isoform specific. To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3β in heart function. We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, double-knockout hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from double-knockout implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. Double-knockout cardiac myocytes showed cell cycle progression resulting in increased DNA content and multinucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe-induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis, and its loss is incompatible with life because of cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy. © 2016 American Heart Association, Inc.

Reversal of cardiac myocyte dysfunction as a unique mechanism of rescue by P2X4 receptors in cardiomyopathy.

PubMed

Shen, Jian-Bing; Shutt, Robin; Agosto, Mariela; Pappano, Achilles; Liang, Bruce T

2009-04-01

Binary cardiac transgenic (Tg) overexpression of P2X(4) receptors (P2X(4)R) improved the survival of the cardiomyopathic calsequestrin (CSQ) mice. Here we studied the mechanism of rescue using binary P2X(4)R/CSQ Tg and CSQ Tg mice as models. Cellular and intact heart properties were determined by simultaneous sarcomere shortening (SS) and Ca(2+) transients in vitro and echocardiography in vivo. Similar to a delay in death, binary mice exhibited a slowed heart failure progression with a greater left ventricular (LV) fractional shortening (FS) and thickness and a concomitant lesser degree of LV dilatation in both systole and diastole at 8 or 12 wk. By 16 wk, binary hearts showed similarly depressed FS and thinned out LV and equal enlargement of LV as did 12-wk-old CSQ hearts. Binary cardiac myocytes showed higher peak basal cell shortening (CS) and SS as well as greater basal rates of shortening and relaxation than did the CSQ myocytes at either 8 or 12 wk. Similar data were obtained in comparing the Ca(2+) transient. At 16 wk, binary myocytes were like the 12-wk-old CSQ myocytes with equally depressed CS, SS, and Ca(2+) transient. CSQ myocytes were longer than myocytes from wild-type and binary mice at 12 wk of age. At 16 wk, the binary myocyte length increased to that of the 12-wk-old CSQ myocyte, parallel to LV dilatation. The data suggest a unique mechanism, which involves a reversal of cardiac myocyte dysfunction and a delay in heart failure progression. It represents an example of targeting the abnormal failing myocyte in treating heart failure.

Sulforaphane protects H9c2 cardiomyocytes from angiotensin II-induced hypertrophy.

PubMed

Wu, Q-Q; Zong, J; Gao, L; Dai, J; Yang, Z; Xu, M; Fang, Y; Ma, Z-G; Tang, Q-Z

2014-05-01

Cardiac hypertrophy is an adaptive process of the heart in response to various stimuli, but sustained cardiac hypertrophy will finally lead to heart failure. Sulforaphane-extracted from cruciferous vegetables of the genus Brassica such as broccoli, brussels sprouts, and cabbage-has been evaluated for its anticarcinogenic and antioxidant effects. To investigate the effect of sulforaphane on angiotensin II (Ang II)-induced cardiac hypertrophy in vitro. Embryonic rat heart-derived H9c2 cells were co-incubated with sulforaphane and Ang II. The cell surface area and mRNA levels of hypertrophic markers were measured to clarify the effect of sulforaphane on cardiac hypertrophy. The underlying mechanism was further investigated by detecting the activation of Akt and NF-κB signaling pathways. We found that H9c2 cells pretreated with sulforaphane were protected from Ang II-induced hypertrophy. The increasing mRNA levels of ANP, BNP, and β-MHC in Ang II-stimulated cells were also down-regulated after sulforaphane treatment. Moreover, sulforaphane repressed the Ang II-induced phosphorylation of Akt, GSK3β, mTOR, eIF4e, as well as of IκBα and NF-κB. Based on our results, sulforaphane attenuates Ang II-induced hypertrophy of H9c2 cardiomyocytes mediated by the inhibition of intracellular signaling pathways including Akt and NF-κB.

T-type Ca2+ channels regulate the exit of cardiac myocytes from the cell cycle after birth

PubMed Central

Wang, Fang; Gao, Hui; Kubo, Hajime; Fan, Xiaoxuan; Zhang, Hongyu; Berretta, Remus; Chen, Xiongwen; Sharp, Thomas; Starosta, Timothy; Makarewich, Catherine; Li, Ying; Molkentin, Jeffrey D.; Houser, Steven R.

2013-01-01

T-type Ca2+ channels (TTCCs) are expressed in the fetal heart and then disappear from ventricular myocytes after birth. The hypothesis examined in this study was the α1G TTCCs' influence in myocyte maturation and their rapid withdrawal from the cell cycle after birth. Methods Cardiac myocytes were isolated from neonatal and adult wild type (WT), α1G−/− and α1G over expressing (α1GDT) mice. Bromodeoxyuridine (BrdU) uptake, myocyte nucleation, cell cycle analysis, and T-type Ca2+ currents were measured. Results All myocytes were mono-nucleated at birth and 35% of WT myocytes expressed functional TTCCs. Very few neonatal myocytes had functional TTCCs in α1G−/− hearts. By the end of the first week after birth no WT or α1G−/− had functional TTCCs. During the first week after birth about 25% of WT myocytes were BrdU+ and became bi-nucleated. Significantly fewer α1G−/− myocytes became bi-nucleated and fewer of these myocytes were BrdU+. Neonatal α1G−/− myocytes were also smaller than WT. Adult WT and α1G−/− hearts were similar in size, but α1G−/− myocytes were smaller and a greater % were mono-nucleated. α1G over expressing hearts were smaller than WT but their myocytes were larger. Conclusions The studies performed show that loss of functional TTCCs is associated with bi-nucleation and myocyte withdrawal from the cell cycle. Loss of α1G TTCCs slowed the transition from mono- to bi-nucleation and resulted in an adult heart with a greater number of small cardiac myocytes. These results suggest that TTCCs are involved in the regulation of myocyte size and the exit of myocytes from the cell cycle during the first week after birth. PMID:23743021

Bucindolol, a nonselective beta 1- and beta 2-adrenergic receptor antagonist, decreases beta-adrenergic receptor density in cultured embryonic chick cardiac myocyte membranes.

PubMed

Asano, K; Zisman, L S; Yoshikawa, T; Headley, V; Bristow, M R; Port, J D

2001-06-01

Bucindolol and carvedilol, nonselective beta1- and beta2-adrenergic receptor antagonists, have been widely used in clinical therapeutic trials of congestive heart failure. The aim of the current study was to investigate long-term effects of bucindolol or carvedilol on beta-adrenergic receptor protein and gene expression in cardiac myocytes. Embryonic chick cardiac myocytes were cultured and incubated with bucindolol (1 microM), carvedilol (1 microM), or norepinephrine (1 microM) for 24 h. 125I-iodocyanopindolol binding assays demonstrated that incubation with norepinephrine or bucindolol, but not carvedilol, significantly decreased beta-adrenergic receptor density in crude membranes prepared from the myocytes. Neither bucindolol nor carvedilol significantly stimulated adenylyl cyclase activity in membranes from drug-untreated cells. Unlike by norepinephrine, the receptor density reduction by bucindolol incubation was not accompanied by a change in beta1-adrenergic receptor messenger RNA abundance. A decrease in membrane beta-adrenergic receptor density without a change in cognate messenger RNA abundance was also observed in hamster DDT1 MF2 cell line incubated with bucindolol (1 microM, 24 h). We conclude that incubation with bucindolol, but not carvedilol, results in true reduction of beta-adrenergic receptor density in chick cardiac myocyte membranes by mechanisms that are distinct from those responsible for receptor density reduction by the agonist norepinephrine.

[Image processing applying in analysis of motion features of cultured cardiac myocyte in rat].

PubMed

Teng, Qizhi; He, Xiaohai; Luo, Daisheng; Wang, Zhengrong; Zhou, Beiyi; Yuan, Zhirun; Tao, Dachang

2007-02-01

Study of mechanism of medicine actions, by quantitative analysis of cultured cardiac myocyte, is one of the cutting edge researches in myocyte dynamics and molecular biology. The characteristics of cardiac myocyte auto-beating without external stimulation make the research sense. Research of the morphology and cardiac myocyte motion using image analysis can reveal the fundamental mechanism of medical actions, increase the accuracy of medicine filtering, and design the optimal formula of medicine for best medical treatments. A system of hardware and software has been built with complete sets of functions including living cardiac myocyte image acquisition, image processing, motion image analysis, and image recognition. In this paper, theories and approaches are introduced for analysis of living cardiac myocyte motion images and implementing quantitative analysis of cardiac myocyte features. A motion estimation algorithm is used for motion vector detection of particular points and amplitude and frequency detection of a cardiac myocyte. Beatings of cardiac myocytes are sometimes very small. In such case, it is difficult to detect the motion vectors from the particular points in a time sequence of images. For this reason, an image correlation theory is employed to detect the beating frequencies. Active contour algorithm in terms of energy function is proposed to approximate the boundary and detect the changes of edge of myocyte.

Effects of rogue ryanodine receptors on Ca2+ sparks in cardiac myocytes

PubMed Central

Chen, Xudong; Feng, Yundi; Tan, Wenchang

2018-01-01

Ca2+ sparks and Ca2+ quarks, arising from clustered and rogue ryanodine receptors (RyRs), are significant Ca2+ release events from the junctional sarcoplasmic reticulum (JSR). Based on the anomalous subdiffusion of Ca2+ in the cytoplasm, a mathematical model was developed to investigate the effects of rogue RyRs on Ca2+ sparks in cardiac myocytes. Ca2+ quarks and sparks from the stochastic opening of rogue and clustered RyRs are numerically reproduced and agree with experimental measurements. It is found that the stochastic opening Ca2+ release units (CRUs) of clustered RyRs are regulated by free Ca2+ concentration in the JSR lumen (i.e. [Ca2+]lumen). The frequency of spontaneous Ca2+ sparks is remarkably increased by the rogue RyRs opening at high [Ca2+]lumen, but not at low [Ca2+]lumen. Hence, the opening of rogue RyRs contributes to the formation of Ca2+ sparks at high [Ca2+]lumen. The interplay of Ca2+ sparks and Ca2+ quarks has been discussed in detail. This work is of significance to provide insight into understanding Ca2+ release mechanisms in cardiac myocytes. PMID:29515864

Effects of rogue ryanodine receptors on Ca2+ sparks in cardiac myocytes.

PubMed

Chen, Xudong; Feng, Yundi; Huo, Yunlong; Tan, Wenchang

2018-02-01

Ca 2+ sparks and Ca 2+ quarks, arising from clustered and rogue ryanodine receptors (RyRs), are significant Ca 2+ release events from the junctional sarcoplasmic reticulum (JSR). Based on the anomalous subdiffusion of Ca 2+ in the cytoplasm, a mathematical model was developed to investigate the effects of rogue RyRs on Ca 2+ sparks in cardiac myocytes. Ca 2+ quarks and sparks from the stochastic opening of rogue and clustered RyRs are numerically reproduced and agree with experimental measurements. It is found that the stochastic opening Ca 2+ release units (CRUs) of clustered RyRs are regulated by free Ca 2+ concentration in the JSR lumen (i.e. [Ca 2+ ] lumen ). The frequency of spontaneous Ca 2+ sparks is remarkably increased by the rogue RyRs opening at high [Ca 2+ ] lumen , but not at low [Ca 2+ ] lumen . Hence, the opening of rogue RyRs contributes to the formation of Ca 2+ sparks at high [Ca 2+ ] lumen . The interplay of Ca 2+ sparks and Ca 2+ quarks has been discussed in detail. This work is of significance to provide insight into understanding Ca 2+ release mechanisms in cardiac myocytes.

Minocycline suppresses oxidative stress and attenuates fetal cardiac myocyte apoptosis triggered by in utero cocaine exposure

PubMed Central

Sinha-Hikim, Indrani; Shen, Ruoqing; Nzenwa, Ify; Gelfand, Robert; Mahata, Sushil K.

2015-01-01

This study investigates the molecular mechanisms by which minocycline, a second generation tetracycline, prevents cardiac myocyte death induced by in utero cocaine exposure. Timed mated pregnant Sprague-Dawley (SD) rats received one of the following treatments twice daily from embryonic (E) day 15–21 (E15–E21): (i) intraperitoneal (IP) injections of saline (control); (ii) IP injections of cocaine (15 mg/kg BW); and (iii) IP injections of cocaine + oral administration of 25 mg/kg BW of minocycline. Pups were killed on postnatal day 15 (P15). Additional pregnant dams received twice daily IP injections of cocaine (from E15–E21) + oral administration of a relatively higher (37.5 mg/kg BW) dose of minocycline. Minocycline treatment continued from E15 until the pups were sacrificed on P15. In utero cocaine exposure resulted in an increase in oxidative stress and fetal cardiac myocyte apoptosis through activation of c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK)-mediated mitochondria-dependent apoptotic pathway. Continued minocycline treatment from E15 through P15 significantly prevented oxidative stress, kinase activation, perturbation of BAX/BCL-2 ratio, cytochrome c release, caspase activation, and attenuated fetal cardiac myocyte apoptosis after prenatal cocaine exposure. These results demonstrate in vivo cardioprotective effects of minocycline in preventing fetal cardiac myocyte death after prenatal cocaine exposure. Given its proven clinical safety and ability to cross the placental barrier and enter into the fetal circulation, minocycline may be an effective therapy for preventing cardiac consequences of in utero cocaine exposure. PMID:21424555

The Permeability Transition Pore Controls Cardiac Mitochondrial Maturation and Myocyte Differentiation

PubMed Central

Hom, Jennifer R.; Quintanilla, Rodrigo A.; Hoffman, David L.; Karen L., de Mesy Bentley; Molkentin, Jeffery D.; Sheu, Shey-Shing; Porter, George A.

2011-01-01

SUMMARY Although mature myocytes rely on mitochondria as the primary source of energy, the role of mitochondria in the developing heart is not well known. Here, we find closure of the mitochondrial permeability transition pore (mPTP) drives maturation of mitochondrial structure and function and myocyte differentiation. Cardiomyocytes at embryonic day (E) 9.5, when compared to E13.5, displayed fragmented mitochondria with few cristae, a less polarized mitochondrial membrane potential, higher reactive oxygen species (ROS) levels, and an open mPTP. Pharmacologic and genetic closing of the mPTP yielded maturation of mitochondrial structure and function, lowered ROS, and increased myocyte differentiation (measured by counting Z-bands). Furthermore, myocyte differentiation was inhibited and enhanced with oxidant and antioxidant treatment, respectively, suggesting that redox signaling pathways lie downstream of mitochondria to regulate cardiac myocyte differentiation. PMID:21920313

Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes.

PubMed

Marian, A J; Yu, Q T; Mann, D L; Graham, F L; Roberts, R

1995-07-01

Mutations in the beta-myosin heavy chain (beta MyHC) induce hypertrophic cardiomyopathy (HCM), cardiac hypertrophy, and sarcomere disarray, with the latter being the characteristic hallmark. Thus, we sought to determine whether expression of mutant beta MyHC in adult feline cardiac myocytes, a species known to develop HCM with a phenotype identical to that in humans, induces sarcomere disarray. A full-length beta MyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the beta MyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant beta MyHC cDNAs were cloned into p delta E1spIB shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/beta MyHC-N and Ad5/CMV/beta MyHC-403) were generated through homologous recombination of p delta E1spIB/CMV/beta MyHC-N or Ad5/CMV/beta MyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-1 cells with the beta MyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was > 95%. Expression of the beta MyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription-PCR. The net total protein and beta-myosin synthesis were determined by using the amount of incorporation of [3H]phenylalanine into total protein and beta-myosin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

PDE1C deficiency antagonizes pathological cardiac remodeling and dysfunction

PubMed Central

Knight, Walter E.; Chen, Si; Zhang, Yishuai; Oikawa, Masayoshi; Wu, Meiping; Zhou, Qian; Miller, Clint L.; Cai, Yujun; Mickelsen, Deanne M.; Moravec, Christine; Small, Eric M.; Abe, Junichi; Yan, Chen

2016-01-01

Cyclic nucleotide phosphodiesterase 1C (PDE1C) represents a major phosphodiesterase activity in human myocardium, but its function in the heart remains unknown. Using genetic and pharmacological approaches, we studied the expression, regulation, function, and underlying mechanisms of PDE1C in the pathogenesis of cardiac remodeling and dysfunction. PDE1C expression is up-regulated in mouse and human failing hearts and is highly expressed in cardiac myocytes but not in fibroblasts. In adult mouse cardiac myocytes, PDE1C deficiency or inhibition attenuated myocyte death and apoptosis, which was largely dependent on cyclic AMP/PKA and PI3K/AKT signaling. PDE1C deficiency also attenuated cardiac myocyte hypertrophy in a PKA-dependent manner. Conditioned medium taken from PDE1C-deficient cardiac myocytes attenuated TGF-β–stimulated cardiac fibroblast activation through a mechanism involving the crosstalk between cardiac myocytes and fibroblasts. In vivo, cardiac remodeling and dysfunction induced by transverse aortic constriction, including myocardial hypertrophy, apoptosis, cardiac fibrosis, and loss of contractile function, were significantly attenuated in PDE1C-knockout mice relative to wild-type mice. These results indicate that PDE1C activation plays a causative role in pathological cardiac remodeling and dysfunction. Given the continued development of highly specific PDE1 inhibitors and the high expression level of PDE1C in the human heart, our findings could have considerable therapeutic significance. PMID:27791092

Berberine treatment prevents cardiac dysfunction and remodeling through activation of 5'-adenosine monophosphate-activated protein kinase in type 2 diabetic rats and in palmitate-induced hypertrophic H9c2 cells.

PubMed

Chang, Wenguang; Zhang, Ming; Meng, Zhaojie; Yu, Yang; Yao, Fan; Hatch, Grant M; Chen, Li

2015-12-15

Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5'-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy. Copyright © 2015 Elsevier B.V. All rights reserved.

Minocycline suppresses oxidative stress and attenuates fetal cardiac myocyte apoptosis triggered by in utero cocaine exposure.

PubMed

Sinha-Hikim, Indrani; Shen, Ruoqing; Nzenwa, Ify; Gelfand, Robert; Mahata, Sushil K; Sinha-Hikim, Amiya P

2011-06-01

This study investigates the molecular mechanisms by which minocycline, a second generation tetracycline, prevents cardiac myocyte death induced by in utero cocaine exposure. Timed mated pregnant Sprague-Dawley (SD) rats received one of the following treatments twice daily from embryonic (E) day 15-21 (E15-E21): (i) intraperitoneal (IP) injections of saline (control); (ii) IP injections of cocaine (15 mg/kg BW); and (iii) IP injections of cocaine + oral administration of 25 mg/kg BW of minocycline. Pups were killed on postnatal day 15 (P15). Additional pregnant dams received twice daily IP injections of cocaine (from E15-E21) + oral administration of a relatively higher (37.5 mg/kg BW) dose of minocycline. Minocycline treatment continued from E15 until the pups were sacrificed on P15. In utero cocaine exposure resulted in an increase in oxidative stress and fetal cardiac myocyte apoptosis through activation of c-Jun-NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK)-mediated mitochondria-dependent apoptotic pathway. Continued minocycline treatment from E15 through P15 significantly prevented oxidative stress, kinase activation, perturbation of BAX/BCL-2 ratio, cytochrome c release, caspase activation, and attenuated fetal cardiac myocyte apoptosis after prenatal cocaine exposure. These results demonstrate in vivo cardioprotective effects of minocycline in preventing fetal cardiac myocyte death after prenatal cocaine exposure. Given its proven clinical safety and ability to cross the placental barrier and enter into the fetal circulation, minocycline may be an effective therapy for preventing cardiac consequences of in utero cocaine exposure.

Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H2O2-induced oxidative stress in H9c2 rat cardiomyoblasts.

PubMed

Chen, Wei-Cheng; Hsieh, Shih-Rong; Chiu, Chun-Hwei; Hsu, Ban-Dar; Liou, Ying-Ming

2014-06-09

Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca2+ in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H2O2-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts. Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 μM H2O2 exposure for 30 min with and/or without 10 to 20 μM EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H2O2-induced injury in H9c2 cells. H2O2 exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca2+ overload, increases in glycolytic protein, α-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H2O2-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3β (GSK-3β)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3β inhibitor (SB 216763) significantly improved the H2O2-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3β (S9), and level of cyclin D1 in cells. Collectively, these findings suggest that EGCG blunts the H2O2-induced oxidative effect on the Akt activity

Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H2O2-induced oxidative stress in H9c2 rat cardiomyoblasts

PubMed Central

2014-01-01

Background Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca2+ in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H2O2-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts. Results Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 μM H2O2 exposure for 30 min with and/or without 10 to 20 μM EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H2O2-induced injury in H9c2 cells. H2O2 exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca2+ overload, increases in glycolytic protein, α-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H2O2-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3β (GSK-3β)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3β inhibitor (SB 216763) significantly improved the H2O2-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3β (S9), and level of cyclin D1 in cells. Conclusions Collectively, these findings suggest that EGCG blunts the H2O2-induced oxidative

Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2.

PubMed

Zoccarato, Anna; Surdo, Nicoletta C; Aronsen, Jan M; Fields, Laura A; Mancuso, Luisa; Dodoni, Giuliano; Stangherlin, Alessandra; Livie, Craig; Jiang, He; Sin, Yuan Yan; Gesellchen, Frank; Terrin, Anna; Baillie, George S; Nicklin, Stuart A; Graham, Delyth; Szabo-Fresnais, Nicolas; Krall, Judith; Vandeput, Fabrice; Movsesian, Matthew; Furlan, Leonardo; Corsetti, Veronica; Hamilton, Graham; Lefkimmiatis, Konstantinos; Sjaastad, Ivar; Zaccolo, Manuela

2015-09-25

Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications. © 2015 American Heart Association, Inc.

Mitochondria-Targeted Antioxidant Prevents Cardiac Dysfunction Induced by Tafazzin Gene Knockdown in Cardiac Myocytes

PubMed Central

He, Quan; Harris, Nicole; Ren, Jun; Han, Xianlin

2014-01-01

Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS) have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress. PMID:25247053

Effects of seasonal acclimatization on action potentials and sarcolemmal K+ currents in roach (Rutilus rutilus) cardiac myocytes.

PubMed

Badr, Ahmed; Hassinen, Minna; El-Sayed, Mohamed F; Vornanen, Matti

2017-03-01

Temperature sensitivity of electrical excitability is a potential limiting factor for high temperature tolerance of ectotherms. The present study examines whether heat resistance of electrical excitability of cardiac myocytes is modified by seasonal thermal acclimatization in roach (Rutilus rutilus), a eurythermal teleost species. To this end, temperature dependencies of ventricular action potentials (APs), and atrial and ventricular K + currents were measured from winter-acclimatized (WiR) and summer-acclimatized (SuR) roach. Under patch-clamp recording conditions, ventricular APs could be triggered over a wide range of temperatures (4-43°C) with prominent changes in resting membrane potential (RMP), AP duration and amplitude. In general, APs of SuR were slightly more tolerant to high temperatures than those of WiR, e.g. the break point temperature (T BP ) of RMP was 37.6±0.4°C in WiR and 41±1°C in SuR (p<0.05). Of the two major cardiac K + currents, the inward rectifier K + current (I K1 ) was particularly heat resistant in both SuR (T BP 39.4±0.4°C) and WiR (T BP 40.0±0.4°C) ventricular myocytes. The delayed rectifier K + current (I Kr ) was not as heat resistant as I K1 . Surprisingly, I Kr of WiR tolerated heat better (T BP 31.9±0.8°C) than I Kr of SuR (T BP 24.1±0.5°C) (p<0.05). I Kr (Erg2) channel transcripts of both atrial and ventricular myocytes were up-regulated in WiR. I K1 (Kir2) channel transcripts were not affected by seasonal acclimatization, although ventricular I K1 current was up-regulated in summer. Collectively, these findings show that thermal tolerance limits of K + currents in isolated myocytes between seasonally acclimatized roach are much less pronounced than the heat sensitivity of ECG variables in intact fish. Copyright © 2016 Elsevier Inc. All rights reserved.

Non-canonical Wnt signaling enhances differentiation of Sca1+/c-kit+ adipose-derived murine stromal vascular cells into spontaneously beating cardiac myocytes.

PubMed

Palpant, Nathan J; Yasuda, So-ichiro; MacDougald, Ormond; Metzger, Joseph M

2007-09-01

Recent reports have described a stem cell population termed stromal vascular cells (SVCs) derived from the stromal vascular fraction of adipose tissue, which are capable of intrinsic differentiation into spontaneously beating cardiomyocytes in vitro. The objective of this study was to further define the cardiac lineage differentiation potential of SVCs in vitro and to establish methods for enriching SVC-derived beating cardiac myocytes. SVCs were isolated from the stromal vascular fraction of murine adipose tissue. Cells were cultured in methylcellulose-based murine stem cell media. Analysis of SVC-derived beating myocytes included Western blot and calcium imaging. Enrichment of acutely isolated SVCs was carried out using antibody-tagged magnetic nanoparticles, and pharmacologic manipulation of Wnt and cytokine signaling. Under initial media conditions, spontaneously beating SVCs expressed both cardiac developmental and adult protein isoforms. Functionally, this specialized population can spontaneously contract and pace under field stimulation and shows the presence of coordinated calcium transients. Importantly, this study provides evidence for two independent mechanisms of enriching the cardiac differentiation of SVCs. First, this study shows that differentiation of SVCs into cardiac myocytes is augmented by non-canonical Wnt agonists, canonical Wnt antagonists, and cytokines. Second, SVCs capable of cardiac lineage differentiation can be enriched by selection for stem cell-specific membrane markers Sca1 and c-kit. Adipose-derived SVCs are a unique population of stem cells that show evidence of cardiac lineage development making them a potential source for stem cell-based cardiac regeneration studies.

Non-canonical Wnt Signaling Enhances differentiation of Sca1+/c-kit+ Adipose-derived Murine Stromal Vascular Cells into Spontaneously Beating Cardiac Myocytes

PubMed Central

Palpant, Nathan J.; Yasuda, So-ichiro; MacDougald, Ormond; Metzger, Joseph M.

2007-01-01

Recent reports have described a stem cell population termed stromal vascular cells (SVCs) derived from the stromal vascular fraction of adipose tissue, which are capable of intrinsic differentiation into spontaneously beating cardiomyocytes in vitro. The objective of this study was to further define the cardiac lineage differentiation potential of SVCs in vitro and to derive methods for enriching SVC-derived beating cardiac myocytes. SVCs were isolated from the stromal vascular fraction of murine adipose tissue. Cells were cultured in methylcellulose-based murine stem cell media. Analysis of SVC-derived beating myocytes included Western blot, and calcium imaging. Enrichment of acutely isolated SVCs was carried out using antibody tagged magnetic nanoparticles, and pharmacologic manipulation of Wnt and cytokine signaling. Under initial media conditions, spontaneously beating SVCs expressed both cardiac developmental and adult protein isoforms. Functionally, this specialized population can spontaneously contract and pace under field stimulation, and shows the presence of coordinated calcium transients. Importantly, this study provides evidence for two independent mechanisms of enriching the cardiac differentiation of SVCs. First, this study shows that differentiation of SVCs into cardiac myocytes is augmented by non-canonical Wnt agonists, canonical Wnt antagonists, and cytokines. Second, SVCs capable of cardiac lineage differentiation can be enriched by selection for stem cell-specific membrane markers Sca1 and c-kit. Adipose-derived SVCs are a unique population of stem cells that show evidence of cardiac lineage development making them a potential source for stem cell-based cardiac regeneration studies. PMID:17706246

Cardiac progenitor cell‑derived exosomes promote H9C2 cell growth via Akt/mTOR activation.

PubMed

Li, Shentang; Jiang, Jie; Yang, Zuocheng; Li, Zhuoying; Ma, Xing; Li, Xin

2018-05-21

Exosomes are cell‑derived vesicles released from a variety of mammalian cells that are involved in cell‑to‑cell signalling. It has been reported that cardiac progenitor cells (CPCs) derived from an adult heart are one of the most promising stem cell types for cardioprotection and repair. The mammalian target of rapamycin (mTOR) signalling pathway is a pivotal regulator in CPCs, therefore, CPC‑derived exosomes were used in the present study to investigate whether it can promote H9C2 cell growth through the protein kinase B (PKB, or Akt)/mTOR signalling pathway. The CPCs were isolated from Sprague‑Dawley hearts. Following treatment with a specific medium, the exosomes were purified and identified by electron micrograph and western blot assays, using CD63 and CD81 as markers. The methyl‑thiazolyl‑tetrazolium and 5‑ethynyl‑2'‑deoxyuridine methods were used to detect H9C2 cell growth. The expression of Akt and mTOR were detected by western blot analysis following treatment with 200 or 400 µg/ml of exosomes for 24 or 48 h, respectively. It was found that, compared with higher concentrations of exosomes, prolonging the duration of exposure promoted cell growth. Accordingly, CPC‑derived exosomes stimulated the expression of Akt to a marked degree; groups treated with exosomes for 48 h showed higher expression of Akt than those treated for 24 h at the same concentration. mTOR was also stimulated by CPC‑derived exosomes. The activation of mTOR increased in accordance with the treatment time at an exosome concentration of 200 µg/ml and decreased with treatment time at an exosome concentration of 400 µg/ml. In conclusion, the present study demonstrated that CPC‑derived exosomes promoted H9C2 cell growth via the activation of Akt/mTOR in a time‑dependent manner at a relatively low exosome concentration, which may provide a novel therapy for cardiovascular disease.

Cardiac P2X purinergic receptors as a new pathway for increasing Na+ entry in cardiac myocytes

PubMed Central

Shen, Jian-Bing; Yang, Ronghua; Pappano, Achilles

2014-01-01

P2X4 receptors (P2X4Rs) are ligand-gated ion channels capable of conducting cations such as Na+. Endogenous cardiac P2X4R can mediate ATP-activated current in adult murine cardiomyocytes. In the present study, we tested the hypothesis that cardiac P2X receptors can induce Na+ entry and modulate Na+ handling. We further determined whether P2X receptor-induced stimulation of the Na+/Ca2+ exchanger (NCX) has a role in modulating the cardiac contractile state. Changes in Na+-K+-ATPase current (Ip) and NCX current (INCX) after agonist stimulation were measured in ventricular myocytes of P2X4 transgenic mice using whole cell patch-clamp techniques. The agonist 2-methylthio-ATP (2-meSATP) increased peak Ip from a basal level of 0.52 ± 0.02 to 0.58 ± 0.03 pA/pF. 2-meSATP also increased the Ca2+ entry mode of INCX (0.55 ± 0.09 pA/pF under control conditions vs. 0.82 ± 0.14 pA/pF with 2-meSATP) at a membrane potential of +50 mV. 2-meSATP shifted the reversal potential of INCX from −14 ± 2.3 to −25 ± 4.1 mV, causing an estimated intracellular Na+ concentration increase of 1.28 ± 0.42 mM. These experimental results were closely mimicked by mathematical simulations based on previously established models. KB-R7943 or a structurally different agent preferentially opposing the Ca2+ entry mode of NCX, YM-244769, could inhibit the 2-meSATP-induced increase in cell shortening in transgenic myocytes. Thus, the Ca2+ entry mode of INCX participates in P2X agonist-stimulated contractions. In ventricular myocytes from wild-type mice, the P2X agonist could increase INCX, and KB-R7943 was able to inhibit the contractile effect of endogenous P2X4Rs, indicating a physiological role of these receptors in wild-type cells. The data demonstrate a novel Na+ entry pathway through ligand-gated P2X4Rs in cardiomyocytes. PMID:25239801

Cloning and characterization of the mouse alpha1C/A-adrenergic receptor gene and analysis of an alpha1C promoter in cardiac myocytes: role of an MCAT element that binds transcriptional enhancer factor-1 (TEF-1).

PubMed

O'Connell, T D; Rokosh, D G; Simpson, P C

2001-05-01

alpha1-Adrenergic receptor (AR) subtypes in the heart are expressed by myocytes but not by fibroblasts, a feature that distinguishes alpha1-ARs from beta-ARs. Here we studied myocyte-specific expression of alpha1-ARs, focusing on the subtype alpha1C (also called alpha1A), a subtype implicated in cardiac hypertrophic signaling in rat models. We first cloned the mouse alpha1C-AR gene, which consisted of two exons with an 18 kb intron, similar to the alpha1B-AR gene. The receptor coding sequence was >90% homologous to that of rat and human. alpha1C-AR transcription in mouse heart was initiated from a single Inr consensus sequence at -588 from the ATG; this and a putative polyadenylation sequence 8.5 kb 3' could account for the predominant 11 kb alpha1C mRNA in mouse heart. A 5'-nontranscribed fragment of 4.4 kb was active as a promoter in cardiac myocytes but not in fibroblasts. Promoter activity in myocytes required a single muscle CAT (MCAT) element, and this MCAT bound in vitro to recombinant and endogenous transcriptional enhancer factor-1. Thus, alpha1C-AR transcription in cardiac myocytes shares MCAT dependence with other cardiac-specific genes, including the alpha- and beta-myosin heavy chains, skeletal alpha-actin, and brain natriuretic peptide. However, the mouse alpha1C gene was not transcribed in the neonatal heart and was not activated by alpha1-AR and other hypertrophic agonists in rat myocytes, and thus differed from other MCAT-dependent genes and the rat alpha1C gene.

Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.

PubMed

Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B Cicero; Zarzoso, Manuel; Ramirez, Rafael J; Sener, Michelle F; Mundada, Lakshmi V; Klos, Matthew; Devaney, Eric J; Vikstrom, Karen L; Herron, Todd J; Jalife, José

2013-11-01

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. © 2013.

Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes.

PubMed

Richards, Mark; Lomas, Oliver; Jalink, Kees; Ford, Kerrie L; Vaughan-Jones, Richard D; Lefkimmiatis, Konstantinos; Swietach, Pawel

2016-06-01

3',5'-Cyclic adenosine monophosphate (cAMP) signals in the heart are often confined to concentration microdomains shaped by cAMP diffusion and enzymatic degradation. While the importance of phosphodiesterases (degradative enzymes) in sculpting cAMP microdomains is well established in cardiomyocytes, less is known about cAMP diffusivity (DcAMP) and factors affecting it. Many earlier studies have reported fast diffusivity, which argues against sharply defined microdomains. [cAMP] dynamics in the cytoplasm of adult rat ventricular myocytes were imaged using a fourth generation genetically encoded FRET-based sensor. The [cAMP]-response to the addition and removal of isoproterenol (β-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read out of DcAMP, a stable [cAMP] gradient was generated using a microfluidic device which delivered agonist to one half of the myocyte only. After accounting for phosphodiesterase activity, DcAMP was calculated to be 32 µm(2)/s; an order of magnitude lower than in water. Diffusivity was independent of the amount of cAMP produced. Saturating cAMP-binding sites with the analogue 6-Bnz-cAMP did not accelerate DcAMP, arguing against a role of buffering in restricting cAMP mobility. cAMP diffused at a comparable rate to chemically unrelated but similar sized molecules, arguing for a common physical cause of restricted diffusivity. Lower mitochondrial density and order in neonatal cardiac myocytes allowed for faster diffusion, demonstrating the importance of mitochondria as physical barriers to cAMP mobility. In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Cardiology.

Disruption of dopamine D1/D2 receptor complex is involved in the function of haloperidol in cardiac H9c2 cells.

PubMed

Lencesova, L; Szadvari, I; Babula, P; Kubickova, J; Chovancova, B; Lopusna, K; Rezuchova, I; Novakova, Z; Krizanova, O; Novakova, M

2017-12-15

Haloperidol is an antipsychotic agent and acts as dopamine D2 receptor (D2R) antagonist, as a prototypical ligand of sigma1 receptors (Sig1R) and it increases expression of type 1 IP 3 receptors (IP 3 R1). However, precise mechanism of haloperidol action on cardiomyocytes through dopaminergic signaling was not described yet. This study investigated a role of dopamine receptors in haloperidol-induced increase in IP 3 R1 and Sig1R, and compared physiological effect of melperone and haloperidol on basic heart parameters in rats. We used differentiated NG-108 cells and H9c2 cells. Gene expression, Western blot and immunofluorescence were used to evaluate haloperidol-induced differences; proximity ligation assay (PLA) and immunoprecipitation to determine interactions of D1/D2 receptors. To evaluate cardiac parameters, Wistar albino male rats were used. We have shown that antagonism of D2R with either haloperidol or melperone results in upregulation of both, IP 3 R1 and Sig1R, which is associated with increased D2R, but reduced D1R expression. Immunofluorescence, immunoprecipitation and PLA support formation of heteromeric D1/D2 complexes in H9c2 cells. Treatment with haloperidol (but not melperone) caused decrease in systolic and diastolic blood pressure and significant increase in heart rate. Because D1R/D2R complexes can engage Gq-like signaling in other experimental systems, these results are consistent with the possibility that disruption of D1R/D2R complex in H9c2 cells might cause a decrease in IP 3 R1 activity, which in turn may account for the increase expression of IP 3 R and Sig1R. D2R is probably not responsible for changes in cardiac parameters, since melperone did not have any effect. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Signaling Pathways in Cardiac Myocyte Apoptosis

PubMed Central

Xia, Peng; Liu, Yuening

2016-01-01

Cardiovascular diseases, the number 1 cause of death worldwide, are frequently associated with apoptotic death of cardiac myocytes. Since cardiomyocyte apoptosis is a highly regulated process, pharmacological intervention of apoptosis pathways may represent a promising therapeutic strategy for a number of cardiovascular diseases and disorders including myocardial infarction, ischemia/reperfusion injury, chemotherapy cardiotoxicity, and end-stage heart failure. Despite rapid growth of our knowledge in apoptosis signaling pathways, a clinically applicable treatment targeting this cellular process is currently unavailable. To help identify potential innovative directions for future research, it is necessary to have a full understanding of the apoptotic pathways currently known to be functional in cardiac myocytes. Here, we summarize recent progress in the regulation of cardiomyocyte apoptosis by multiple signaling molecules and pathways, with a focus on the involvement of these pathways in the pathogenesis of heart disease. In addition, we provide an update regarding bench to bedside translation of this knowledge and discuss unanswered questions that need further investigation. PMID:28101515

Length dependence of force generation exhibit similarities between rat cardiac myocytes and skeletal muscle fibres.

PubMed

Hanft, Laurin M; McDonald, Kerry S

2010-08-01

According to the Frank-Starling relationship, increased ventricular volume increases cardiac output, which helps match cardiac output to peripheral circulatory demand. The cellular basis for this relationship is in large part the myofilament length-tension relationship. Length-tension relationships in maximally calcium activated preparations are relatively shallow and similar between cardiac myocytes and skeletal muscle fibres. During twitch activations length-tension relationships become steeper in both cardiac and skeletal muscle; however, it remains unclear whether length dependence of tension differs between striated muscle cell types during submaximal activations. The purpose of this study was to compare sarcomere length-tension relationships and the sarcomere length dependence of force development between rat skinned left ventricular cardiac myocytes and fast-twitch and slow-twitch skeletal muscle fibres. Muscle cell preparations were calcium activated to yield 50% maximal force, after which isometric force and rate constants (k(tr)) of force development were measured over a range of sarcomere lengths. Myofilament length-tension relationships were considerably steeper in fast-twitch fibres compared to slow-twitch fibres. Interestingly, cardiac myocyte preparations exhibited two populations of length-tension relationships, one steeper than fast-twitch fibres and the other similar to slow-twitch fibres. Moreover, myocytes with shallow length-tension relationships were converted to steeper length-tension relationships by protein kinase A (PKA)-induced myofilament phosphorylation. Sarcomere length-k(tr) relationships were distinct between all three cell types and exhibited patterns markedly different from Ca(2+) activation-dependent k(tr) relationships. Overall, these findings indicate cardiac myocytes exhibit varied length-tension relationships and sarcomere length appears a dominant modulator of force development rates. Importantly, cardiac myocyte length

Cardiac P2X purinergic receptors as a new pathway for increasing Na⁺ entry in cardiac myocytes.

PubMed

Shen, Jian-Bing; Yang, Ronghua; Pappano, Achilles; Liang, Bruce T

2014-11-15

P2X4 receptors (P2X4Rs) are ligand-gated ion channels capable of conducting cations such as Na(+). Endogenous cardiac P2X4R can mediate ATP-activated current in adult murine cardiomyocytes. In the present study, we tested the hypothesis that cardiac P2X receptors can induce Na(+) entry and modulate Na(+) handling. We further determined whether P2X receptor-induced stimulation of the Na(+)/Ca(2+) exchanger (NCX) has a role in modulating the cardiac contractile state. Changes in Na(+)-K(+)-ATPase current (Ip) and NCX current (INCX) after agonist stimulation were measured in ventricular myocytes of P2X4 transgenic mice using whole cell patch-clamp techniques. The agonist 2-methylthio-ATP (2-meSATP) increased peak Ip from a basal level of 0.52 ± 0.02 to 0.58 ± 0.03 pA/pF. 2-meSATP also increased the Ca(2+) entry mode of INCX (0.55 ± 0.09 pA/pF under control conditions vs. 0.82 ± 0.14 pA/pF with 2-meSATP) at a membrane potential of +50 mV. 2-meSATP shifted the reversal potential of INCX from -14 ± 2.3 to -25 ± 4.1 mV, causing an estimated intracellular Na(+) concentration increase of 1.28 ± 0.42 mM. These experimental results were closely mimicked by mathematical simulations based on previously established models. KB-R7943 or a structurally different agent preferentially opposing the Ca(2+) entry mode of NCX, YM-244769, could inhibit the 2-meSATP-induced increase in cell shortening in transgenic myocytes. Thus, the Ca(2+) entry mode of INCX participates in P2X agonist-stimulated contractions. In ventricular myocytes from wild-type mice, the P2X agonist could increase INCX, and KB-R7943 was able to inhibit the contractile effect of endogenous P2X4Rs, indicating a physiological role of these receptors in wild-type cells. The data demonstrate a novel Na(+) entry pathway through ligand-gated P2X4Rs in cardiomyocytes. Copyright © 2014 the American Physiological Society.

UNCOUPLING THE COUPLED CALCIUM AND ZINC DYSHOMEOSTASIS IN CARDIAC MYOCYTES AND MITOCHONDRIA SEEN IN ALDOSTERONISM

PubMed Central

Kamalov, German; Ahokas, Robert A.; Zhao, Wenyuan; Zhao, Tieqiang; Shahbaz, Atta U.; Johnson, Patti L.; Bhattacharya, Syamal K.; Sun, Yao; Gerling, Ivan C.; Weber, Karl T.

2010-01-01

Intracellular [Ca2+]i overloading in cardiomyocytes is a fundamental pathogenic event associated with chronic aldosterone/salt treatment (ALDOST) and accounts for an induction of oxidative stress that leads to necrotic cell death and consequent myocardial scarring. This prooxidant response to Ca2+ overloading in cardiac myocytes and mitochondria is intrinsically coupled to simultaneous increased Zn2+ entry serving as an antioxidant. Herein, we investigated whether Ca2+ and Zn2+ dyshomeostasis and prooxidant:antioxidant dysequilibrium seen at 4 wks, the pathologic stage of ALDOST, could be uncoupled in favor of antioxidants, using cotreatment with a ZnSO4 supplement, pyrrolidine dithiocarbamate (PDTC), a Zn2+ ionophore, or ZnSO4 in combination with amlodipine (Amlod), a Ca2+ channel blocker. We monitored and compared responses in cardiomyocyte free [Ca2+]i and [Zn2+]i together with biomarkers of oxidative stress in cardiac myocytes and mitochondria. At wk 4 ALDOST and compared to controls, we found: i) an elevation in [Ca2+]i coupled with [Zn2+]i; and ii) increased mitochondrial H2O2 production, and increased mitochondrial and cardiac 8-isoprostane levels. Cotreatment with the ZnSO4 supplement alone, PDTC, or ZnSO4+Amlod augmented the rise in cardiomyocyte [Zn2+]i beyond that seen with ALDOST alone, while attenuating the rise in [Ca2+]i which together served to reduce oxidative stress. Thus, a coupled dyshomeostasis of intracellular Ca2+ and Zn2+ was demonstrated in cardiac myocytes and mitochondria during 4 wks ALDOST, where prooxidants overwhelm antioxidant defenses. This intrinsically coupled Ca2+ and Zn2+ dyshomeostasis could be uncoupled in favor of antioxidant defenses by selectively increasing free [Zn2+]i and/or reducing [Ca2+]i using cotreatment with ZnSO4 or PDTC alone or ZnSO4+Amlod in combination. PMID:20051880

Ultrasonic destruction of albumin microbubbles enhances gene transfection and expression in cardiac myocytes.

PubMed

Wang, Guo-zhong; Liu, Jing-hua; Lü, Shu-zheng; Lü, Yun; Guo, Cheng-jun; Zhao, Dong-hui; Fang, Dong-ping; He, Dong-fang; Zhou, Yuan; Ge, Chang-jiang

2011-05-01

It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes. The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed. The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95 ± 2.41) U/g or (29.28 ± 3.65) U/g vs. (0.84 ± 0.21) U/g, P < 0.01), and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA ((51.95 ± 2.41) U/g vs. (29.28 ± 3.65) U/g, P < 0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced β-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35 ± 11.21) U/g protein vs. (14.13 ± 2.58) U/g protein, P < 0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63 ± 7.65) U/g vs. (14.13 ± 2.58) U/g, P < 0.05). Ultrasonic

Purification of cardiac myocytes from human heart biopsies for gene expression analysis.

PubMed

Kosloski, L M; Bales, I K; Allen, K B; Walker, B L; Borkon, A M; Stuart, R S; Pak, A F; Wacker, M J

2009-09-01

The collection of gene expression data from human heart biopsies is important for understanding the cellular mechanisms of arrhythmias and diseases such as cardiac hypertrophy and heart failure. Many clinical and basic research laboratories conduct gene expression analysis using RNA from whole cardiac biopsies. This allows for the analysis of global changes in gene expression in areas of the heart, while eliminating the need for more complex and technically difficult single-cell isolation procedures (such as flow cytometry, laser capture microdissection, etc.) that require expensive equipment and specialized training. The abundance of fibroblasts and other cell types in whole biopsies, however, can complicate gene expression analysis and the interpretation of results. Therefore, we have designed a technique to quickly and easily purify cardiac myocytes from whole cardiac biopsies for RNA extraction. Human heart tissue samples were collected, and our purification method was compared with the standard nonpurification method. Cell imaging using acridine orange staining of the purified sample demonstrated that >98% of total RNA was contained within identifiable cardiac myocytes. Real-time RT-PCR was performed comparing nonpurified and purified samples for the expression of troponin T (myocyte marker), vimentin (fibroblast marker), and alpha-smooth muscle actin (smooth muscle marker). Troponin T expression was significantly increased, and vimentin and alpha-smooth muscle actin were significantly decreased in the purified sample (n = 8; P < 0.05). Extracted RNA was analyzed during each step of the purification, and no significant degradation occurred. These results demonstrate that this isolation method yields a more purified cardiac myocyte RNA sample suitable for downstream applications, such as real-time RT-PCR, and allows for more accurate gene expression changes in cardiac myocytes from heart biopsies.

Effect of oxidative insult on young and adult cardiac muscle cells in vitro.

PubMed

Nag, A C; Sreepathi, P; Lee, M L; Reddan, J R

1996-01-01

The effect of hydrogen peroxide on cultured neonatal and adult cardiac myocytes was investigated. On neonatal cardiac myocytes the effect was very pronounced at a low concentration (0.03 mM), whereas the adult cardiac myocytes were resistant to the same concentration of H2O2. Dividing neonatal cardiac myocytes were more susceptible to H2O2 insult than the non-dividing adult cardiac myocytes. At a concentration of 0.1 mM H2O2, the neonatal cardiac myocytes were significantly damaged compared with the adult cardiac myocytes. Cardiac muscle cells from neonatal and adult hearts at high density culture were more tolerant to the oxidative insult by H2O2 than cells in low density culture. The damaging effect of H2O2 was very selective on F-actin in neonatal and adult cardiac muscle cells. The effect of H2O2 on myosin, titin, alpha-actinin, desmin or tubulin was not pronounced. Microscopical studies suggested a more marked protection by catalase than by glutathione reductase in the neonatal cells.

Sphingosine mediates the immediate negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian cardiac myocyte.

PubMed

Oral, H; Dorn, G W; Mann, D L

1997-02-21

To determine whether activation of the neutral sphingomyelinase pathway was responsible for the immediate (<30 min) negative inotropic effects of tumor necrosis factor-alpha (TNF-alpha), we examined sphingosine levels in diluent and TNF-alpha-stimulated cardiac myocytes. TNF-alpha stimulation of adult feline cardiac myocytes provoked a rapid (<15 min) increase in the hydrolysis of [14C]sphingomyelin in cell-free extracts, as well as an increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF-alpha-stimulated cardiac myocytes showed that TNF-alpha stimulation produced a rapid (<30 min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF-alpha on intracellular calcium homeostasis, as well as the negative inotropic effects of TNF-alpha in isolated contracting myocytes; time course studies showed that exogenous D-sphingosine produced abnormalities in cell shortening that were maximal at 5 min. Finally, blocking sphingosine production using an inhibitor of ceramidase, n-oleoylethanolamine, completely abrogated the negative inotropic effects of TNF-alpha in isolated contracting cardiac myocytes. Additional studies employing biologically active ceramide analogs and sphingosine 1-phosphate suggested that neither the immediate precursor of sphingosine nor the immediate metabolite of sphingosine, respectively, were likely to be responsible for the immediate negative inotropic effects of TNF-alpha. Thus, these studies suggest that sphingosine mediates the immediate negative inotropic effects of TNF-alpha in isolated cardiac myocytes.

Repressive histone methylation regulates cardiac myocyte cell cycle exit.

PubMed

El-Nachef, Danny; Oyama, Kyohei; Wu, Yun-Yu; Freeman, Miles; Zhang, Yiqiang; Robb MacLellan, W

2018-05-22

Mammalian cardiac myocytes (CMs) stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart's regenerative potential after injury. The molecular events that mediate CM cell cycle exit are poorly understood. To determine the epigenetic mechanisms limiting CM cycling in adult CMs (ACMs) and whether trimethylation of lysine 9 of histone H3 (H3K9me3), a histone modification associated with repressed chromatin, is required for the silencing of cell cycle genes, we developed a transgenic mouse model where H3K9me3 is specifically removed in CMs by overexpression of histone demethylase, KDM4D. Although H3K9me3 is found across the genome, its loss in CMs preferentially disrupts cell cycle gene silencing. KDM4D binds directly to cell cycle genes and reduces H3K9me3 levels at these promotors. Loss of H3K9me3 preferentially leads to increased cell cycle gene expression resulting in enhanced CM cycling. Heart mass was increased in KDM4D overexpressing mice by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in KDM4D expressing hearts, suggesting CM hyperplasia accounts for the increased heart mass. Inducing KDM4D after normal development specifically in ACMs resulted in increased cell cycle gene expression and cycling. We demonstrated that H3K9me3 is required for CM cell cycle exit and terminal differentiation in ACMs. Depletion of H3K9me3 in adult hearts prevents and reverses permanent cell cycle exit and allows hyperplastic growth in adult hearts in vivo. Copyright © 2017. Published by Elsevier Ltd.

Comparative Effects of Urocortins and Stresscopin on Cardiac Myocyte Contractility

PubMed Central

Makarewich, Catherine A.; Troupes, Constantine D.; Schumacher, Sarah M.; Gross, Polina; Koch, Walter J.; Crandall, David L.; Houser, Steven R.

2015-01-01

Rationale There is a current need for development of new therapies for patients with heart failure. Objective To test the effects of members of the Corticotropin-Releasing Factor (CRF) family of peptides on myocyte contractility to validate them as potential heart failure therapeutics. Methods and Results Adult feline left ventricular myocytes (AFMs) were isolated and contractility was assessed in the presence and absence of CRF peptides Urocortin 2 (UCN2), Urocortin 3 (UCN3), Stresscopin (SCP), and the β-adrenergic agonist isoproterenol (Iso). An increase in fractional shortening and peak Ca2+ transient amplitude was seen in the presence of all CRF peptides. A decrease in Ca2+ decay rate (Tau) was also observed at all concentrations tested. cAMP generation was measured by ELISA in isolated AFMs in response to the CRF peptides and Iso and significant production was seen at all concentrations and time points tested. Conclusions The CRF family of peptides effectively increases cardiac contractility and should be evaluated as potential novel therapeutics for heart failure patients. PMID:26231084

Endocytosis‒Mediated Invasion and Pathogenicity of Streptococcus agalactiae in Rat Cardiomyocyte (H9C2).

PubMed

Pooja, Sharma; Pushpanathan, Muthuirulan; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

2015-01-01

Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae internalization, we chemically inhibited discrete parts of cellular uptake system in H9C2 cells using genistein, chlorpromazine, nocodazole and cytochalasin B. Chemical inhibition of microtubule and actin formation by nocodazole and cytochalasin B impaired S. agalactiae internalization into H9C2 cells. Consistently, reverse‒ transcription PCR (RT‒PCR) and quantitative real time‒PCR (RT-qPCR) analyses also detected higher levels of transcripts for cytoskeleton forming genes, Acta1 and Tubb5 in S. agalactiae‒infected H9C2 cells, suggesting the requirement of functional cytoskeleton in pathogenesis. Host survival assay demonstrated that S. agalactiae internalization induced cytotoxicity in H9C2 cells. S. agalactiae cells grown with benzyl penicillin reduced its ability to internalize and induce cytotoxicity in H9C2 cells, which could be attributed with the removal of surface lipoteichoic acid (LTA) from S. agalactiae. Further, the LTA extracted from S. agalactiae also exhibited dose‒dependent cytotoxicity in H9C2 cells. Taken together, our data suggest that S. agalactiae cells internalized H9C2 cells through energy‒dependent endocytic processes and the LTA of S. agalactiae play major role in host cell internalization and cytotoxicity induction.

Endocytosis‒Mediated Invasion and Pathogenicity of Streptococcus agalactiae in Rat Cardiomyocyte (H9C2)

PubMed Central

Pooja, Sharma; Pushpanathan, Muthuirulan; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

2015-01-01

Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae internalization, we chemically inhibited discrete parts of cellular uptake system in H9C2 cells using genistein, chlorpromazine, nocodazole and cytochalasin B. Chemical inhibition of microtubule and actin formation by nocodazole and cytochalasin B impaired S. agalactiae internalization into H9C2 cells. Consistently, reverse‒ transcription PCR (RT‒PCR) and quantitative real time‒PCR (RT-qPCR) analyses also detected higher levels of transcripts for cytoskeleton forming genes, Acta1 and Tubb5 in S. agalactiae‒infected H9C2 cells, suggesting the requirement of functional cytoskeleton in pathogenesis. Host survival assay demonstrated that S. agalactiae internalization induced cytotoxicity in H9C2 cells. S. agalactiae cells grown with benzyl penicillin reduced its ability to internalize and induce cytotoxicity in H9C2 cells, which could be attributed with the removal of surface lipoteichoic acid (LTA) from S. agalactiae. Further, the LTA extracted from S. agalactiae also exhibited dose‒dependent cytotoxicity in H9C2 cells. Taken together, our data suggest that S. agalactiae cells internalized H9C2 cells through energy‒dependent endocytic processes and the LTA of S. agalactiae play major role in host cell internalization and cytotoxicity induction. PMID:26431539

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators.

PubMed Central

Berlin, J R; Konishi, M

1993-01-01

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2

The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy

PubMed Central

Aronsen, Jan Magnus; Ferrini, Arianna; Brien, Patrick; Alkass, Kanar; Tomasso, Antonio; Agrawal, Asmita; Bergmann, Olaf; Reik, Wolf; Roderick, Hywel Llewelyn

2016-01-01

Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure. In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes. In hypertrophy, H3K9me2 was reduced following a miR-217–mediated decrease in expression of the H3K9 dimethyltransferases EHMT1 and EHMT2 (EHMT1/2). miR-217–mediated, genetic, or pharmacological inactivation of EHMT1/2 was sufficient to promote pathological hypertrophy and fetal gene reexpression, while suppression of this pathway protected against pathological hypertrophy both in vitro and in mice. Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy. These results suggest that targeting miR-217 and EHMT1/2 to prevent H3K9 methylation loss is a viable therapeutic approach for the treatment of heart disease. PMID:27893464

Mechanically induced orientation of adult rat cardiac myocytes in vitro

NASA Technical Reports Server (NTRS)

Samuel, J.-L.; Vandenburgh, H. H.

1990-01-01

The present study describes the spatial orientation of a population of freshly isolated adult rat cardiac myocytes using a computerized mechanical cell stimulator device for tissue cultured cells. A continuous unidirectional stretch of the substratum at 60 to 400 microns/min for 120 to 30 min, respectively, during the cell attachment period in a serum-free medium was found to induce a significant threefold increase in the number of rod-shaped myocytes oriented parallel to the direction of movement. The myocytes orient less well with unidirectional substratum stretching after their adhesion to the substratum. Adult myocytes plated onto a substratum undergoing continuous 10-percent stretch-relaxation cycling show no significant change in the myocyte orientation or cytoskeletal organization. In addition to the type of mechanical activity, orientation of rod-shaped myocytes is dependent on the speed of the substratum, the final stretch amplitude, and the timing between initiation of substratum stretching and adhesion of myocytes to the substratum.

Interactions between benzylamiloride and fura-2: studies in vitro and in cardiac myocytes.

PubMed

Hudson, C A; Rojas, J D; Sarvazyan, N; Wesson, D E; Martínez-Zaguilán, R

1998-08-01

Amiloride derivatives are commonly used inhibitors of Na+/H+- and Na+/Ca2+-exchange. Because they are fluorescent molecules the use of benzylamiloride (BZA), an inhibitor of Na+/Ca2+ exchange, in conjunction with Fura-2, a commonly used fluorescent Ca2+ indicator, might complicate interpretation of fluorescence data obtained. In vitro data show that BZA decreases the Fura-2 fluorescence at all useful wavelengths in a concentration-dependent manner. The Fura-2 ratio 340/380 (used to estimate intracellular Ca2+ ([Ca2+]in)) also decreased with increasing BZA concentrations. The Stern-Volmer relation suggests that this phenomenon is due to either static or dynamic quenching. Varying temperatures from 4 to 37 degreesC did not alter Stern-Volmer constants, consistent instead with fluorescence resonance energy transfer (FRET). The in situ relevance of these interactions was evaluated in adult rat cardiac myocytes which exhibit Na+/Ca2+ exchange reflected by rapid [Ca2+]in increase following Na+ removal. Pretreatment with BZA >/= 25 microM decreased the magnitude of Fura-2 changes induced by Na+ removal. Analysis of the individual Fura-2 useful wavelengths indicated that >/= 25 microM BZA altered the Fura-2 signal in a manner consistent with the quenching effects noted in vitro. Together, these data show that BZA interacts with Fura-2 in vitro and in situ and suggest caution when interpreting Fura-2 fluorescence data derived in conjunction with BZA. Copyright 1998 Academic Press.

Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition

PubMed Central

Chia, Karin K. M.; Liu, Chia-Chi; Hamilton, Elisha J.; Garcia, Alvaro; Fry, Natasha A.; Hannam, William; Figtree, Gemma A.

2015-01-01

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1-Na+-K+ pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na+-K+ pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na+-K+ pump current (Ip). Coexposure to 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1-Na+-K+ pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced β1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47phox NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22phox subunit, and it decreased O2·−-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47phox indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22phox and p47phox NADPH oxidase subunits and decrease β1-Na+-K+ pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in β1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo. PMID:26084308

Stimulation of the p38 Mitogen-activated Protein Kinase Pathway in Neonatal Rat Ventricular Myocytes by the G Protein–coupled Receptor Agonists, Endothelin-1 and Phenylephrine: A Role in Cardiac Myocyte Hypertrophy?

PubMed Central

Clerk, Angela; Michael, Ashour; Sugden, Peter H.

1998-01-01

We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein–coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by ∼12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 μM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response. PMID:9679149

Matrix elasticity regulates the optimal cardiac myocyte shape for contractility

PubMed Central

McCain, Megan L.; Yuan, Hongyan; Pasqualini, Francesco S.; Campbell, Patrick H.

2014-01-01

Concentric hypertrophy is characterized by ventricular wall thickening, fibrosis, and decreased myocyte length-to-width aspect ratio. Ventricular thickening is considered compensatory because it reduces wall stress, but the functional consequences of cell shape remodeling in this pathological setting are unknown. We hypothesized that decreases in myocyte aspect ratio allow myocytes to maximize contractility when the extracellular matrix becomes stiffer due to conditions such as fibrosis. To test this, we engineered neonatal rat ventricular myocytes into rectangles mimicking the 2-D profiles of healthy and hypertrophied myocytes on hydrogels with moderate (13 kPa) and high (90 kPa) elastic moduli. Actin alignment was unaffected by matrix elasticity, but sarcomere content was typically higher on stiff gels. Microtubule polymerization was higher on stiff gels, implying increased intracellular elastic modulus. On moderate gels, myocytes with moderate aspect ratios (∼7:1) generated the most peak systolic work compared with other cell shapes. However, on stiffer gels, low aspect ratios (∼2:1) generated the most peak systolic work. To compare the relative contributions of intracellular vs. extracellular elasticity to contractility, we developed an analytical model and used our experimental data to fit unknown parameters. Our model predicted that matrix elasticity dominates over intracellular elasticity, suggesting that the extracellular matrix may potentially be a more effective therapeutic target than microtubules. Our data and model suggest that myocytes with lower aspect ratios have a functional advantage when the elasticity of the extracellular matrix decreases due to conditions such as fibrosis, highlighting the role of the extracellular matrix in cardiac disease. PMID:24682394

An integrative model of the cardiac ventricular myocyte incorporating local control of Ca2+ release.

PubMed Central

Greenstein, Joseph L; Winslow, Raimond L

2002-01-01

The local control theory of excitation-contraction (EC) coupling in cardiac muscle asserts that L-type Ca(2+) current tightly controls Ca(2+) release from the sarcoplasmic reticulum (SR) via local interaction of closely apposed L-type Ca(2+) channels (LCCs) and ryanodine receptors (RyRs). These local interactions give rise to smoothly graded Ca(2+)-induced Ca(2+) release (CICR), which exhibits high gain. In this study we present a biophysically detailed model of the normal canine ventricular myocyte that conforms to local control theory. The model formulation incorporates details of microscopic EC coupling properties in the form of Ca(2+) release units (CaRUs) in which individual sarcolemmal LCCs interact in a stochastic manner with nearby RyRs in localized regions where junctional SR membrane and transverse-tubular membrane are in close proximity. The CaRUs are embedded within and interact with the global systems of the myocyte describing ionic and membrane pump/exchanger currents, SR Ca(2+) uptake, and time-varying cytosolic ion concentrations to form a model of the cardiac action potential (AP). The model can reproduce both the detailed properties of EC coupling, such as variable gain and graded SR Ca(2+) release, and whole-cell phenomena, such as modulation of AP duration by SR Ca(2+) release. Simulations indicate that the local control paradigm predicts stable APs when the L-type Ca(2+) current is adjusted in accord with the balance between voltage- and Ca(2+)-dependent inactivation processes as measured experimentally, a scenario where common pool models become unstable. The local control myocyte model provides a means for studying the interrelationship between microscopic and macroscopic behaviors in a manner that would not be possible in experiments. PMID:12496068

Sarcomere length dependence of rat skinned cardiac myocyte mechanical properties: dependence on myosin heavy chain

PubMed Central

Korte, F Steven; McDonald, Kerry S

2007-01-01

The effects of sarcomere length (SL) on sarcomeric loaded shortening velocity, power output and rates of force development were examined in rat skinned cardiac myocytes that contained either α-myosin heavy chain (α-MyHC) or β-MyHC at 12 ± 1°C. When SL was decreased from 2.3 μm to 2.0 μm submaximal isometric force decreased ∼40% in both α-MyHC and β-MyHC myocytes while peak absolute power output decreased 55% in α-MyHC myocytes and 70% in β-MyHC myocytes. After normalization for the fall in force, peak power output decreased about twice as much in β-MyHC as in α-MyHC myocytes (41%versus 20%). To determine whether the fall in normalized power was due to the lower force levels, [Ca2+] was increased at short SL to match force at long SL. Surprisingly, this led to a 32% greater peak normalized power output at short SL compared to long SL in α-MyHC myocytes, whereas in β-MyHC myocytes peak normalized power output remained depressed at short SL. The role that interfilament spacing plays in determining SL dependence of power was tested by myocyte compression at short SL. Addition of 2% dextran at short SL decreased myocyte width and increased force to levels obtained at long SL, and increased peak normalized power output to values greater than at long SL in both α-MyHC and β-MyHC myocytes. The rate constant of force development (ktr) was also measured and was not different between long and short SL at the same [Ca2+] in α-MyHC myocytes but was greater at short SL in β-MyHC myocytes. At short SL with matched force by either dextran or [Ca2+], ktr was greater than at long SL in both α-MyHC and β-MyHC myocytes. Overall, these results are consistent with the idea that an intrinsic length component increases loaded crossbridge cycling rates at short SL and β-MyHC myocytes exhibit a greater sarcomere length dependence of power output. PMID:17347271

The antioxidant edaravone prevents cardiac dysfunction by suppressing oxidative stress in type 1 diabetic rats and in high-glucose-induced injured H9c2 cardiomyoblasts.

PubMed

Ji, Lei; Liu, Yingying; Zhang, Ying; Chang, Wenguang; Gong, Junli; Wei, Shengnan; Li, Xudong; Qin, Ling

2016-09-01

Edaravone, a radical scavenger, has been recognized as a potential protective agent for cardiovascular diseases. However, little is known about the effect of edaravone in cardiac complications associated with diabetes. Here, we have demonstrated that edaravone prevents cardiac dysfunction and apoptosis in the streptozotocin-induced type 1 diabetic rat heart. Mechanistic studies revealed that edaravone treatment improved cardiac function and restored superoxide dismutase levels. In addition, treatment of diabetic animals by edaravone increased protein expressions of sirtuin-1 (SIRT-1), peroxisome proliferator activated receptor γ coactivator α (PGC-1α), nuclear factor like-2 (NRF-2), and B cell lymphoma 2 (Bcl-2), and reduced protein expressions of Bax and Caspase-3 compared to the control group. High glucose incubation resulted in the production of reactive oxygen species (ROS) and cell death. Treatment of high-glucose-incubated H9c2 cells by edaravone reduced ROS production and cell death. In addition, the treatment of high-glucose-incubated H9c2 cells by edaravone increased the activity of antioxidative stress by increasing SIRT-1, PGC-1α, and NRF-2, and this treatment also reduced apoptosis by increasing Bcl-2 expression and reducing Bax and Caspase-3 expressions. Knockdown SIRT-1 with small interferer RNA abolished the effects of edaravone. Overall, our data demonstrated that edaravone may be an effective agent against the development of diabetic cardiomyopathy.

The NF-κB Subunit c-Rel Stimulates Cardiac Hypertrophy and Fibrosis

PubMed Central

Gaspar-Pereira, Silvia; Fullard, Nicola; Townsend, Paul A.; Banks, Paul S.; Ellis, Elizabeth L.; Fox, Christopher; Maxwell, Aidan G.; Murphy, Lindsay B.; Kirk, Adam; Bauer, Ralf; Caamaño, Jorge H.; Figg, Nichola; Foo, Roger S.; Mann, Jelena; Mann, Derek A.; Oakley, Fiona

2012-01-01

Cardiac remodeling and hypertrophy are the pathological consequences of cardiovascular disease and are correlated with its associated mortality. Activity of the transcription factor NF-κB is increased in the diseased heart; however, our present understanding of how the individual subunits contribute to cardiovascular disease is limited. We assign a new role for the c-Rel subunit as a stimulator of cardiac hypertrophy and fibrosis. We discovered that c-Rel-deficient mice have smaller hearts at birth, as well as during adulthood, and are protected from developing cardiac hypertrophy and fibrosis after chronic angiotensin infusion. Results of both gene expression and cross-linked chromatin immunoprecipitation assay analyses identified transcriptional activators of hypertrophy, myocyte enhancer family, Gata4, and Tbx proteins as Rel gene targets. We suggest that the p50 subunit could limit the prohypertrophic actions of c-Rel in the normal heart, because p50 overexpression in H9c2 cells repressed c-Rel levels and the absence of cardiac p50 was associated with increases in both c-Rel levels and cardiac hypertrophy. We report for the first time that c-Rel is highly expressed and confined to the nuclei of diseased adult human hearts but is restricted to the cytoplasm of normal cardiac tissues. We conclude that c-Rel-dependent signaling is critical for both cardiac remodeling and hypertrophy. Targeting its activities could offer a novel therapeutic strategy to limit the effects of cardiac disease. PMID:22210479

MicroRNA-449a Inhibition Protects H9C2 Cells Against Hypoxia/Reoxygenation-Induced Injury by Targeting the Notch-1 Signaling Pathway.

PubMed

Cheng, Jing; Wu, Qianfu; Lv, Rong; Huang, Li; Xu, Banglong; Wang, Xianbao; Chen, Aihua; He, Fei

2018-05-07

The present study aimed to detect the expression of miR-449a and investigate the effect of miR-449a on cell injury in cardiomyocytes subjected to hypoxia/ reoxygenation (H/R) and its underlying mechanisms. The expression of miR-449a was determined using reverse transcription-polymerase chain reaction in both neonatal rat ventricular myocytes and H9C2 cells. For gain-of-function and loss-of-function studies, H9C2 cells were transfected with either miR-449a mimics or miR-449a inhibitor. The target gene of miR-449a was confirmed by a dual-luciferase reporter assay. Apoptosis was analyzed by both flow cytometry using Annexin V and propidium iodide and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL). Necrosis was confirmed by the detection of lactate dehydrogenase release. The cell viability was measured using the methylthiotetrazole method. The protein levels of Notch-1, Notch-1 intracellular domain, hairy and enhancer of split-1 (Hes-1), and apoptosis-related genes were measured by Western blot analysis. MiR-449a was significantly upregulated in both neonatal rat ventricular myocytes and H9C2 cells subjected to H/R. However, H/R-induced cell apoptosis and necrosis were markedly reduced by miR-449a inhibition. By targeting Notch-1, miR-449a regulated the Notch-1/ Hes-1 signaling pathway. The blockade of the Notch signaling pathway partly abolished the protective effect of miR-449a suppression against H/R injury, whereas the overexpression of Notch-1 intracellular domain partly reversed the effect of miR-449a overexpression on H/R-induced cell injury. The present study suggested that miR-449a inhibition protected H9C2 cells against H/R-induced cell injury by targeting the Notch-1 signaling pathway, providing a novel insight into the molecular basis of myocardial ischemia-reperfusion injury and a potential therapeutic target. © 2018 The Author(s). Published by S. Karger AG, Basel.

Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes

PubMed Central

Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B. Cicero; Zarzoso, Manuel; Ramirez, Rafael J.; Sener, Michelle F.; Mundada, Lakshmi V.; Klos, Matthew; Devaney, Eric J.; Vikstrom, Karen L.; Herron, Todd J.; Jalife, José

2014-01-01

Applications of human induced pluripotent stemcell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricularmyocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. PMID:24095945

Negative Inotropic Effects of High Mobility Box Group 1 Protein in Isolated Contracting Cardiac Myocytes

PubMed Central

Tzeng, Huei-Ping; Fan, Jinping; Vallejo, Jesus G.; Dong, Jian Wen; Chen, Xiongwen; Houser, Steven R.; Mann, Douglas L.

2013-01-01

HMGB1 released from necrotic cells or macrophages functions as a late inflammatory mediator, and has been shown to induce cardiovascular collapse during sepsis. Thus far, however, the effect(s) of HMGB1 in the heart are not known. We determined the effects of HMGB1 on isolated feline cardiac myocytes by measuring sarcomere shortening in contracting cardiac myocytes, intracellular Ca2+ transients using fluo-3, and L-type calcium currents using whole cell perforate configuration of the patch clamp technique. Treatment of isolated myocytes with HMGB1 (100 ng/ml) resulted in a 70% decrease in sarcomere shortening and a 50% decrease in the height of the peak Ca++ transient within 5 min (p <0.01). The immediate negative inotropic effects HMGB1 on cell contractility and calcium homeostasis were partially reversible upon washout of HMGB1. A significant inhibition of the inward L-type calcium currents also was documented by the patch clamp technique. HMGB1 induced the PKCε translocation and a PKC inhibitor significantly attenuated the negative inotropic effects of HMGB1. These studies show for the first time that HMGB1 impairs sarcomere shortening by decreasing calcium availability in cardiac myocytes through modulating membrane calcium influx, and suggest that HMGB1 maybe act as a novel myocardial depressant factor during cardiac injury. PMID:18223193

N-acetylcysteine reverses cardiac myocyte dysfunction in a rodent model of behavioral stress

PubMed Central

Chen, Fangping; Hadfield, Jessalyn M.; Berzingi, Chalak; Hollander, John M.; Miller, Diane B.; Nichols, Cody E.

2013-01-01

Compelling clinical reports reveal that behavioral stress alone is sufficient to cause reversible myocardial dysfunction in selected individuals. We developed a rodent stress cardiomyopathy model by a combination of prenatal and postnatal behavioral stresses (Stress). We previously reported a decrease in percent fractional shortening by echo, both systolic and diastolic dysfunction by catheter-based hemodynamics, as well as attenuated hemodynamic and inotropic responses to the β-adrenergic agonist, isoproterenol (ISO) in Stress rats compared with matched controls (Kan H, Birkle D, Jain AC, Failinger C, Xie S, Finkel MS. J Appl Physiol 98: 77–82, 2005). We now report enhanced catecholamine responses to behavioral stress, as evidenced by increased circulating plasma levels of norepinephrine (P < 0.01) and epinephrine (P < 0.01) in Stress rats vs. controls. Cardiac myocytes isolated from Stress rats also reveal evidence of oxidative stress, as indicated by decreased ATP, increased GSSG, and decreased GSH-to-GSSG ratio in the presence of increased GSH peroxidase and catalase activities (P < 0.01, for each). We also report blunted inotropic and intracellular Ca2+ concentration responses to extracellular Ca2+ (P < 0.05), as well as altered inotropic responses to the intracellular calcium regulator, caffeine (20 mM; P < 0.01). Treatment of cardiac myocytes with N-acetylcysteine (NAC) (10−3 M) normalized calcium handling in response to ISO and extracellular Ca2+ concentration and inotropic response to caffeine (P < 0.01, for each). NAC also attenuated the blunted inotropic response to ISO and Ca2+ (P < 0.01, for each). Surprisingly, NAC did not reverse the changes in GSH, GSSG, or GSH-to-GSSG ratio. These data support a GSH-independent salutary effect of NAC on intracellular calcium signaling in this rodent model of stress-induced cardiomyopathy. PMID:23722706

Metabolic coupling of glutathione between mouse and quail cardiac myocytes and its protective role against oxidative stress.

PubMed

Nakamura, T Y; Yamamoto, I; Kanno, Y; Shiba, Y; Goshima, K

1994-05-01

Cultured quail myocytes were much more resistant to H2O2 toxicity than cultured mouse myocytes. The intracellular concentration of glutathione ([GSH]i) and the activity of gamma-glutamylcysteine synthetase (gamma-GCS) in quail heart cells were about five and three times higher, respectively, than in mouse heart cells, although catalase and glutathione peroxidase (GSHpx) activity was similar in both. Preloading of gamma-glutamylcysteine monoethyl ester (gamma-GCE), a membrane-permeating GSH precursor, increased the H2O2 resistance of cultured mouse myocytes. These observations suggest that the high [GSH]i and the high activity of gamma-GCS in quail myocytes are responsible for their high resistance to H2O2. Both H2O2 sensitivity and [GSH]i of mosaic sheets composed of equal amounts of mouse and quail myocytes approximated those of sheets composed entirely of quail myocytes. From these observations, it is hypothesized that GSH was transferred from quail myocytes to mouse myocytes, probably through gap junctions between them, and that quail myocytes resynthesized GSH by a feedback mechanism, thus maintaining their intracellular GSH levels. When the fluorescent dye lucifer yellow was injected into a beating quail myocyte in a mosaic sheet, it spread to neighboring mouse myocytes but not to neighboring L cells (a cell line derived from mouse connective tissue). These observations indicate that existence of gap junctions in the region of cell contact between mouse and quail myocytes but not between quail myocytes and L cells. When quail myocytes preloaded with [3H]gamma-GCE were cocultured with mouse myocytes and L cells, the radioactivity was transmitted to neighboring mouse myocytes but not L cells. These observations show that GSH and/or its precursors can be transmitted from quail myocytes to mouse myocytes through gap junctions and that this can protect mouse myocytes from H2O2 toxicity. Mouse myocyte sheets composed of 10(4) cells or more showed higher resistance

An anomalous subdiffusion model for calcium spark in cardiac myocytes

NASA Astrophysics Data System (ADS)

Tan, Wenchang; Fu, Chaoqi; Fu, Ceji; Xie, Wenjun; Cheng, Heping

2007-10-01

The elementary events of excitation-contraction coupling in heart muscle are Ca2+ sparks, which arise from ryanodine receptors in the sarcoplasmic reticulum (SR). Here, an anomalous subdiffusion model is developed to explore Ca2+ spark formation in cardiac myocytes. Numerical simulations reproduce the brightness, the time course, and spatial size of a typical cardiac Ca2+ spark. It is suggested that the diffusion of Ca2+ spark in the cytoplasm may no longer obey Fickian second law, but the anomalous space subdiffusion. The physical reason is perhaps due to the effects of the electric field of the calcium ions and the viscoelasticity of the cytoplasm and its complex structures.

Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro

PubMed Central

Tolmachov, Oleg; Ma, Yu-Ling; Themis, Michael; Patel, Pravina; Spohr, Hilmar; MacLeod, Kenneth T; Ullrich, Nina D; Kienast, Yvonne; Coutelle, Charles; Peters, Nicholas S

2006-01-01

Background Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. Methods To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin® and the polycation transduction enhancer Transfectam®. The EGFP-positive transduced cells were then enriched by flow cytometry. Results More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than

Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro.

PubMed

Tolmachov, Oleg; Ma, Yu-Ling; Themis, Michael; Patel, Pravina; Spohr, Hilmar; Macleod, Kenneth T; Ullrich, Nina D; Kienast, Yvonne; Coutelle, Charles; Peters, Nicholas S

2006-06-06

Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin and the polycation transduction enhancer Transfectam. The EGFP-positive transduced cells were then enriched by flow cytometry. More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non

Direct, Differential Effects of Tamoxifen, 4-Hydroxytamoxifen, and Raloxifene on Cardiac Myocyte Contractility and Calcium Handling

PubMed Central

Asp, Michelle L.; Martindale, Joshua J.; Metzger, Joseph M.

2013-01-01

Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT), and related drug raloxifene (Ral) on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca2+ transient amplitude. Effects were primarily observed at 5 and 10 μM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca2+ transient amplitudes. At 10 μM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca2+ handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy. PMID:24205315

Temporal responses to intrinsically coupled calcium and zinc dyshomeostasis in cardiac myocytes and mitochondria during aldosteronism

PubMed Central

Kamalov, German; Ahokas, Robert A.; Zhao, Wenyuan; Shahbaz, Atta U.; Bhattacharya, Syamal K.; Sun, Yao; Gerling, Ivan C.

2010-01-01

Intracellular Ca2+ overloading, coupled to induction of oxidative stress, is present at 4-wk aldosterone/salt treatment (ALDOST). This prooxidant reaction in cardiac myocytes and mitochondria accounts for necrotic cell death and subsequent myocardial scarring. It is intrinsically linked to increased intracellular zinc concentration ([Zn2+]i) serving as an antioxidant. Herein, we addressed the temporal responses in coupled Ca2+ and Zn2+ dyshomeostasis, reflecting the prooxidant-antioxidant equilibrium, by examining preclinical (week 1) and pathological (week 4) stages of ALDOST to determine whether endogenous antioxidant defenses would be ultimately overwhelmed to account for this delay in cardiac remodeling. We compared responses in cardiomyocyte free [Ca2+]i and [Zn2+]i and mitochondrial total [Ca2+]m and [Zn2+]m, together with biomarkers of oxidative stress and antioxidant defenses, during 1- and 4-wk ALDOST. At week 1 and compared with controls, we found: 1) elevations in [Ca2+]i and [Ca2+]m were coupled with [Zn2+]i and [Zn2+]m; 2) increased mitochondrial H2O2 production, cardiomyocyte xanthine oxidase activity, and cardiac and mitochondrial 8-isoprostane levels, counterbalanced by increased activity of antioxidant proteins, enzymes, and the nonenzymatic antioxidants that can be considered as cumulative antioxidant capacity; some of these enzymes and proteins (e.g., metallothionein-1, Cu/Zn-superoxide, glutathione synthase) are regulated by metal-responsive transcription factor-1; and 3) although these augmented antioxidant defenses were sustained at week 4, they fell short in combating the persistent intracellular Ca2+ overloading and marked rise in cardiac tissue 8-isoprostane and mitochondrial transition pore opening. Thus a coupled Ca2+ and Zn2+ dyshomeostasis occurs early during ALDOST in cardiac myocytes and mitochondria that regulate redox equilibrium until week 4 when ongoing intracellular Ca2+ overloading and prooxidants overwhelm antioxidant defenses

Beta1-adrenoceptor antagonist, metoprolol attenuates cardiac myocyte Ca2+ handling dysfunction in rats with pulmonary artery hypertension.

PubMed

Fowler, Ewan D; Drinkhill, Mark J; Norman, Ruth; Pervolaraki, Eleftheria; Stones, Rachel; Steer, Emma; Benoist, David; Steele, Derek S; Calaghan, Sarah C; White, Ed

2018-07-01

Right heart failure is the major cause of death in Pulmonary Artery Hypertension (PAH) patients but is not a current, specific therapeutic target. Pre-clinical studies have shown that adrenoceptor blockade can improve cardiac function but the mechanisms of action within right ventricular (RV) myocytes are unknown. We tested whether the β 1 -adrenoceptor blocker metoprolol could improve RV myocyte function in an animal model of PAH, by attenuating adverse excitation-contraction coupling remodeling. PAH with RV failure was induced in rats by monocrotaline injection. When PAH was established, animals were given 10 mg/kg/day metoprolol (MCT + BB) or vehicle (MCT). The median time to the onset of heart failure signs was delayed from 23 days (MCT), to 31 days (MCT + BB). At 23 ± 1 days post-injection, MCT + BB showed improved in vivo cardiac function, measured by echocardiography. RV hypertrophy was reduced despite persistent elevated afterload. RV myocyte contractility during field stimulation was improved at higher pacing frequencies in MCT + BB. Preserved t-tubule structure, more uniform evoked Ca 2+ release, increased SERCA2a expression and faster ventricular repolarization (measured in vivo by telemetry) may account for the improved contractile function. Sarcoplasmic reticulum Ca 2+ overload was prevented in MCT + BB myocytes resulting in fewer spontaneous Ca 2+ waves, with a lower pro-arrhythmic potential. Our novel finding of attenuation of defects in excitation contraction coupling by β 1 -adrenoceptor blockade with delays in the onset of HF, identifies the RV as a promising therapeutic target in PAH. Moreover, our data suggest existing therapies for left ventricular failure may also be beneficial in PAH induced RV failure. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture

NASA Technical Reports Server (NTRS)

Clark, W. A.; Decker, M. L.; Behnke-Barclay, M.; Janes, D. M.; Decker, R. S.

1998-01-01

Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists

Neuropeptide Y rapidly enhances [Ca2+]i transients and Ca2+ sparks in adult rat ventricular myocytes through Y1 receptor and PLC activation.

PubMed

Heredia, María del Puy; Delgado, Carmen; Pereira, Laetitia; Perrier, Romain; Richard, Sylvain; Vassort, Guy; Bénitah, Jean-Pierre; Gómez, Ana María

2005-01-01

Neuropeptide Y (NPY) is the most abundant peptide in the mammalian heart, but its cardiac actions are not fully understood. Here we investigate the effect of NPY in intracellular Ca2+ release, using isolated rat cardiac myocytes and confocal microscopy. Cardiac myocytes were field-stimulated at 1 Hz. The evoked [Ca2+]i transient was of higher amplitude and of faster decay in the presence of 100 nM NPY. Cell contraction was also increased by NPY. We analyzed the occurrence of Ca2+ sparks and their characteristics after NPY application. NPY significantly increased Ca2+ sparks frequency in quiescent cells. The Ca2+ spark amplitude was enhanced by NPY but the other characteristics of Ca2+ sparks were not significantly altered. Because cardiac myocytes express both Y1 and Y2 NPY receptors, we repeated the experiments in the presence of the receptor blockers, BIBP3226 and BIIE0246. We found that Y1 NPY receptor blockade completely inhibited NPY effects on [Ca2+]i transient. PTX-sensitive G-proteins and/or phospholypase C (PLC) have been invoked to mediate NPY effects in other cell types. We tested these two hypotheses. In PTX-treated myocytes NPY was still effective, which suggests that the observed NPY actions are not mediated by PTX-sensitive G-proteins. In contrast, the increase in [Ca2+]i transient by NPY was completely inhibited by the PLC inhibitor U73122. In conclusion, we find that NPY has a positive inotropic effect in isolated rat cardiac myocytes, which involves increase in Ca2+ release after activation of Y1 NPY receptor and subsequent stimulation of PLC.

A beta1-adrenergic receptor CaM kinase II-dependent pathway mediates cardiac myocyte fetal gene induction.

PubMed

Sucharov, Carmen C; Mariner, Peter D; Nunley, Karin R; Long, Carlin; Leinwand, Leslie; Bristow, Michael R

2006-09-01

Beta-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathies. Chronic activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress ultimately harms the failing heart by mechanisms that include alterations in gene expression. Here, we show that stimulation of beta-ARs with isoproterenol in neonate rat ventricular myocytes causes a "fetal" response in the relative activities of the human cardiac fetal and/or adult gene promoters that includes repression of the human and rat alpha-myosin heavy chain (alpha-MyHC) promoters with simultaneous activation of the human atrial natriuretic peptide (ANP) and rat beta-MyHC promoters. We also show that the promoter changes correlate with changes in endogenous gene expression as measured by mRNA expression. Furthermore, we show that these changes are specifically mediated by the beta1-AR, but not the beta2-AR, and are independent of alpha1-AR stimulation. We also demonstrate that the fetal gene response is independent of cAMP and protein kinase A, whereas inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK) pathway blocks isoproterenol-mediated fetal gene program induction. Finally, we show that induction of the fetal program is dependent on activation of the L-type Ca2+ channel. We conclude that in neonatal rat cardiac myocytes, agonist-occupied beta1-AR mobilizes Ca2+ stores to activate fetal gene induction through cAMP independent pathways that involve CaMK.

Formation and infrared absorption of protonated naphthalenes (1-C10H9+ and 2-C10H9+) and their neutral counterparts in solid para-hydrogen.

PubMed

Bahou, Mohammed; Wu, Yu-Jong; Lee, Yuan-Pern

2013-02-14

Protonated naphthalene (C(10)H(9)(+)) and its neutral counterparts (hydronaphthyl radicals, C(10)H(9)) are important intermediates in the reactions of aromatic compounds and in understanding the unidentified infrared (IR) emissions from interstellar media. We report the IR spectra of 1-C(10)H(9)(+), 2-C(10)H(9)(+), 1-C(10)H(9), and 2-C(10)H(9) trapped in solid para-hydrogen (p-H(2)); the latter three are new. These species were produced upon electron bombardment of a mixture of naphthalene (C(10)H(8)) and p-H(2) during matrix deposition. The intensities of IR features of 1-C(10)H(9)(+) decreased after the matrix was maintained in darkness for 19 h, whereas those of 1-C(10)H(9) and 2-C(10)H(9) increased. Irradiation of this matrix sample with light at 365 nm diminished lines of 1-C(10)H(9)(+) and 2-C(10)H(9) and enhanced lines of 1-C(10)H(9) and 2-C(10)H(9)(+); the latter species was unstable and converted to 1-C(10)H(9)(+) in less than 30 min and 2-C(10)H(9) was converted to 1-C(10)H(9) at 365 nm. Observed wavenumbers and relative intensities of these species agree satisfactorily with the anharmonic vibrational wavenumbers and IR intensities predicted with the B3PW91/6-311++G(2d,2p) method. Compared with spectra recorded previously with IR photodissociation of Ar-tagged C(10)H(9)(+) or IR multiphoton dissociation of C(10)H(9)(+), our method has the advantages of producing high-resolution IR spectra with a wide spectral coverage, true IR intensity and excellent ratio of signal to noise; both protonated species and their neutral counterparts are produced with little interference from other fragments. With these advantages, the IR spectra of 1-C(10)H(9)(+), 2-C(10)H(9)(+), 1-C(10)H(9), and 2-C(10)H(9) are here clearly characterized.

Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols

PubMed Central

Jeziorowska, Dorota; Fontaine, Vincent; Jouve, Charlène; Villard, Eric; Dussaud, Sébastien; Akbar, David; Letang, Valérie; Cervello, Pauline; Itier, Jean-Michiel; Pruniaux, Marie-Pierre; Hulot, Jean-Sébastien

2017-01-01

Human induced pluripotent stem cells (iPSCs) represent a powerful human model to study cardiac disease in vitro, notably channelopathies and sarcomeric cardiomyopathies. Different protocols for cardiac differentiation of iPSCs have been proposed either based on embroid body formation (3D) or, more recently, on monolayer culture (2D). We performed a direct comparison of the characteristics of the derived cardiomyocytes (iPSC-CMs) on day 27 ± 2 of differentiation between 3D and 2D differentiation protocols with two different Wnt-inhibitors were compared: IWR1 (inhibitor of Wnt response) or IWP2 (inhibitor of Wnt production). We firstly found that the level of Troponin T (TNNT2) expression measured by FACS was significantly higher for both 2D protocols as compared to the 3D protocol. In the three methods, iPSC-CM show sarcomeric structures. However, iPSC-CM generated in 2D protocols constantly displayed larger sarcomere lengths as compared to the 3D protocol. In addition, mRNA and protein analyses reveal higher cTNi to ssTNi ratios in the 2D protocol using IWP2 as compared to both other protocols, indicating a higher sarcomeric maturation. Differentiation of cardiac myocytes with 2D monolayer-based protocols and the use of IWP2 allows the production of higher yield of cardiac myocytes that have more suitable characteristics to study sarcomeric cardiomyopathies. PMID:28587156

High-Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure

PubMed Central

Rosa-Garrido, Manuel; Chapski, Douglas J.; Schmitt, Anthony D.; Kimball, Todd H.; Karbassi, Elaheh; Monte, Emma; Balderas, Enrique; Pellegrini, Matteo; Shih, Tsai-Ting; Soehalim, Elizabeth; Liem, David; Ping, Peipei; Galjart, Niels J.; Ren, Shuxun; Wang, Yibin; Ren, Bing

2017-01-01

Background: Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. Methods: To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload–induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Results: Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. Conclusions: These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy. PMID

High-Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure.

PubMed

Rosa-Garrido, Manuel; Chapski, Douglas J; Schmitt, Anthony D; Kimball, Todd H; Karbassi, Elaheh; Monte, Emma; Balderas, Enrique; Pellegrini, Matteo; Shih, Tsai-Ting; Soehalim, Elizabeth; Liem, David; Ping, Peipei; Galjart, Niels J; Ren, Shuxun; Wang, Yibin; Ren, Bing; Vondriska, Thomas M

2017-10-24

Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload-induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy. © 2017 The Authors.

Myocyte-Derived Hsp90 Modulates Collagen Upregulation via Biphasic Activation of STAT-3 in Fibroblasts during Cardiac Hypertrophy

PubMed Central

Datta, Ritwik; Bansal, Trisha; Rana, Santanu; Datta, Kaberi; Datta Chaudhuri, Ratul; Chawla-Sarkar, Mamta

2016-01-01

ABSTRACT Signal transducer and activator of transcription 3 (STAT-3)-mediated signaling in relation to upregulated collagen expression in fibroblasts during cardiac hypertrophy is well defined. Our recent findings have identified heat shock protein 90 (Hsp90) to be a critical modulator of fibrotic signaling in cardiac fibroblasts in this disease milieu. The present study was therefore intended to analyze the role of Hsp90 in the STAT-3-mediated collagen upregulation process. Our data revealed a significant difference between in vivo and in vitro results, pointing to a possible involvement of myocyte-fibroblast cross talk in this process. Cardiomyocyte-targeted knockdown of Hsp90 in rats (Rattus norvegicus) in which the renal artery was ligated showed downregulated collagen synthesis. Furthermore, the results obtained with cardiac fibroblasts conditioned with Hsp90-inhibited hypertrophied myocyte supernatant pointed toward cardiomyocytes' role in the regulation of collagen expression in fibroblasts during hypertrophy. Our study also revealed a novel signaling mechanism where myocyte-derived Hsp90 orchestrates not only p65-mediated interleukin-6 (IL-6) synthesis but also its release in exosomal vesicles. Such myocyte-derived exosomes and myocyte-secreted IL-6 are responsible in unison for the biphasic activation of STAT-3 signaling in cardiac fibroblasts that culminates in excess collagen synthesis, leading to severely compromised cardiac function during cardiac hypertrophy. PMID:28031326

Myocyte-Derived Hsp90 Modulates Collagen Upregulation via Biphasic Activation of STAT-3 in Fibroblasts during Cardiac Hypertrophy.

PubMed

Datta, Ritwik; Bansal, Trisha; Rana, Santanu; Datta, Kaberi; Datta Chaudhuri, Ratul; Chawla-Sarkar, Mamta; Sarkar, Sagartirtha

2017-03-15

Signal transducer and activator of transcription 3 (STAT-3)-mediated signaling in relation to upregulated collagen expression in fibroblasts during cardiac hypertrophy is well defined. Our recent findings have identified heat shock protein 90 (Hsp90) to be a critical modulator of fibrotic signaling in cardiac fibroblasts in this disease milieu. The present study was therefore intended to analyze the role of Hsp90 in the STAT-3-mediated collagen upregulation process. Our data revealed a significant difference between in vivo and in vitro results, pointing to a possible involvement of myocyte-fibroblast cross talk in this process. Cardiomyocyte-targeted knockdown of Hsp90 in rats ( Rattus norvegicus ) in which the renal artery was ligated showed downregulated collagen synthesis. Furthermore, the results obtained with cardiac fibroblasts conditioned with Hsp90-inhibited hypertrophied myocyte supernatant pointed toward cardiomyocytes' role in the regulation of collagen expression in fibroblasts during hypertrophy. Our study also revealed a novel signaling mechanism where myocyte-derived Hsp90 orchestrates not only p65-mediated interleukin-6 (IL-6) synthesis but also its release in exosomal vesicles. Such myocyte-derived exosomes and myocyte-secreted IL-6 are responsible in unison for the biphasic activation of STAT-3 signaling in cardiac fibroblasts that culminates in excess collagen synthesis, leading to severely compromised cardiac function during cardiac hypertrophy. Copyright © 2017 American Society for Microbiology.

Long Noncoding RNA MHRT Protects Cardiomyocytes against H2O2-Induced Apoptosis

PubMed Central

Zhang, Jianying; Gao, Caihua; Meng, Meijuan; Tang, Hongxia

2016-01-01

Acute myocardial infarction (AMI) remains a leading cause of morbidity and mortality worldwide. The exploration of new biomarkers with high sensitivity and specificity for early diagnosis of AMI therefore becomes one of the primary task. In the current study, we aim to detect whether there is any heart specific long noncoding RNA (lncRNA) releasing into the circulation during AMI, and explore its function in the neonatal rat cardiac myocytes injury induced by H2O2. Our results revealed that the cardiac-specific lncRNA MHRT (Myosin Heavy Chain Associated RNA Transcripts) was significantly elevated in the blood from AMI patients compared with the healthy control (*p<0.05). Using an in vitro neonatal rat cardiac myocytes injury model, we demonstrated that lncRNA MHRT was upregulated in the cardiac myocytes after treatment with hydrogen peroxide (H2O2) via real-time RT-PCR (qRT-PCR). Furthermore, we knockdowned the MHRT gene by siRNA to confirm its roles in the H2O2-induced cardiac cell apoptosis, and found that knockdown of MHRT led to significant more apoptotic cells than the non-target control (**p<0.01), indicating that the lncRNA MHRT is a protective factor for cardiomyocyte and the plasma concentration of MHRT may serve as a biomarker for myocardial infarction diagnosis in humans AMI. PMID:26759697

Targeted Gene Silencing of Tumor Necrosis Factor Attenuates the Negative Inotropic Effects of Lipopolysaccharide in Isolated Contracting Cardiac Myocytes

PubMed Central

Ramabadran, R. S.; Chancey, Amanda; Vallejo, Jesus G.; Barger, Philip M.; Sivasubramanian, Natarajan; Mann, Douglas L.

2008-01-01

Bacterial endotoxin (lipopolysaccharide) depresses cardiovascular function; however, the mediators and signaling pathways that are responsible for the negative inotropic effects of lipopolysaccharide are not fully known. We used RNA interference to determine the relative role of tumor necrosis factor with respect to mediating the negative inotropic effects of lipopolysaccharide in isolated cardiac myocytes. Cardiac myocyte cultures were treated with lipopolysaccharide in the presence or absence of small interfering RNAs (siRNA) for tumor necrosis factor. We examined the effects of tumor necrosis factor siRNA on lipopolysaccharide-induced tumor necrosis factor messenger RNA (mRNA) and protein biosynthesis, as well as the negative inotropic effects of lipopolysaccharide in isolated contracting cardiac myocytes. Treatment of adult cardiac myocyte cultures with tumor necrosis factor siRNA significantly attenuated lipopolysaccharide-induced tumor necrosis factor mRNA and protein biosynthesis, whereas transfection with a double-stranded RNA that does not target mammalian mRNA had no effect. Pretreatment with tumor necrosis factor siRNA significantly attenuated, but did not abrogate, the lipopolysaccharide-induced decrease in sarcomere shortening in isolated contracting cardiac myocytes. In contrast, tumor necrosis factor siRNA had a comparatively smaller effect on improving sarcomere shortening once the negative inotropic effects of lipopolysaccharide were fully established. These results suggest that tumor necrosis factor plays an important upstream role in lipopolysaccharide-induced negative inotropic effects in isolated contracting cardiac myocytes and that other molecular mechanisms are responsible for the decrease in sarcomere shortening after sustained lipopolysaccharide signaling. PMID:18427645

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes.

PubMed

Lin, Z X; Holtzer, S; Schultheiss, T; Murray, J; Masaki, T; Fischman, D A; Holtzer, H

1989-06-01

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes

PubMed Central

1989-01-01

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating

A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart

PubMed Central

Ackers-Johnson, Matthew; Li, Peter Yiqing; Holmes, Andrew P.; O’Brien, Sian-Marie; Pavlovic, Davor; Foo, Roger S.

2018-01-01

Rationale Cardiovascular disease represents a global pandemic. The advent of and recent advances in mouse genomics, epigenomics, and transgenics offer ever-greater potential for powerful avenues of research. However, progress is often constrained by unique complexities associated with the isolation of viable myocytes from the adult mouse heart. Current protocols rely on retrograde aortic perfusion using specialized Langendorff apparatus, which poses considerable logistical and technical barriers to researchers and demands extensive training investment. Objective To identify and optimize a convenient, alternative approach, allowing the robust isolation and culture of adult mouse cardiac myocytes using only common surgical and laboratory equipment. Methods and Results Cardiac myocytes were isolated with yields comparable to those in published Langendorff-based methods, using direct needle perfusion of the LV ex vivo and without requirement for heparin injection. Isolated myocytes can be cultured antibiotic free, with retained organized contractile and mitochondrial morphology, transcriptional signatures, calcium handling, responses to hypoxia, neurohormonal stimulation, and electric pacing, and are amenable to patch clamp and adenoviral gene transfer techniques. Furthermore, the methodology permits concurrent isolation, separation, and coculture of myocyte and nonmyocyte cardiac populations. Conclusions We present a novel, simplified method, demonstrating concomitant isolation of viable cardiac myocytes and nonmyocytes from the same adult mouse heart. We anticipate that this new approach will expand and accelerate innovative research in the field of cardiac biology. PMID:27502479

Curcumin induces the apoptotic intrinsic pathway via upregulation of reactive oxygen species and JNKs in H9c2 cardiac myoblasts.

PubMed

Zikaki, Kyriaki; Aggeli, Ioanna-Katerina; Gaitanaki, Catherine; Beis, Isidoros

2014-06-01

Curcumin derived from the rhizome of turmeric (Curcuma longa L.), is a well known coloring culinary agent, that has therapeutic properties against diverse pathologies such as cancer, atherosclerosis and heart failure. Given the salutary potential of curcumin, deciphering its mode of action particularly in cardiac cells, is of outstanding value. Accumulating evidence implicates curcumin in the regulation of multiple signaling pathways leading to cell survival or apoptosis. Therefore, the present study aimed at elucidating the molecular mechanisms triggered by curcumin in H9c2 cells. Curcumin was found to activate p38-mitogen-activated protein kinase (p38-MAPK) as well as c-jun NH2 terminal kinases (JNKs), in a dose- and time-dependent manner. We also observed curcumin to impair cell survival by promoting apoptosis, evidenced by chromatin condensation, poly(ADP-ribose) polymerase (PARP) and caspase-3 cleavage, as well as Bax translocation and cytochrome c release into the cytosol. Curcumin-induced apoptosis was ascribed to JNKs, since hindering their activity abolished PARP fragmentation. Furthermore, we identified curcumin to exert a pro-oxidative activity, with 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining revealing up-regulation of reactive oxygen species (ROS) levels and anti-oxidants found to abrogate PARP cleavage. In conclusion, curcumin was found to stimulate the apoptotic cell death of H9c2 cells by upregulating ROS generation and triggering activation of JNKs. With reports underscoring the capacity of curcumin to perturb the cellular redox balance ensuring survival or enhancing apoptosis, determination of its mode of action appears to be critical. Future studies should assess the appropriate administration conditions of curcumin, so as to optimize its therapeutic potential against cardiovascular pathologies.

Intrinsic H+ ion mobility in the rabbit ventricular myocyte

PubMed Central

Vaughan-Jones, R D; Peercy, B E; Keener, J P; Spitzer, K W

2002-01-01

The intrinsic mobility of intracellular H+ ions was investigated by confocally imaging the longitudinal movement of acid inside rabbit ventricular myocytes loaded with the acetoxymethyl ester (AM) form of carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1). Acid was diffused into one end of the cell through a patch pipette filled with an isotonic KCl solution of pH 3.0. Intracellular H+ mobility was low, acid taking 20-30 s to move 40 μm down the cell. Inhibiting sarcolemmal Na+-H+ exchange with 1 mm amiloride had no effect on this time delay. Net Hi+ movement was associated with a longitudinal intracellular pH (pHi) gradient of up to 0.4 pH units. Hi+ movement could be modelled using the equations for diffusion, assuming an apparent diffusion coefficient for H+ ions (DappH) of 3.78 × 10−7 cm2 s−1, a value more than 300-fold lower than the H+ diffusion coefficient in a dilute, unbuffered solution. Measurement of the intracellular concentration of SNARF (≈400 μM) and its intracellular diffusion coefficient (0.9 × 10−7 cm2 s−1) indicated that the fluorophore itself exerted an insignificant effect (between 0.6 and 3.3 %) on the longitudinal movement of H+ equivalents inside the cell. The longitudinal movement of intracellular H+ is discussed in terms of a diffusive shuttling of H+ equivalents on high capacity mobile buffers which comprise about half (≈11 mm) of the total intrinsic buffering capacity within the myocyte (the other half being fixed buffer sites on low mobility, intracellular proteins). Intrinsic Hi+ mobility is consistent with an average diffusion coefficient for the intracellular mobile buffers (Dmob) of ≈9 × 10−7 cm2 s−1. PMID:12015426

Electrical coupling of single cardiac rat myocytes to field-effect and bipolar transistors.

PubMed

Kind, Thomas; Issing, Matthias; Arnold, Rüdiger; Müller, Bernt

2002-12-01

A novel bipolar transistor for extracellular recording the electrical activity of biological cells is presented, and the electrical behavior compared with the field-effect transistor (FET). Electrical coupling is examined between single cells separated from the heart of adults rats (cardiac myocytes) and both types of transistors. To initiate a local extracellular voltage, the cells are periodically stimulated by a patch pipette in voltage clamp and current clamp mode. The local extracellular voltage is measured by the planar integrated electronic sensors: the bipolar and the FET. The small signal transistor currents correspond to the local extracellular voltage. The two types of sensor transistors used here were developed and manufactured in the laboratory of our institute. The manufacturing process and the interfaces between myocytes and transistors are described. The recordings are interpreted by way of simulation based on the point-contact model and the single cardiac myocyte model.

Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function

PubMed Central

Vandenboom, Rene; Herron, Todd; Favre, Elizabeth; Albayya, Faris P.

2011-01-01

The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a “headless” myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC + endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P < 0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules. PMID:21112946

Liganded Peroxisome Proliferator-Activated Receptors (PPARs) Preserve Nuclear Histone Deacetylase 5 Levels in Endothelin-Treated Sprague-Dawley Rat Cardiac Myocytes

PubMed Central

Zhang, Haining; Shao, Zongjun; Alibin, Caroline P.; Acosta, Crystal; Anderson, Hope D.

2014-01-01

Ligand activation of peroxisome proliferator-activated receptors (PPARs) prevents cardiac myocyte hypertrophy, and we previously reported that diacylglycerol kinase zeta (DGKζ) is critically involved. DGKζ is an intracellular lipid kinase that catalyzes phosphorylation of diacylglycerol; by attenuating DAG signaling, DGKζ suppresses protein kinase C (PKC) and G-protein signaling. Here, we investigated how PPAR-DGKζ signaling blocks activation of the hypertrophic gene program. We focused on export of histone deacetylase 5 (HDAC5) from the nucleus, a key event during hypertrophy, since crosstalk occurs between PPARs and other members of the HDAC family. Using cardiac myocytes isolated from Sprague-Dawley rats, we determined that liganded PPARs disrupt endothelin-1 (ET1)-induced nuclear export of HDAC5 in a manner that is dependent on DGKζ. When DGKζ-mediated PKC inhibition was circumvented using a constitutively-active PKCε mutant, PPARs failed to block ET1-induced nuclear retention of HDAC5. Liganded PPARs also prevented (i) activation of protein kinase D (the downstream effector of PKC), (ii) HDAC5 phosphorylation at 14-3-3 protein chaperone binding sites (serines 259 and 498), and (iii) physical interaction between HDAC5 and 14-3-3, all of which are consistent with blockade of nucleo-cytoplasmic shuttling of HDAC5. Finally, the ability of PPARs to prevent neutralization of HDAC5 activity was associated with transcriptional repression of hypertrophic genes. This occurred by first, reduced MEF2 transcriptional activity and second, augmented deacetylation of histone H3 associated with hypertrophic genes expressing brain natriuretic peptide, β-myosin heavy chain, skeletal muscle α-actin, and cardiac muscle α-actin. Our findings identify spatial regulation of HDAC5 as a target for liganded PPARs, and to our knowledge, are the first to describe a mechanistic role for nuclear DGKζ in cardiac myocytes. In conclusion, these results implicate modulation of HDAC5

Higenamine protects ischemia/reperfusion induced cardiac injury and myocyte apoptosis through activation of β2-AR/PI3K/AKT signaling pathway

PubMed Central

Wu, Mei-ping; Zhang, Yi-shuai; Zhou, Qian-mei; Xiong, Jian; Dong, Yao-rong; Yan, Chen

2016-01-01

Cardiomyocyte apoptosis contributes to ischemic cardiac injury and the development of heart failure. Higenamine is a key component of the Chinese herb aconite root that has been prescribed for treating symptoms of heart failure for thousands of years in the oriental Asian countries. It has been shown that higenamine has anti-apoptotic effects in a few cell types including cardiomyocytes. However, the pharmacological target and molecular mechanism of higenamine in the heart are still not fully illustrated. Herein, we report that higenamine protected myocyte apoptosis and ischemia/reperfusion (I/R) injury through selective activation of beta2-adrenergic receptor (β2-AR). In particular, we show that higenamine significantly reduced I/R-induced myocardial infarction in mice. In both primary neonatal rat and adult mouse ventricular myocytes, we show higenamine inhibited cell apoptosis and also reduced biochemical markers of apoptosis such as cleaved caspase 3 and 9. More importantly, we show that the anti-apoptotic effects of higenamine in cardiomyocytes were completely abolished by β2-AR but not β1-AR antagonism. Furthermore, we confirmed that higenamine attenuated I/R-induced myocardial injury and reduced cleaved caspases in a β2-AR dependent manner in intact mouse hearts. Higenamine stimulated AKT phosphorylation and required PI3K activation for the anti-apoptotic effect in cardiomyocytes. These findings together suggest that anti-apoptotic and cardiac protective effects of higenamine are mediated by the β2-AR/PI3K/AKT cascade. PMID:26746354

Synthesis, structure and reactivity of rare-earth metallacarborane alkyls [η(1):η(5)-O(CH2)2C2B9H9]Ln(σ:η(1)-CH2C6H4-o-NMe2)(THF)2.

PubMed

Yang, Jingying; Xie, Zuowei

2015-04-14

Rare-earth metallacarborane alkyls can be stabilized by the incorporation of a functional sidearm into both π and σ ligands. Reaction of [Me3NH][7,8-O(CH2)2-7,8-C2B9H10] with one equiv. of Ln(CH2C6H4-o-NMe2)3 gave metallacarborane alkyls [η(1):η(5)-O(CH2)2C2B9H9]Ln(σ:η(1)-CH2C6H4-o-NMe2)(THF)2 (Ln = Y (), Gd (), Er ()) via alkane elimination. They represent the first examples of rare-earth metallacarborane alkyls. Treatment of with RN[double bond, length as m-dash]C[double bond, length as m-dash]NR (R = Cy, (i)Pr) or 2-benzoylpyridine afforded the corresponding mono-insertion products [η(1):η(5)-O(CH2)2C2B9H9]Y[η(2)-(RN)2C(CH2C6H4-o-NMe2)](DME) (R = Cy (), (i)Pr ()) or [η(1):η(5)-O(CH2)2C2B9H9]Y[C5H4NC(Ph)(CH2C6H4-o-NMe2)O](THF)2 (), respectively. Complex also reacted with ArNCO or ArNC (Ar = 2,6-diisopropylphenyl, 2,6-dimethylphenyl) to give di-insertion products [η(1):η(5)-O(CH2)2C2B9H9]Y[OC([double bond, length as m-dash]NC6H3Me2)N(C6H3Me2)C(CH2C6H4-o-NMe2)O](THF)2 () or [η(1):η(5)-O(CH2)2C2B9H9]Y[C([double bond, length as m-dash]NC6H3(i)Pr2)C([double bond, length as m-dash]NC6H3(i)Pr2)(CH2C6H4-o-NMe2)](DME) (). These results showed that the reactivity pattern of the Ln-C σ bond in rare-earth metallacarborane alkyls was dependent on the nature of the unsaturated organic molecules. New complexes were characterized by various spectroscopic techniques and elemental analysis. Some were further confirmed by single-crystal X-ray analysis.

Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

PubMed Central

Haj Slimane, Zeineb; Bedioune, Ibrahim; Lechêne, Patrick; Varin, Audrey; Lefebvre, Florence; Mateo, Philippe; Domergue-Dupont, Valérie; Dewenter, Matthias; Richter, Wito; Conti, Marco; El-Armouche, Ali; Zhang, Jin; Fischmeister, Rodolphe; Vandecasteele, Grégoire

2014-01-01

Aims The cAMP-dependent protein kinase (PKA) mediates β-adrenoceptor (β-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. Methods and results Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. β-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. Conclusion Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to β-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to β-AR stimulation. PMID:24550350

Optimisation of a Generic Ionic Model of Cardiac Myocyte Electrical Activity

PubMed Central

Guo, Tianruo; Al Abed, Amr; Lovell, Nigel H.; Dokos, Socrates

2013-01-01

A generic cardiomyocyte ionic model, whose complexity lies between a simple phenomenological formulation and a biophysically detailed ionic membrane current description, is presented. The model provides a user-defined number of ionic currents, employing two-gate Hodgkin-Huxley type kinetics. Its generic nature allows accurate reconstruction of action potential waveforms recorded experimentally from a range of cardiac myocytes. Using a multiobjective optimisation approach, the generic ionic model was optimised to accurately reproduce multiple action potential waveforms recorded from central and peripheral sinoatrial nodes and right atrial and left atrial myocytes from rabbit cardiac tissue preparations, under different electrical stimulus protocols and pharmacological conditions. When fitted simultaneously to multiple datasets, the time course of several physiologically realistic ionic currents could be reconstructed. Model behaviours tend to be well identified when extra experimental information is incorporated into the optimisation. PMID:23710254

Exploring Regulatory Mechanisms of Atrial Myocyte Hypertrophy of Mitral Regurgitation through Gene Expression Profiling Analysis: Role of NFAT in Cardiac Hypertrophy

PubMed Central

Chang, Tzu-Hao; Chen, Mien-Cheng; Chang, Jen-Ping; Huang, Hsien-Da; Ho, Wan-Chun; Lin, Yu-Sheng; Pan, Kuo-Li; Huang, Yao-Kuang; Liu, Wen-Hao; Wu, Chia-Chen

2016-01-01

Background Left atrial enlargement in mitral regurgitation (MR) predicts a poor prognosis. The regulatory mechanisms of atrial myocyte hypertrophy of MR patients remain unknown. Methods and Results This study comprised 14 patients with MR, 7 patients with aortic valve disease (AVD), and 6 purchased samples from normal subjects (NC). We used microarrays, enrichment analysis and quantitative RT-PCR to study the gene expression profiles in the left atria. Microarray results showed that 112 genes were differentially up-regulated and 132 genes were differentially down-regulated in the left atria between MR patients and NC. Enrichment analysis of differentially expressed genes demonstrated that “NFAT in cardiac hypertrophy” pathway was not only one of the significant associated canonical pathways, but also the only one predicted with a non-zero score of 1.34 (i.e. activated) through Ingenuity Pathway Analysis molecule activity predictor. Ingenuity Pathway Analysis Global Molecular Network analysis exhibited that the highest score network also showed high association with cardiac related pathways and functions. Therefore, 5 NFAT associated genes (PPP3R1, PPP3CB, CAMK1, MEF2C, PLCE1) were studies for validation. The mRNA expressions of PPP3CB and MEF2C were significantly up-regulated, and CAMK1 and PPP3R1 were significantly down-regulated in MR patients compared to NC. Moreover, MR patients had significantly increased mRNA levels of PPP3CB, MEF2C and PLCE1 compared to AVD patients. The atrial myocyte size of MR patients significantly exceeded that of the AVD patients and NC. Conclusions Differentially expressed genes in the “NFAT in cardiac hypertrophy” pathway may play a critical role in the atrial myocyte hypertrophy of MR patients. PMID:27907007

Sympathetic neurons are a powerful driver of myocyte function in cardiovascular disease.

PubMed

Larsen, Hege E; Lefkimmiatis, Konstantinos; Paterson, David J

2016-12-14

Many therapeutic interventions in disease states of heightened cardiac sympathetic activity are targeted to the myocytes. However, emerging clinical data highlights a dominant role in disease progression by the neurons themselves. Here we describe a novel experimental model of the peripheral neuro-cardiac axis to study the neuron's ability to drive a myocyte cAMP phenotype. We employed a co-culture of neonatal ventricular myocytes and sympathetic stellate neurons from normal (WKY) and pro-hypertensive (SHR) rats that are sympathetically hyper-responsive and measured nicotine evoked cAMP responses in the myocytes using a fourth generation FRET cAMP sensor. We demonstrated the dominant role of neurons in driving the myocyte ß-adrenergic phenotype, where SHR cultures elicited heightened myocyte cAMP responses during neural activation. Moreover, cross-culturing healthy neurons onto diseased myocytes rescued the diseased cAMP response of the myocyte. Conversely, healthy myocytes developed a diseased cAMP response if diseased neurons were introduced. Our results provide evidence for a dominant role played by the neuron in driving the adrenergic phenotype seen in cardiovascular disease. We also highlight the potential of using healthy neurons to turn down the gain of neurotransmission, akin to a smart pre-synaptic ß-blocker.

Characterization and inhibition of beta-adrenergic receptor kinase in intact myocytes.

PubMed

Laugwitz, K L; Kronsbein, K; Schmitt, M; Hoffmann, K; Seyfarth, M; Schömig, A; Ungerer, M

1997-08-01

beta-Adrenergic receptor kinase (beta ARK) phosphorylates and thereby inactivates agonist-occupied beta-adrenergic receptors (beta AR). beta ARK is thought to play an important role in the regulation of cardiac function. Therefore, we studied beta ARK activation and its inhibition in intact smooth muscle cells and in cardiomyoblasts. beta AR agonist-stimulated translocation of beta ARK was monitored by immunofluorescence labelling with specific antibodies and confocal laser scanning microscopy in DDT-MF 2 hamster smooth muscle cells and in H9c2 rat cardiomyoblasts. In unstimulated cells. beta ARK was mainly located in the cytosol. After beta AR agonist stimulation, the beta ARK signal was partially translocated to the membranes. Liposomal gene transfer of the COOH-terminus of beta ARK ('beta ARKmini') as a beta ARK inhibitor led to functional expression of this protein in both cell lines with high efficiency. Western blots with beta ARK antibodies showed a gene concentration-dependent immunoreactivity of the 'beta ARKmini' protein. 'beta ARKmini'-transfected myocytes demonstrated reduced membrane targeting of the beta ARK immuno-fluorescence signal. Additionally, the effect of 'beta ARKmini' on beta AR-induced desensitization of myocytic cAMP accumulation was investigated. In control cells, desensitization with isoproterenol led to a subsequent reduction of beta AR-induced cAMP accumulation. In 'beta ARKmini'-transfected myocytes, this beta AR-induced desensitization was significantly diminished, whereas normal beta AR-induced cAMP accumulation was unaffected. A gene concentration of 2 micrograms 'beta ARKmini' DNA/100,000 cardiomyoblasts, and of 0.7 microgram 'beta ARKmini' DNA/100,000 DDT-MF2 smooth muscle cells led to approximately 5.9- and approximately 5.6-fold overexpressions of 'beta ARKmini' vs. native beta ARK, respectively. These gene doses proved sufficient to attenuate beta-adrenergic desensitization significantly. (1) beta ARK translocation was

Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a 'function follows form' model of differentiation.

PubMed

Kankeu, Cynthia; Clarke, Kylie; Van Haver, Delphi; Gevaert, Kris; Impens, Francis; Dittrich, Anna; Roderick, H Llewelyn; Passante, Egle; Huber, Heinrich J

2018-05-17

The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a 'function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes.

Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morton, M.J., E-mail: michael.morton@astrazeneca.com; Armstrong, D.; Abi Gerges, N.

2014-09-01

Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity inmore » the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.« less

Cardiac Myocyte-specific Knock-out of Calcium-independent Phospholipase A2γ (iPLA2γ) Decreases Oxidized Fatty Acids during Ischemia/Reperfusion and Reduces Infarct Size *

PubMed Central

Moon, Sung Ho; Mancuso, David J.; Sims, Harold F.; Liu, Xinping; Nguyen, Annie L.; Yang, Kui; Guan, Shaoping; Dilthey, Beverly Gibson; Jenkins, Christopher M.; Weinheimer, Carla J.; Kovacs, Attila; Abendschein, Dana; Gross, Richard W.

2016-01-01

Calcium-independent phospholipase A2γ (iPLA2γ) is a mitochondrial enzyme that produces lipid second messengers that facilitate opening of the mitochondrial permeability transition pore (mPTP) and contribute to the production of oxidized fatty acids in myocardium. To specifically identify the roles of iPLA2γ in cardiac myocytes, we generated cardiac myocyte-specific iPLA2γ knock-out (CMiPLA2γKO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA2γKO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA2γKO mice demonstrated attenuated Ca2+-induced mPTP opening that could be rapidly restored by the addition of palmitate and substantially reduced production of oxidized polyunsaturated fatty acids (PUFAs). Furthermore, myocardial ischemia/reperfusion (I/R) in CMiPLA2γKO mice (30 min of ischemia followed by 30 min of reperfusion in vivo) dramatically decreased oxidized fatty acid production in the ischemic border zones. Moreover, CMiPLA2γKO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-at-risk in comparison with their WT littermates. Furthermore, we found that membrane depolarization in murine heart mitochondria was sensitized to Ca2+ by the presence of oxidized PUFAs. Because mitochondrial membrane depolarization and calcium are known to activate iPLA2γ, these results are consistent with salvage of myocardium after I/R by iPLA2γ loss of function through decreasing mPTP opening, diminishing production of proinflammatory oxidized fatty acids, and attenuating the deleterious effects of abrupt increases in calcium ion on membrane potential during reperfusion. PMID:27453526

Chlorogenic acid analogues from Gynura nepalensis protect H9c2 cardiomyoblasts against H2O2-induced apoptosis

PubMed Central

Yu, Bang-wei; Li, Jin-long; Guo, Bin-bin; Fan, Hui-min; Zhao, Wei-min; Wang, He-yao

2016-01-01

Aim: Chlorogenic acid has shown protective effect on cardiomyocytes against oxidative stress-induced damage. Herein, we evaluated nine caffeoylquinic acid analogues (1–9) isolated from the leaves of Gynura nepalensis for their protective effect against H2O2-induced H9c2 cardiomyoblast damage and explored the underlying mechanisms. Methods: H9c2 cardiomyoblasts were exposed to H2O2 (0.3 mmol/L) for 3 h, and cell viability was detected with MTT assay. Hoechst 33342 staining was performed to evaluate cell apoptosis. MMPs (mitochondrial membrane potentials) were measured using a JC-1 assay kit, and ROS (reactive oxygen species) generation was measured using CM-H2 DCFDA. The expression levels of relevant proteins were detected using Western blot analysis. Results: Exposure to H2O2 markedly decreased the viability of H9c2 cells and catalase activity, and increased LDH release and intracellular ROS production; accompanied by a loss of MMP and increased apoptotic rate. Among the 9 chlorogenic acid analogues as well as the positive control drug epigallocatechin gallate (EGCG) tested, compound 6 (3,5-dicaffeoylquinic acid ethyl ester) was the most effective in protecting H9c2 cells from H2O2-induced cell death. Pretreatment with compound 6 (1.56–100 μmol/L) dose-dependently alleviated all the H2O2-induced detrimental effects. Moreover, exposure to H2O2 significantly increased the levels of Bax, p53, cleaved caspase-8, and cleaved caspase-9, and decreased the level of Bcl-2, resulting in cell apoptosis. Exposure to H2O2 also significantly increased the phosphorylation of p38, JNK and ERK in the H9c2 cells. Pretreatment with compound 6 (12.5 and 25 μmol/L) dose-dependently inhibited the H2O2-induced increase in the level of cleaved caspase-9 but not of cleaved caspase-8. It also dose-dependently suppressed the H2O2-induced phosphorylation of JNK and ERK but not that of p38. Conclusion: Compound 6 isolated from the leaves of Gynura nepalensis potently protects H9c2

Rapid frequency‐dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes

PubMed Central

Wüst, Rob C. I.; Helmes, Michiel; Martin, Jody L.; van der Wardt, Thomas J. T.; Musters, René J. P.; van der Velden, Jolanda

2017-01-01

Key points Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle.The magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiac myocytes are largely unknown.Rapid stimulation frequency‐dependent increases but relatively slow decreases in free mitochondrial calcium concentration were observed in rat cardiac myocytes. This asymmetry caused a rise in the mitochondrial calcium concentration with stimulation frequency.These results provide insight into the mechanisms of mitochondrial calcium uptake and release that are important in healthy and diseased myocardium. Abstract Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially targeted Förster resonance energy transfer (FRET)‐based calcium indicator (4mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca2+]m) was measured at different stimulation frequencies (0.1–4 Hz) and external calcium concentrations (1.8–3.6 mm) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura‐4AM. The increases in [Ca2+]m during electrical stimulation at 0.1 Hz were rapid (rise time = 49 ± 2 ms), while the decreases in [Ca2+]m occurred more slowly (decay half time = 1.17 ± 0.07 s). Model calculations confirmed that this asymmetry caused the rise in [Ca2+]m during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium–calcium exchanger (mNCE) resulted in a rise in [Ca2+]m at baseline and, paradoxically, in an

Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca2+ signaling in ventricular myocytes.

PubMed

Park, Seon-Ah; Hong, Bing-Zhe; Ha, Ki-Chan; Kim, Uh-Hyun; Han, Myung-Kwan; Kwak, Yong-Geun

2017-06-02

Cyclic ADP-ribose (cADPR) releases Ca 2+ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca 2+ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca 2+ ([Ca 2+ ] i ) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca 2+ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca 2+ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

Protective effect of Dendrobium officinale polysaccharides on H2O2-induced injury in H9c2 cardiomyocytes.

PubMed

Zhao, Xiaoyan; Dou, Mengmeng; Zhang, Zhihao; Zhang, Duoduo; Huang, Chengzhi

2017-10-01

The preliminary studies have shown that Dendrobium officinale possessed therapeutic effects on hypertension and atherosclerosis. Studies also reported that Dendrobium officinale polysaccharides showed antioxidant capabilities. However, little is known about its effects on myocardial cells under oxidative stress. The present study was designed to study the protective effect of Dendrobium officinale polysaccharides against H 2 O 2 -induced oxidative stress in H9c2 cells. MTT assay was carried out to determine the cell viability of H9c2 cells when pretreated with Dendrobium officinale polysaccharides. Fluorescent microscopy measurements were performed for evaluating the apoptosis in H9c2 cells. Furthermore, effects of Dendrobium officinale polysaccharides on the activities of antioxidative indicators (malondialdehyde, superoxide dismutase), reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) levels were analyzed. Dendrobium officinale polysaccharides attenuated H 2 O 2 -induced cell death, as determined by the MTT assay. Dendrobium officinale polysaccharides decreased malondialdehyde levels, increased superoxide dismutase activities, and inhibited the generation of intracellular ROS. Moreover, pretreatment with Dendrobium officinale polysaccharides also inhibited apoptosis and increased the MMP levels in H9c2 cells. These results suggested the protective effects of Dendrobium officinale polysaccharides against H 2 O 2 -induced injury in H9c2 cells. The results also indicated the anti-oxidative capability of Dendrobium officinale polysaccharides. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Alpinate Oxyphyllae Fructus Inhibits IGFII-Related Signaling Pathway to Attenuate Ang II-Induced Pathological Hypertrophy in H9c2 Cardiomyoblasts.

PubMed

Tsai, Chuan-Te; Chang, Yung-Ming; Lin, Shu-Luan; Chen, Yueh-Sheng; Yeh, Yu-Lan; Padma, Viswanadha Vijaya; Tsai, Chin-Chuan; Chen, Ray-Jade; Ho, Tsung-Jung; Huang, Chih-Yang

2016-03-01

Angiotensin II (Ang II) is a very important cardiovascular disease inducer and may cause cardiac pathological hypertrophy and remodeling. We evaluated a Chinese traditional medicine, alpinate oxyphyllae fructus (AOF), for therapeutic efficacy for treating Ang II-induced cardiac hypertrophy. AOF has been used to treat patients with various symptoms accompanying hypertension and cerebrovascular disorders in Korea. We investigated its protective effect against Ang II-induced cytoskeletal change and hypertrophy in H9c2 cells. The results showed that treating cells with Ang II resulted in pathological hypertrophy, such as increased expression of transcription factors NFAT-3/p-NFAT-3, hypertrophic response genes (atrial natriuretic peptide [ANP] and b-type natriuretic peptide [BNP]), and Gαq down-stream effectors (PLCβ3 and calcineurin). Pretreatment with AOF (60-100 μg/mL) led to significantly reduced hypertrophy. We also found that AOF pretreatment significantly suppressed the cardiac remodeling proteins, metalloproteinase (MMP9 and MMP2), and tissue plasminogen activator (tPA), induced by Ang II challenge. In conclusion, we provide evidence that AOF protects against Ang II-induced pathological hypertrophy by specifically inhibiting the insulin-like growth factor (IGF) II/IIR-related signaling pathway in H9c2 cells. AOF might be a candidate for cardiac hypertrophy and ventricular remodeling prevention in chronic cardiovascular diseases.

Confinement of β1- and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

PubMed Central

Valentine, Cathleen D.; Haggie, Peter M.

2011-01-01

The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β1- and β2AR, are structurally similar but mediate distinct signaling responses. Scaffold protein–mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β1- and β2AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)–domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β2AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β2AR confinement. For both β1- and β2AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β1- or β2AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes. PMID:21680711

Confinement of β(1)- and β(2)-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae.

PubMed

Valentine, Cathleen D; Haggie, Peter M

2011-08-15

The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β(1)- and β(2)AR, are structurally similar but mediate distinct signaling responses. Scaffold protein-mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β(1)- and β(2)AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)-domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β(2)AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β(2)AR confinement. For both β(1)- and β(2)AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β(1)- or β(2)AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes.

Cyclin D2 induces proliferation of cardiac myocytes and represses hypertrophy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Busk, Peter K.; Hinrichsen, Rebecca; Bartkova, Jirina

2005-03-10

The myocytes of the adult mammalian heart are considered unable to divide. Instead, mitogens induce cardiomyocyte hypertrophy. We have investigated the effect of adenoviral overexpression of cyclin D2 on myocyte proliferation and morphology. Cardiomyocytes in culture were identified by established markers. Cyclin D2 induced DNA synthesis and proliferation of cardiomyocytes and impaired hypertrophy induced by angiotensin II and serum. At the molecular level, cyclin D2 activated CDK4/6 and lead to pRB phosphorylation and downregulation of the cell cycle inhibitors p21{sup Waf1/Cip1} and p27{sup Kip1}. Expression of the CDK4/6 inhibitor p16 inhibited proliferation and cyclin D2 overexpressing myocytes became hypertrophic undermore » such conditions. Inhibition of hypertrophy by cyclin D2 correlated with downregulation of p27{sup Kip1}. These data show that hypertrophy and proliferation are highly related processes and suggest that cardiomyocyte hypertrophy is due to low amounts of cell cycle activators unable to overcome the block imposed by cell cycle inhibitors. Cell cycle entry upon hypertrophy may be converted to cell division by increased expression of activators such as cyclin D2.« less

The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor beta.

PubMed Central

Pinsky, D J; Cai, B; Yang, X; Rodriguez, C; Sciacca, R R; Cannon, P J

1995-01-01

Inducible nitric oxide (NO) produced by macrophages is cytotoxic to invading organisms and has an important role in host defense. Recent studies have demonstrated inducible NO production within the heart, and that cytokine-induced NO mediates alterations in cardiac contractility, but the cytotoxic potential of nitric oxide with respect to the heart has not been defined. To evaluate the role of inducible nitric oxide synthase (iNOS) on cardiac myocyte cytotoxicity, we exposed adult rat cardiac myocytes to either cytokines alone or to activated J774 macrophages in coculture. Increased expression of both iNOS message and protein was seen in J774 macrophages treated with IFN gamma and LPS and cardiac myocytes treated with TNF-alpha, IL-1 beta, and IFN gamma. Increased NO synthesis was confirmed in both the coculture and isolated myocyte preparations by increased nitrite production. Increased NO synthesis was associated with a parallel increase in myocyte death as measured by CPK release into the culture medium as well as by loss of membrane integrity, visualized by trypan blue staining. Addition of the competitive NO synthase inhibitor L-NMMA to the culture medium prevented both the increased nitrite production and the cytotoxicity observed after cytokine treatment in both the isolated myocyte and the coculture experiments. Because transforming growth-factor beta modulates iNOS expression in other cell types, we evaluated its effects on cardiac myocyte iNOS expression and NO-mediated myocyte cytotoxicity. TGF-beta reduced expression of cardiac myocyte iNOS message and protein, reduced nitrite production, and reduced NO-mediated cytotoxicity in parallel. Taken together, these experiments show the cytotoxic potential of endogenous NO production within the heart, and suggest a role for TGF-beta or NO synthase antagonists to mute these lethal effects. These findings may help explain the cardiac response to sepsis or allograft rejection, as well as the progression of

Neutrophil adherence to isolated adult canine myocytes. Evidence for a CD18-dependent mechanism.

PubMed

Entman, M L; Youker, K; Shappell, S B; Siegel, C; Rothlein, R; Dreyer, W J; Schmalstieg, F C; Smith, C W

1990-05-01

Cardiac myocytes were isolated from adult dogs and incubated with isolated canine neutrophils (PMN). Intercellular adhesion was low and unchanged by stimulation of the PMN with zymosan activated serum or platelet activating factor (PAF) at concentrations that significantly enhance PMN adhesion to protein-coated glass and canine endothelial cell monolayers. Intercellular adhesion was significantly increased only when both myocytes and PMN were stimulated (e.g., myocytes incubated with IL-1, tumor necrosis factor, or phorbol myristate acetate, and PMN were chemotactically stimulated). Inhibitors of protein synthesis diminished the IL-1 beta-induced effect by greater than 80%. The IL-1 beta, PAF-stimulated PMN-myocyte adhesion was associated with substantial H2O2 production. Under conditions with low PMN-myocyte adhesion (i.e., IL-1 beta alone, PAF alone, or no stimulus) H2O2 production was generally less than 5% of that occurring with high adhesion. An anti-CD18 monoclonal antibody (R15.7) inhibited stimulated PMN-myocyte adhesion by greater than 95% and reduced H2O2 production by greater than 90%. Control isotype-matched, binding, and nonbinding antibodies were without effect on adherence or H2O2 production. The results indicate that cytokine stimulation of adult myocytes induces expression of a ligand involved in CD18-dependent adherence of canine neutrophils.

Polymer microfiber meshes facilitate cardiac differentiation of c-kit{sup +} human cardiac stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kan, Lijuan; Thayer, Patrick; Fan, Huimin

Electrospun microfiber meshes have been shown to support the proliferation and differentiation of many types of stem cells, but the phenotypic fate of c-kit{sup +} human cardiac stem cells (hCSCs) have not been explored. To this end, we utilized thin (~5 µm) elastomeric meshes consisting of aligned 1.7 µm diameter poly (ester-urethane urea) microfibers as substrates to examine their effect on hCSC viability, morphology, proliferation, and differentiation relative to cells cultured on tissue culture polystyrene (TCPS). The results showed that cells on microfiber meshes displayed an elongated morphology aligned in the direction of fiber orientation, lower proliferation rates, but increasedmore » expressions of genes and proteins majorly associated with cardiomyocyte phenotype. The early (NK2 homeobox 5, Nkx2.5) and late (cardiac troponin I, cTnI) cardiomyocyte genes were significantly increased on meshes (Nkx=2.5 56.2±13.0, cTnl=2.9±0.56,) over TCPS (Nkx2.5=4.2±0.9, cTnl=1.6±0.5, n=9, p<0.05 for both groups) after differentiation. In contrast, expressions of smooth muscle markers, Gata6 and myosin heavy chain (SM-MHC), were decreased on meshes. Immunocytochemical analysis with cardiac antibody exhibited the similar pattern of above cardiac differentiation. We conclude that aligned microfiber meshes are suitable for guiding cardiac differentiation of hCSCs and may facilitate stem cell-based therapies for treatment of cardiac diseases. - Highlights: • First study to characterize c-kit{sup +} human cardiac stem cells on microfiber meshes. • Microfiber meshes seem reducing cell proliferation, but no effect on cell viability. • Microfiber meshes facilitate the elongation of human cardiac stem cells in culture. • Cardiac but not smooth muscle differentiation were enhanced on microfiber meshes. • Microfiber meshes may be used as cardiac patches in cell-based cardiac therapy.« less

Experimental generation and computational modeling of intracellular pH gradients in cardiac myocytes.

PubMed

Swietach, Pawel; Leem, Chae-Hun; Spitzer, Kenneth W; Vaughan-Jones, Richard D

2005-04-01

It is often assumed that pH(i) is spatially uniform within cells. A double-barreled microperfusion system was used to apply solutions of weak acid (acetic acid, CO(2)) or base (ammonia) to localized regions of an isolated ventricular myocyte (guinea pig). A stable, longitudinal pH(i) gradient (up to 1 pH(i) unit) was observed (using confocal imaging of SNARF-1 fluorescence). Changing the fractional exposure of the cell to weak acid/base altered the gradient, as did changing the concentration and type of weak acid/base applied. A diffusion-reaction computational model accurately simulated this behavior of pH(i). The model assumes that H(i)(+) movement occurs via diffusive shuttling on mobile buffers, with little free H(+) diffusion. The average diffusion constant for mobile buffer was estimated as 33 x 10(-7) cm(2)/s, consistent with an apparent H(i)(+) diffusion coefficient, D(H)(app), of 14.4 x 10(-7) cm(2)/s (at pH(i) 7.07), a value two orders of magnitude lower than for H(+) ions in water but similar to that estimated recently from local acid injection via a cell-attached glass micropipette. We conclude that, because H(i)(+) mobility is so low, an extracellular concentration gradient of permeant weak acid readily induces pH(i) nonuniformity. Similar concentration gradients for weak acid (e.g., CO(2)) occur across border zones during regional myocardial ischemia, raising the possibility of steep pH(i) gradients within the heart under some pathophysiological conditions.

Kinetics of Mechanical Stretch-Induced Nitric Oxide Production in Rat Ventricular Cardiac Myocytes.

PubMed

Shim, A L; Mitrokhin, V M; Gorbacheva, L R; Savinkova, I G; Pustovit, K B; Mladenov, M I; Kamkin, A G

2017-09-01

Discrete mechanical stretch of isolated spontaneously contracting cardiac myocytes was employed to examine the kinetics of NO production in these cells. NO oscillations were detected with fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. The mechanisms underlying stretch-induced changes in NO concentration remain unclear and further studies are needed to evaluate the role of NO oscillation in the regulation of cardiomyocyte function.

Oxidative Stress-Responsive Apoptosis Inducing Protein (ORAIP) Plays a Critical Role in High Glucose-Induced Apoptosis in Rat Cardiac Myocytes and Murine Pancreatic β-Cells.

PubMed

Yao, Takako; Fujimura, Tsutomu; Murayama, Kimie; Okumura, Ko; Seko, Yoshinori

2017-10-18

We previously identified a novel apoptosis-inducing humoral factor in the conditioned medium of hypoxic/reoxygenated-cardiac myocytes. We named this novel post-translationally-modified secreted-form of eukaryotic translation initiation factor 5A Oxidative stress-Responsive Apoptosis-Inducing Protein (ORAIP). We confirmed that myocardial ischemia/reperfusion markedly increased plasma ORAIP levels and rat myocardial ischemia/reperfusion injury was clearly suppressed by neutralizing anti-ORAIP monoclonal antibodies (mAbs) in vivo. In this study, to investigate the mechanism of cell injury of cardiac myocytes and pancreatic β-cells involved in diabetes mellitus (DM), we analyzed plasma ORAIP levels in DM model rats and the role of ORAIP in high glucose-induced apoptosis of cardiac myocytes in vitro. We also examined whether recombinant-ORAIP induces apoptosis in pancreatic β-cells. Plasma ORAIP levels in DM rats during diabetic phase were about 18 times elevated as compared with non-diabetic phase. High glucose induced massive apoptosis in cardiac myocytes (66.2 ± 2.2%), which was 78% suppressed by neutralizing anti-ORAIP mAb in vitro. Furthermore, recombinant-ORAIP clearly induced apoptosis in pancreatic β-cells in vitro. These findings strongly suggested that ORAIP plays a pivotal role in hyperglycemia-induced myocardial injury and pancreatic β-cell injury in DM. ORAIP will be a biomarker and a critical therapeutic target for cardiac injury and progression of DM itself.

An Experimental Model Using Cultured Cardiac Myocytes for a Study of the Generation of Premature Ventricular Contractions Under Ultrasound Exposure

NASA Astrophysics Data System (ADS)

Kudo, Nobuki; Yamamoto, Masaya

2011-09-01

It is known that use of a contrast agents in echocardiography increases the probability of generation of premature ventricular contractions (PVCs). As a basic study to elucidate the mechanisms and to reduce adverse effects, the generation of PVCs was investigated using cultured cardiac myocytes instead of the intact heart in vivo. Cardiac myocytes were isolated from neonatal rats and cultured on a cover slip. The myocyte sample was exposed to pulsed ultrasound with microbubbles adjacent to the myocytes, and generation of PVCs was examined with ultrasound exposure at various delay times after onset of myocyte contraction. The experimental results showed that generation of PVCs had a stable threshold delay time and that PVCs were generated only when myocytes were exposed to ultrasound with delay times longer than the threshold. The results indicate that the model used in this study is useful for revealing the mechanisms by which PVCs are induced by ultrasound exposure.

Pre-Conditioning with CDP-Choline Attenuates Oxidative Stress-Induced Cardiac Myocyte Death in a Hypoxia/Reperfusion Model

PubMed Central

González-Pacheco, Héctor; Méndez-Domínguez, Aurelio; Hernández, Salomón; López-Marure, Rebeca; Vazquez-Mellado, Maria J.; Aguilar, Cecilia; Rocha-Zavaleta, Leticia

2014-01-01

Background. CDP-choline is a key intermediate in the biosynthesis of phosphatidylcholine, which is an essential component of cellular membranes, and a cell signalling mediator. CDP-choline has been used for the treatment of cerebral ischaemia, showing beneficial effects. However, its potential benefit for the treatment of myocardial ischaemia has not been explored yet. Aim. In the present work, we aimed to evaluate the potential use of CDP-choline as a cardioprotector in an in vitro model of ischaemia/reperfusion injury. Methods. Neonatal rat cardiac myocytes were isolated and subjected to hypoxia/reperfusion using the coverslip hypoxia model. To evaluate the effect of CDP-choline on oxidative stress-induced reperfusion injury, the cells were incubated with H2O2 during reperfusion. The effect of CDP-choline pre- and postconditioning was evaluated using the cell viability MTT assay, and the proportion of apoptotic and necrotic cells was analyzed using the Annexin V determination by flow cytometry. Results. Pre- and postconditioning with 50 mg/mL of CDP-choline induced a significant reduction of cells undergoing apoptosis after hypoxia/reperfusion. Preconditioning with CDP-choline attenuated postreperfusion cell death induced by oxidative stress. Conclusion. CDP-choline administration reduces cell apoptosis induced by oxidative stress after hypoxia/reperfusion of cardiac myocytes. Thus, it has a potential as cardioprotector in ischaemia/reperfusion-injured cardiomyocytes. PMID:24578622

β-Myosin Heavy Chain Is Induced by Pressure Overload in a Minor Sub-Population of Smaller Mouse Cardiac Myocytes

PubMed Central

López, Javier E.; Myagmar, Bat-Erdene; Swigart, Philip M.; Montgomery, Megan D.; Haynam, Stephen; Bigos, Marty; Rodrigo, Manoj C.; Simpson, Paul C.

2011-01-01

Rationale Induction of the fetal hypertrophic marker gene beta-myosin heavy chain (β-MyHC) is a signature feature of pressure overload hypertrophy in rodents. β-MyHC is assumed present in all or most enlarged myocytes. Objective To quantify the number and size of myocytes expressing endogenous β-MyHC using a flow cytometry approach. Methods and Results Myocytes were isolated from the LV of male C57Bl/6J mice after transverse aortic constriction (TAC), and the fraction of cells expressing endogenous β-MyHC was quantified by flow cytometry on 10,000–20,000 myocytes, using a validated β-MyHC antibody. Side scatter by flow cytometry in the same cells was validated as an index of myocyte size. β-MyHC-positive myocytes were 3±1% of myocytes in control hearts (n=12), increasing to 25±10% at 3d-6w after TAC (n=24, p<0.01). β-MyHC-positive myocytes did not enlarge with TAC, and were smaller at all times than myocytes without β-MyHC (~70% as large, p<0.001). β-MyHC-positive myocytes arose by addition of β-MyHC to α-MyHC, and had more total MyHC after TAC than did the hypertrophied myocytes that had α-MyHC only. Myocytes positive for β-MyHC were found in discrete regions of the LV, in 3 patterns, peri-vascular, in areas with fibrosis, and in apparently normal myocardium. Conclusion β-MyHC protein is induced by pressure overload in a minor sub-population of smaller cardiac myocytes. The hypertrophied myocytes after TAC have α-MyHC only. These data challenge the current paradigm of the fetal hypertrophic gene program, and identify a new sub-population of smaller working ventricular myocytes with more myosin. PMID:21778428

Redox signaling in cardiac myocytes

PubMed Central

Santos, Celio X.C.; Anilkumar, Narayana; Zhang, Min; Brewer, Alison C.; Shah, Ajay M.

2011-01-01

The heart has complex mechanisms that facilitate the maintenance of an oxygen supply–demand balance necessary for its contractile function in response to physiological fluctuations in workload as well as in response to chronic stresses such as hypoxia, ischemia, and overload. Redox-sensitive signaling pathways are centrally involved in many of these homeostatic and stress-response mechanisms. Here, we review the main redox-regulated pathways that are involved in cardiac myocyte excitation–contraction coupling, differentiation, hypertrophy, and stress responses. We discuss specific sources of endogenously generated reactive oxygen species (e.g., mitochondria and NADPH oxidases of the Nox family), the particular pathways and processes that they affect, the role of modulators such as thioredoxin, and the specific molecular mechanisms that are involved—where this knowledge is available. A better understanding of this complex regulatory system may allow the development of more specific therapeutic strategies for heart diseases. PMID:21236334

Contractile reserve and calcium regulation are depressed in myocytes from chronically unloaded hearts

NASA Technical Reports Server (NTRS)

Ito, Kenta; Nakayama, Masaharu; Hasan, Faisal; Yan, Xinhua; Schneider, Michael D.; Lorell, Beverly H.

2003-01-01

BACKGROUND: Chronic cardiac unloading of the normal heart results in the reduction of left ventricular (LV) mass, but effects on myocyte contractile function are not known. METHODS AND RESULTS: Cardiac unloading and reduction in LV mass were induced by heterotopic heart transplantation to the abdominal aorta in isogenic rats. Contractility and [Ca(2+)](i) regulation in LV myocytes were studied at both 2 and 5 weeks after transplantation. Native in situ hearts from recipient animals were used as the controls for all experiments. Contractile function indices in myocytes from 2-week unloaded and native (control) hearts were similar under baseline conditions (0.5 Hz, 1.2 mmol/L [Ca(2+)](o), and 36 degrees C) and in response to stimulation with high [Ca(2+)](o) (range 2.5 to 4.0 mmol/L). In myocytes from 5-week unloaded hearts, there were no differences in fractional cell shortening and peak-systolic [Ca(2+)](i) at baseline; however, time to 50% relengthening and time to 50% decline in [Ca(2+)](i) were prolonged compared with controls. Severe defects in fractional cell shortening and peak-systolic [Ca(2+)](i) were elicited in myocytes from 5-week unloaded hearts in response to high [Ca(2+)](o). However, there were no differences in the contractile response to isoproterenol between myocytes from unloaded and native hearts. In 5-week unloaded hearts, but not in 2-week unloaded hearts, LV protein levels of phospholamban were increased (345% of native heart values). Protein levels of sarcoplasmic reticulum Ca(2+) ATPase and the Na(+)/Ca(2+) exchanger were not changed. CONCLUSIONS: Chronic unloading of the normal heart caused a time-dependent depression of myocyte contractile function, suggesting the potential for impaired performance in states associated with prolonged cardiac atrophy.

Maturation status of sarcomere structure and function in human iPSC-derived cardiac myocytes.

PubMed

Bedada, Fikru B; Wheelwright, Matthew; Metzger, Joseph M

2016-07-01

Human heart failure due to myocardial infarction is a major health concern. The paucity of organs for transplantation limits curative approaches for the diseased and failing adult heart. Human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) have the potential to provide a long-term, viable, regenerative-medicine alternative. Significant progress has been made with regard to efficient cardiac myocyte generation from hiPSCs. However, directing hiPSC-CMs to acquire the physiological structure, gene expression profile and function akin to mature cardiac tissue remains a major obstacle. Thus, hiPSC-CMs have several hurdles to overcome before they find their way into translational medicine. In this review, we address the progress that has been made, the void in knowledge and the challenges that remain. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Modeling beta-adrenergic control of cardiac myocyte contractility in silico.

PubMed

Saucerman, Jeffrey J; Brunton, Laurence L; Michailova, Anushka P; McCulloch, Andrew D

2003-11-28

The beta-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating beta-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of beta 1-adrenergic receptor, and manipulating the affinity of Gs alpha for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6x increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the beta-adrenergic signaling network may be understood within the context of integrative cellular physiology.

Modeling beta-adrenergic control of cardiac myocyte contractility in silico

NASA Technical Reports Server (NTRS)

Saucerman, Jeffrey J.; Brunton, Laurence L.; Michailova, Anushka P.; McCulloch, Andrew D.; McCullough, A. D. (Principal Investigator)

2003-01-01

The beta-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating beta-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of beta 1-adrenergic receptor, and manipulating the affinity of Gs alpha for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6x increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the beta-adrenergic signaling network may be understood within the context of integrative cellular physiology.

Integrins and Integrin-Associated Proteins in the Cardiac Myocyte

PubMed Central

Ross, Robert S.

2014-01-01

Integrins are heterodimeric, transmembrane receptors that are expressed in all cells, including those in the heart. They participate in multiple critical cellular processes including adhesion, extracellular matrix organization, signaling, survival, and proliferation. Particularly relevant for a contracting muscle cell, integrins are mechanotransducers, translating mechanical to biochemical information. While it is likely that cardiovascular clinicians and scientists have highest recognition of integrins in the cardiovascular system from drugs used to inhibit platelet aggregation, the focus of this article will be on the role of integrins specifically in the cardiac myocyte. Following a general introduction to integrin biology, the manuscript will discuss important work on integrin signaling, mechanotransduction, and lessons learned about integrin function from a range of model organisms. Then we will detail work on integrin-related proteins in the myocyte, how integrins may interact with ion channels and mediate viral uptake into cells, and also play a role in stem cell biology. Finally, we will discuss directions for future study. PMID:24481847

Negative feedback exerted by cAMP-dependent protein kinase and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes: an in vivo study using adenovirus-mediated expression of CNG channels.

PubMed

Rochais, Francesca; Vandecasteele, Grégoire; Lefebvre, Florence; Lugnier, Claire; Lum, Hazel; Mazet, Jean-Luc; Cooper, Dermot M F; Fischmeister, Rodolphe

2004-12-10

Intracardiac cAMP levels are modulated by hormones and neuromediators with specific effects on contractility and metabolism. To understand how the same second messenger conveys different information, mutants of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit CNGA2, encoded into adenoviruses, were used to monitor cAMP in adult rat ventricular myocytes. CNGA2 was not found in native myocytes but was strongly expressed in infected cells. In whole cell patch-clamp experiments, the forskolin analogue L-858051 (L-85) elicited a non-selective, Mg2+ -sensitive current observed only in infected cells, which was thus identified as the CNG current (ICNG). The beta-adrenergic agonist isoprenaline (ISO) also activated ICNG, although the maximal efficiency was approximately 5 times lower than with L-85. However, ISO and L-85 exerted a similar maximal increase of the L-type Ca2+ current. The use of a CNGA2 mutant with a higher sensitivity for cAMP indicated that this difference is caused by the activation of a localized fraction of CNG channels by ISO. cAMP-dependent protein kinase (PKA) blockade with H89 or PKI, or phosphodiesterase (PDE) inhibition with IBMX, dramatically potentiated ISO- and L-85-stimulated ICNG. A similar potentiation of beta-adrenergic stimulation occurred when PDE4 was blocked, whereas PDE3 inhibition had a smaller effect (by 2-fold). ISO and L-85 increased total PDE3 and PDE4 activities in cardiomyocytes, although this effect was insensitive to H89. However, in the presence of IBMX, H89 had no effect on ISO stimulation of ICNG. This study demonstrates that subsarcolemmal cAMP levels are dynamically regulated by a negative feedback involving PKA stimulation of subsarcolemmal cAMP-PDE.

Volume properties and refraction of aqueous solutions of bisadducts of light fullerene C60 and essential amino acids lysine, threonine, and oxyproline (C60(C6H13N2O2)2, C60(C4H8NO3)2, and C60(C5H9NO2)2) at 25°C

NASA Astrophysics Data System (ADS)

Semenov, K. N.; Ivanova, N. M.; Charykov, N. A.; Keskinov, V. A.; Kalacheva, S. S.; Duryagina, N. N.; Garamova, P. V.; Kulenova, N. A.; Nabieva, A.

2017-02-01

Concentration dependences of the density of aqueous solutions of bisadducts of light fullerene C60 and essential amino acids are studied by pycnometry. Concentration dependences of the average molar volumes and partial volumes of components (H2O and corresponding bisadducts) are calculated for C60(C6H13N2O2)2-H2O, C60(C4H8NO3)2-H2O, and C60(C5H9NO2)2-H2O binary systems at 25°C. Concentration dependences of the indices of refraction of C60(C6H13N2O2)2-H2O, C60(C4H8NO3)2-H2O, and C60(C5H9NO2)2-H2O binary systems are determined at 25°C. The concentration dependences of specific refraction and molar refraction of bisadducts and aqueous solutions of them are calculated.

NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Chuen-Mao, E-mail: chuenmao@mail.cgu.edu.tw; Heart Failure Center, Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital at Keelung, Keelung, Taiwan; Lee, I-Ta

TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)],more » MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47{sup phox}, p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF

Profound regulation of Na/K pump activity by transient elevations of cytoplasmic calcium in murine cardiac myocytes

PubMed Central

Lu, Fang-Min; Deisl, Christine; Hilgemann, Donald W

2016-01-01

Small changes of Na/K pump activity regulate internal Ca release in cardiac myocytes via Na/Ca exchange. We now show conversely that transient elevations of cytoplasmic Ca strongly regulate cardiac Na/K pumps. When cytoplasmic Na is submaximal, Na/K pump currents decay rapidly during extracellular K application and multiple results suggest that an inactivation mechanism is involved. Brief activation of Ca influx by reverse Na/Ca exchange enhances pump currents and attenuates current decay, while repeated Ca elevations suppress pump currents. Pump current enhancement reverses over 3 min, and results are similar in myocytes lacking the regulatory protein, phospholemman. Classical signaling mechanisms, including Ca-activated protein kinases and reactive oxygen, are evidently not involved. Electrogenic signals mediated by intramembrane movement of hydrophobic ions, such as hexyltriphenylphosphonium (C6TPP), increase and decrease in parallel with pump currents. Thus, transient Ca elevation and Na/K pump inactivation cause opposing sarcolemma changes that may affect diverse membrane processes. DOI: http://dx.doi.org/10.7554/eLife.19267.001 PMID:27627745

Rate Coefficients of C2H with C2H4, C2H6, and H2 from 150 to 359 K

NASA Technical Reports Server (NTRS)

Opansky, Brian J.; Leone, Stephen R.

1996-01-01

Rate coefficients for the reactions C2H with C2H4, C2H6, and H2 are measured over the temperature range 150-359 K using transient infrared laser absorption spectroscopy. The ethynyl radical is formed by photolysis of C2H2 with a pulsed excimer laser at 193 nm, and its transient absorption is monitored with a color center laser on the Q(sub 11)(9) line of the A(sup 2) Pi-Chi(sup 2) Sigma transition at 3593.68 cm(exp -1). Over the experimental temperature range 150-359 K the rate constants of C2H with C2H4, C2H6, and H2 can be fitted to the Arrhenius expressions k(sub C2H4) = (7.8 +/- 0.6) x 10(exp -11) exp[(134 +/- 44)/T], k(sub C2H6) = (3.5 +/- 0.3) x 10(exp -11) exp[(2.9 +/- 16)/T], and k(sub H2) = (1.2 +/- 0.3) x 10(exp -11) exp[(-998 +/- 57)]/T cm(exp 3) molecule(exp -1) sec(exp -1). The data for C2H with C2H4 and C2H6 indicate a negligible activation energy to product formation shown by the mild negative temperature dependence of both reactions. When the H2 data are plotted together with the most recent high-temperature results from 295 to 854 K, a slight curvature is observed. The H2 data can be fit to the non-Arrhenius form k(sub H2) = 9.2 x 10(exp -18) T(sup 2.17 +/- 0.50) exp[(-478 +/- 165)/T] cm(exp 3) molecules(exp -1) sec(exp -1). The curvature in the Arrhenius plot is discussed in terms of both quantum mechanical tunneling of the H atom from H2 to the C2H radical and bending mode contributions to the partition function.

Modulation of cytosolic and intra-sarcoplasmic reticulum calcium waves by calsequestrin in rat cardiac myocytes

PubMed Central

Kubalova, Zuzana; Györke, Inna; Terentyeva, Radmila; Viatchenko-Karpinski, Serge; Terentyev, Dmitry; Williams, Simon C; Györke, Sandor

2004-01-01

Waves of Ca2+-induced Ca2+ release occur in various cell types and are involved in the pathology of certain forms of cardiac arrhythmia. These arrhythmias include catecholaminergic polymorphic ventricular tachycardia (CPVT), certain cases of which are associated with mutations in the cardiac calsequestrin gene (CASQ2). To explore the mechanisms of Ca2+ wave generation and unravel the underlying causes of CPVT, we investigated the effects of adenoviral-mediated changes in CASQ2 protein levels on the properties of cytosolic and sarcoplasmic reticulum (SR) Ca2+ waves in permeabilized rat ventricular myocytes. The free [Ca2+] inside the sarcoplasmic reticulum ([Ca2+]SR) was monitored by fluo-5N entrapped into the SR, and cytosolic Ca2+ was imaged using fluo-3. Overexpression of CASQ2 resulted in significant increases in the amplitude of Ca2+ waves and interwave intervals, whereas reduced CASQ2 levels caused drastic reductions in the amplitude and period of Ca2+ waves. CASQ2 abundance had no impact on resting diastolic [Ca2+]SR or on the amplitude of the [Ca2+]SR depletion signal during the Ca2+ wave. However, the recovery dynamics of [Ca2+]SR following Ca2+ release were dramatically altered as the rate of [Ca2+]SR recovery increased ∼3-fold in CASQ2-overexpressing myocytes and decreased to 30% of control in CASQ2-underexpressing myocytes. There was a direct linear relationship between Ca2+ wave period and the half-time of basal [Ca2+]SR recovery following Ca2+ release. Loading the SR with the low affinity exogenous Ca2+ buffer citrate exerted effects quantitatively similar to those observed on overexpressing CASQ2. We conclude that free intra-SR [Ca2+] is a critical determinant of cardiac Ca2+ wave generation. Our data indicate that reduced intra-SR Ca2+ binding activity promotes the generation of Ca2+ waves by accelerating the dynamics of attaining a threshold free [Ca2+]SR required for Ca2+ wave initiation, potentially accounting for arrythmogenesis in CPVT linked

Ubiquitin-proteasome system impairment caused by a missense cardiac myosin-binding protein C mutation and associated with cardiac dysfunction in hypertrophic cardiomyopathy.

PubMed

Bahrudin, Udin; Morisaki, Hiroko; Morisaki, Takayuki; Ninomiya, Haruaki; Higaki, Katsumi; Nanba, Eiji; Igawa, Osamu; Takashima, Seiji; Mizuta, Einosuke; Miake, Junichiro; Yamamoto, Yasutaka; Shirayoshi, Yasuaki; Kitakaze, Masafumi; Carrier, Lucie; Hisatome, Ichiro

2008-12-26

The ubiquitin-proteasome system is responsible for the disappearance of truncated cardiac myosin-binding protein C, and the suppression of its activity contributes to cardiac dysfunction. This study investigated whether missense cardiac myosin-binding protein C gene (MYBPC3) mutation in hypertrophic cardiomyopathy (HCM) leads to destabilization of its protein, causes UPS impairment, and is associated with cardiac dysfunction. Mutations were identified in Japanese HCM patients using denaturing HPLC and sequencing. Heterologous expression was investigated in COS-7 cells as well as neonatal rat cardiac myocytes to examine protein stability and proteasome activity. The cardiac function was measured using echocardiography. Five novel MYBPC3 mutations -- E344K, DeltaK814, Delta2864-2865GC, Q998E, and T1046M -- were identified in this study. Compared with the wild type and other mutations, the E334K protein level was significantly lower, it was degraded faster, it had a higher level of polyubiquination, and increased in cells pretreated with the proteasome inhibitor MG132 (50 microM, 6 h). The electrical charge of its amino acid at position 334 influenced its stability, but E334K did not affect its phosphorylation. The E334K protein reduced cellular 20 S proteasome activity, increased the proapoptotic/antiapoptotic protein ratio, and enhanced apoptosis in transfected Cos-7 cells and neonatal rat cardiac myocytes. Patients carrying the E334K mutation presented significant left ventricular dysfunction and dilation. The conclusion is the missense MYBPC3 mutation E334K destabilizes its protein through UPS and may contribute to cardiac dysfunction in HCM through impairment of the ubiquitin-proteasome system.

Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes.

PubMed Central

Farese, R V; Cooper, D R; Konda, T S; Nair, G; Standaert, M L; Davis, J S; Pollet, R J

1988-01-01

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC

[Ca2+]i Elevation and Oxidative Stress Induce KCNQ1 Protein Translocation from the Cytosol to the Cell Surface and Increase Slow Delayed Rectifier (IKs) in Cardiac Myocytes*

PubMed Central

Wang, Yuhong; Zankov, Dimitar P.; Jiang, Min; Zhang, Mei; Henderson, Scott C.; Tseng, Gea-Ny

2013-01-01

Our goals are to simultaneously determine the three-dimensional distribution patterns of KCNQ1 and KCNE1 in cardiac myocytes and to study the mechanism and functional implications for variations in KCNQ1/KCNE1 colocalization in myocytes. We monitored the distribution patterns of KCNQ1, KCNE1, and markers for subcellular compartments/organelles using immunofluorescence/confocal microscopy and confirmed the findings in ventricular myocytes by directly observing fluorescently tagged KCNQ1-GFP and KCNE1-dsRed expressed in these cells. We also monitored the effects of stress on KCNQ1-GFP and endoplasmic reticulum (ER) remodeling during live cell imaging. The data showed that 1) KCNE1 maintained a stable cell surface localization, whereas KCNQ1 exhibited variations in the cytosolic compartment (striations versus vesicles) and the degree of presence on the cell surface; 2) the degree of cell surface KCNQ1/KCNE1 colocalization was positively correlated with slow delayed rectifier (IKs) current density; 3) KCNQ1 and calnexin (an ER marker) shared a cytosolic compartment; and 4) in response to stress ([Ca2+]i elevation, oxidative overload, or AT1R stimulation), KCNQ1 exited the cytosolic compartment and trafficked to the cell periphery in vesicles. This was accompanied by partial ER fragmentation. We conclude that the cellular milieu regulates KCNQ1 distribution in cardiac myocytes and that stressful conditions can increase IKs by inducing KCNQ1 movement to the cell surface. This represents a hitherto unrecognized mechanism by which IKs fulfills its function as a repolarization reserve in ventricular myocytes. PMID:24142691

Characterization of human septic sera induced gene expression modulation in human myocytes

PubMed Central

Hussein, Shaimaa; Michael, Paul; Brabant, Danielle; Omri, Abdelwahab; Narain, Ravin; Passi, Kalpdrum; Ramana, Chilakamarti V.; Parrillo, Joseph E.; Kumar, Anand; Parissenti, Amadeo; Kumar, Aseem

2009-01-01

To gain a better understanding of the gene expression changes that occurs during sepsis, we have performed a cDNA microarray study utilizing a tissue culture model that mimics human sepsis. This study utilized an in vitro model of cultured human fetal cardiac myocytes treated with 10% sera from septic patients or 10% sera from healthy volunteers. A 1700 cDNA expression microarray was used to compare the transcription profile from human cardiac myocytes treated with septic sera vs normal sera. Septic sera treatment of myocytes resulted in the down-regulation of 178 genes and the up-regulation of 4 genes. Our data indicate that septic sera induced cell cycle, metabolic, transcription factor and apoptotic gene expression changes in human myocytes. Identification and characterization of gene expression changes that occur during sepsis may lead to the development of novel therapeutics and diagnostics. PMID:19684886

G protein-coupled receptor kinase 2 promotes cardiac hypertrophy

PubMed Central

Tscheschner, Henrike; Gao, Erhe; Schumacher, Sarah M.; Yuan, Ancai; Backs, Johannes; Most, Patrick; Wieland, Thomas; Koch, Walter J.; Katus, Hugo A.; Raake, Philip W.

2017-01-01

The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3β), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase γ (PI3Kγ). Moreover, inhibition of PI3Kγ as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3Kγ mediated Akt phosphorylation and subsequent inactivation of GSK3β, resulting in enhanced NFAT activity. PMID:28759639

Kinetics of the R + NO2 reactions (R = i-C3H7, n-C3H7, s-C4H9, and t-C4H9) in the temperature range 201-489 K.

PubMed

Rissanen, Matti P; Arppe, Suula L; Eskola, Arkke J; Tammi, Matti M; Timonen, Raimo S

2010-04-15

The bimolecular rate coefficients of four alkyl radical reactions with NO(2) have been measured in direct time-resolved experiments. Reactions were studied under pseudo-first-order conditions in a temperature-controlled tubular flow reactor coupled to a laser photolysis/photoionization mass spectrometer (LP-PIMS). The measured reaction rate coefficients are independent of helium bath gas pressure within the experimental ranges covered and exhibit negative temperature dependence. For i-C(3)H(7) + NO(2) and t-C(4)H(9) + NO(2) reactions, the dependence of ordinate (logarithm of reaction rate coefficients) on abscissa (1/T or log(T)) was nonlinear. The obtained results (in cm(3) s(-1)) can be expressed by the following equations: k(n-C(3)H(7) + NO(2)) = ((4.34 +/- 0.08) x 10(-11)) (T/300 K)(-0.14+/-0.08) (203-473 K, 1-7 Torr), k(i-C(3)H(7) + NO(2)) = ((3.66 +/- 2.54) x 10(-12)) exp(656 +/- 201 K/T)(T/300 K)(1.26+/-0.68) (220-489 K, 1-11 Torr), k(s-C(4)H(9) + NO(2)) = ((4.99 +/- 0.16) x 10(-11))(T/300 K)(-1.74+/-0.12) (241-485 K, 2 - 12 Torr) and k(t-C(4)H(9) + NO(2)) = ((8.64 +/- 4.61) x 10(-12)) exp(413 +/- 154 K/T)(T/300 K)(0.51+/-0.55) (201-480 K, 2-11 Torr), where the uncertainties shown refer only to the 1 standard deviations obtained from the fitting procedure. The estimated overall uncertainty in the determined bimolecular rate coefficients is about +/-20%.

Fluoride induces apoptosis in H9c2 cardiomyocytes via the mitochondrial pathway.

PubMed

Yan, Xiaoyan; Wang, Lu; Yang, Xia; Qiu, Yulan; Tian, Xiaolin; Lv, Yi; Tian, Fengjie; Song, Guohua; Wang, Tong

2017-09-01

Numerous studies have shown that chronic excessive fluoride intake can adversely affect different organ systems. In particular, the cardiovascular system is susceptible to disruption by a high concentration of fluoride. The objectives of this study were to explore the mechanism of apoptosis by detecting the toxic effects of different concentrations of sodium fluoride (NaF) in H9c2 cells exposed for up to 96 h. NaF not only inhibited H9c2 cell proliferation but also induced apoptosis and morphological damage. With increasing NaF concentrations, early apoptosis of H9c2 cells was increased while the mitochondrial membrane potential was decreased. Compared with the control group, the mRNA levels of caspase-3, caspase-9, and cytochrome c all increased with increasing concentrations of NaF. In summary, these data suggest that apoptosis is involved in NaF-induced H9c2 cell toxicity and that activation of the mitochondrial pathway may occur. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Multi-Domain Fibroblast/Myocyte Coupling in the Cardiac Tissue: A Theoretical Study.

PubMed

Greisas, Ariel; Zlochiver, Sharon

2016-09-01

Cardiac fibroblast proliferation and concomitant collagenous matrix accumulation (fibrosis) develop during multiple cardiac pathologies. Recent studies have demonstrated direct electrical coupling between myocytes and fibroblasts in vitro, and assessed the electrophysiological implications of such coupling. However, in the living tissues, such coupling has not been demonstrated, and only indirect coupling via the extracellular space is likely to exist. In this study we employed a multi-domain model to assess the modulation of the cardiac electrophysiological properties by neighboring fibroblasts assuming only indirect coupling. Numerical simulations in 1D and 2D human atrial models showed that extracellular coupling sustains a significant impact on conduction velocity (CV) and a less significant effect on the action potential duration. Both CV and the slope of the CV restitution increased with increasing fibroblast density. This effect was more substantial for lower extracellular conductance. In 2D, spiral waves exhibited reduced frequency with increasing fibroblast density, and the propensity of wavebreaks and complex dynamics at high pacing rates significantly increased.

Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes.

PubMed

Mali, Vishal R; Deshpande, Mandar; Pan, Guodong; Thandavarayan, Rajarajan A; Palaniyandi, Suresh S

2016-02-01

Reactive oxygen species (ROS)-mediated reactive aldehydes induce cellular stress. In cardiovascular diseases such as ischemia-reperfusion injury, lipid-peroxidation derived reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are known to contribute to the pathogenesis. 4HNE is involved in ROS formation, abnormal calcium handling and more importantly defective mitochondrial respiration. Aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)(+)-dependent isozymes which can detoxify endogenous and exogenous aldehydes into non-toxic carboxylic acids. Therefore we hypothesize that 4HNE afflicts mitochondrial respiration and leads to cell death by impairing ALDH2 activity in cultured H9C2 cardiomyocyte cell lines. H9C2 cardiomyocytes were treated with 25, 50 and 75 μM 4HNE and its vehicle, ethanol as well as 25, 50 and 75 μM disulfiram (DSF), an inhibitor of ALDH2 and its vehicle (DMSO) for 4 h. 4HNE significantly decreased ALDH2 activity, ALDH2 protein levels, mitochondrial respiration and mitochondrial respiratory reserve capacity, and increased 4HNE adduct formation and cell death in cultured H9C2 cardiomyocytes. ALDH2 inhibition by DSF and ALDH2 siRNA attenuated ALDH2 activity besides reducing ALDH2 levels, mitochondrial respiration and mitochondrial respiratory reserve capacity and increased cell death. Our results indicate that ALDH2 impairment can lead to poor mitochondrial respiration and increased cell death in cultured H9C2 cardiomyocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

Modeling the Effects of β1-Adrenergic Receptor Blockers and Polymorphisms on Cardiac Myocyte Ca2+ Handling

PubMed Central

Amanfu, Robert K.

2014-01-01

β-Adrenergic receptor blockers (β-blockers) are commonly used to treat heart failure, but the biologic mechanisms governing their efficacy are still poorly understood. The complexity of β-adrenergic signaling coupled with the influence of receptor polymorphisms makes it difficult to intuit the effect of β-blockers on cardiac physiology. While some studies indicate that β-blockers are efficacious by inhibiting β-adrenergic signaling, other studies suggest that they work by maintaining β-adrenergic responsiveness. Here, we use a systems pharmacology approach to test the hypothesis that in ventricular myocytes, these two apparently conflicting mechanisms for β-blocker efficacy can occur concurrently. We extended a computational model of the β1-adrenergic pathway and excitation-contraction coupling to include detailed receptor interactions for 19 ligands. Model predictions, validated with Ca2+ and Förster resonance energy transfer imaging of adult rat ventricular myocytes, surprisingly suggest that β-blockers can both inhibit and maintain signaling depending on the magnitude of receptor stimulation. The balance of inhibition and maintenance of β1-adrenergic signaling is predicted to depend on the specific β-blocker (with greater responsiveness for metoprolol than carvedilol) and β1-adrenergic receptor Arg389Gly polymorphisms. PMID:24867460

Conversion of (η(5)-C2B9H10R)TaX3 (X = Me, NMe2) to (η(6)-C2B9H10R)TaX' (X' = NMe2, azaallyl) in the absence of a reducing agent: synthesis and structure of tantallacarboranes incorporating an arachno-η(6)-C2B9(4-) ligand.

PubMed

Xiang, Li; Xie, Zuowei

2014-08-04

Heating a benzene solution of [η(5)-(Me2NCH2CH2)C2B9H10] Ta(NMe2)3 (1) in the presence of pyridine gave an unprecedented complex [η(1):η(6)-(Me2NCH2CH2)C2B9H10]Ta (NMe2)(NC5H5) (2). On the other hand, reaction of (η(5)-C2B9H11)TaMe3 with adamantly isonitrile (AdNC) in dimethoxyethane (DME) at room temperature afforded another unexpected complex (η(6)-C2B9H11)Ta[η(3)-C,C,N-CH2C(CH3)NAd](DME) (4). These results show that pyridine and DME are essential for the formation of 2 and 4, respectively. It is suggested that the nido-η(5)-C2B9H10R(2−) ligand in tantallacarboranes takes up two electrons released by reductive elimination to form an arachno-η(6)-C2B9H10R(4−) fragment via the cage C–C bond cleavage.

Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte.

PubMed

Dvornikov, Alexey V; Dewan, Sukriti; Alekhina, Olga V; Pickett, F Bryan; de Tombe, Pieter P

2014-05-01

The zebrafish (Danio rerio) has been used extensively in cardiovascular biology, but mainly in the study of heart development. The relative ease of its genetic manipulation may indicate the suitability of this species as a cost-effective model system for the study of cardiac contractile biology. However, whether the zebrafish heart is an appropriate model system for investigations pertaining to mammalian cardiac contractile structure-function relationships remains to be resolved. Myocytes were isolated from adult zebrafish hearts by enzymatic digestion, attached to carbon rods, and twitch force and intracellular Ca(2+) were measured. We observed the modulation of twitch force, but not of intracellular Ca(2+), by both extracellular [Ca(2+)] and sarcomere length. In permeabilized cells/myofibrils, we found robust myofilament length-dependent activation. Moreover, modulation of myofilament activation-relaxation and force redevelopment kinetics by varied Ca(2+) activation levels resembled that found previously in mammalian myofilaments. We conclude that the zebrafish is a valid model system for the study of cardiac contractile structure-function relationships.

Modulation of the caveolin-3 localization to caveolae and STAT3 to mitochondria by catecholamine-induced cardiac hypertrophy in H9c2 cardiomyoblasts

PubMed Central

Jeong, Kyuho; Kwon, Hayeong; Min, Chanhee

2009-01-01

We investigated the effect of phenylephrine (PE)- and isoproterenol (ISO)-induced cardiac hypertrophy on subcellular localization and expression of caveolin-3 and STAT3 in H9c2 cardiomyoblast cells. Caveolin-3 localization to plasma membrane was attenuated and localization of caveolin-3 to caveolae in the plasma membrane was 24.3% reduced by the catecholamine-induced hypertrophy. STAT3 and phospho-STAT3 were up-regulated but verapamil and cyclosporin A synergistically decreased the STAT3 and phospho-STAT3 levels in PE- and ISO-induced hypertrophic cells. Both expression and activation of STAT3 were increased in the nucleus by the hypertrophy. Immunofluorescence analysis revealed that the catecholamine-induced hypertrophy promoted nuclear localization of pY705-STAT3. Of interest, phosphorylation of pS727-STAT3 in mitochondria was significantly reduced by catecholamine-induced hypertrophy. In addition, mitochondrial complexes II and III were greatly down-regulated in the hypertrophic cells. Our data suggest that the alterations in nuclear and mitochondrial activation of STAT3 and caveolae localization of caveolin-3 are related to the development of the catecholamine-induced cardiac hypertrophy. PMID:19299911

Connective tissue growth factor induces cardiac hypertrophy through Akt signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayata, Nozomi; Fujio, Yasushi; Yamamoto, Yasuhiro

2008-05-30

In the process of cardiac remodeling, connective tissue growth factor (CTGF/CCN2) is secreted from cardiac myocytes. Though CTGF is well known to promote fibroblast proliferation, its pathophysiological effects in cardiac myocytes remain to be elucidated. In this study, we examined the biological effects of CTGF in rat neonatal cardiomyocytes. Cardiac myocytes stimulated with full length CTGF and its C-terminal region peptide showed the increase in cell surface area. Similar to hypertrophic ligands for G-protein coupled receptors, such as endothelin-1, CTGF activated amino acid uptake; however, CTGF-induced hypertrophy is not associated with the increased expression of skeletal actin or BNP, analyzedmore » by Northern-blotting. CTGF treatment activated ERK1/2, p38 MAPK, JNK and Akt. The inhibition of Akt by transducing dominant-negative Akt abrogated CTGF-mediated increase in cell size, while the inhibition of MAP kinases did not affect the cardiac hypertrophy. These findings indicate that CTGF is a novel hypertrophic factor in cardiac myocytes.« less

pPKCδ activates SC35 splicing factor during H9c2 myoblastic differentiation.

PubMed

Zara, Susi; Falconi, Mirella; Rapino, Monica; Zago, Michela; Orsini, Giovanna; Mazzotti, Giovanni; Cataldi, Amelia; Teti, Gabriella

2011-01-01

Although Protein Kinase C (PKC) isoforms' role in the neonatal and adult cardiac tissue development and ageing has been widely described "in vivo", the interaction of such enzymes with specific nuclear substrates needs to be investigated. The aim of our research has been the study of the expression, localization and interaction with the splicing factor SC35 of PKC isoforms (α, δ, ε, ζ) and their potential role in modulating the transcription machinery. H9c2 cells induced to myoblast differentiation in the presence of 1% Horse Serum (HS) have represented our experimental model. The expression of PKC isoforms, their distribution and interaction with SC35 have been evaluated by western blotting, co-immunoprecipitation and double gold immunolabeling for transmission and scanning electron microscopy. Our results show PKCδ as the most expressed isoform in differentiated cells. Surprisingly, the distribution of PKCδ and SC35 does not show any significant modification between 10%FBS and 1%HS treated samples and no co-localization is observed. Moreover the interaction between the phosphorylated form of PKCδ (pPKCδ) and SC35 increases, is distributed and co-localizes within the nucleus of differentiated H9c2. These data represent reasonable evidence of pPKCδ mediated SC35 splicing factor activation, suggesting its direct effect on transcription via interaction with the transcription machinery. Furthermore, this co-localization represents a crucial event resulting in downstream changes in transcription of components which determine the morphological modifications related to cardiomyoblast differentiated phenotype.

The IGF-1 Receptor Identifies a Pool of Human Cardiac Stem Cells with Superior Therapeutic Potential for Myocardial Regeneration

PubMed Central

D’Amario, Domenico; Cabral-Da-Silva, Mauricio; Zheng, Hanqiao; Fiorini, Claudia; Goichberg, Polina; Steadman, Elisabeth; Ferreira-Martins, João; Sanada, Fumihiro; Piccoli, Marco; Cappetta, Donato; D’Alessandro, David A.; Michler, Robert E.; Hosoda, Toru; Anastasia, Luigi; Rota, Marcello; Leri, Annarosa; Anversa, Piero; Kajstura, Jan

2012-01-01

Rationale Age and coronary artery disease may negatively affect the function of human cardiac stem cells (hCSCs) and their potential therapeutic efficacy for autologous cell transplantation in the failing heart. Objective Insulin-like growth factor 1 (IGF-1) and 2 (IGF-2), and angiotensin II (Ang II) and their receptors, IGF-1R, IGF-2R and AT1R, were characterized in c-kit-positive-hCSCs to establish whether these systems would allow us to separate hCSC classes with different growth reserve in the aging and diseased myocardium. Methods and Results C-kit-positive-hCSCs were collected from myocardial samples obtained from 24 patients, 48 to 86 years of age, undergoing elective cardiac surgery for coronary artery disease. The expression of IGF-1R in hCSCs recognized a young cell phenotype defined by long telomeres, high telomerase activity, enhanced cell proliferation and attenuated apoptosis. In addition to IGF-1, IGF-1R-positive-hCSCs secreted IGF-2 that promoted myocyte differentiation. Conversely, the presence of IGF-2R and AT1R, in the absence of IGF-1R, identified senescent hCSCs with impaired growth reserve and increased susceptibility to apoptosis. The ability of IGF-1R-positive-hCSCs to regenerate infarcted myocardium was then compared with that of unselected c-kit-positive-hCSCs. IGF-1R-positive-hCSCs improved cardiomyogenesis and vasculogenesis. Pretreatment of IGF-1R-positive-hCSCs with IGF-2 resulted in the formation of more mature myocytes and superior recovery of ventricular structure. Conclusions hCSCs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which are potent modulators of stem cell replication, commitment to the myocyte lineage and myocyte differentiation, pointing to this hCSC subset as the ideal candidate cell for the management of human heart failure. PMID:21546606

Transcriptional deregulation of oncogenic myocyte enhancer factor 2C in T-cell acute lymphoblastic leukemia.

PubMed

Nagel, Stefan; Venturini, Letizia; Meyer, Corinna; Kaufmann, Maren; Scherr, Michaela; Drexler, Hans G; Macleod, Roderick A F

2011-02-01

Myocyte enhancer factor 2C (MEF2C) encodes a transcription factor which is ectopically expressed in T-cell acute lymphoblastic leukemia (T-ALL) cell lines, deregulated directly by ectopically expressed homeodomain protein NKX2-5 or by loss of promoter regions via del(5)(q14). Here, we analyzed the MEF2C 5'-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix proteins, STAT5, and HOXA9/HOXA10. Chromatin immunoprecipitation and overexpression analyses demonstrated direct activation by GFI1B and LYL1 and inhibition by STAT5. HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of regulation via NMYC interactor (NMI) and STAT5. Lacking comma: Chromosomal deletion of the STAT5 binding site in LOUCY cells reduced protein levels of STAT5 in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5 signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that the expression of MEF2C in T-ALL cells is principally deregulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory developmental STAT5 signaling.

Glucose-6-phosphate dehydrogenase and NADPH redox regulates cardiac myocyte L-type calcium channel activity and myocardial contractile function.

PubMed

Rawat, Dhwajbahadur K; Hecker, Peter; Watanabe, Makino; Chettimada, Sukrutha; Levy, Richard J; Okada, Takao; Edwards, John G; Gupte, Sachin A

2012-01-01

We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca(2+) currents (I(Ca-L)) in cardiac myocytes and inhibits myocardial contractility. Because 17-ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit I(Ca-L) and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca(2+) channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and I(Ca-L) were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of -80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO(2) and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak I(Ca-L) amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of I(Ca-L) by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca(2+) channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure).

Effects of Kaempferia parviflora Wall. Ex. Baker and sildenafil citrate on cGMP level, cardiac function, and intracellular Ca2+ regulation in rat hearts.

PubMed

Weerateerangkul, Punate; Palee, Siripong; Chinda, Kroekkiat; Chattipakorn, Siriporn C; Chattipakorn, Nipon

2012-09-01

Although Kaempferia parviflora extract (KPE) and its flavonoids have positive effects on the nitric oxide (NO) signaling pathway, its mechanisms on the heart are still unclear. Because our previous studies demonstrated that KPE decreased defibrillation efficacy in swine similar to that of sildenafil citrate, the phosphodiesterase-5 inhibitor, it is possible that KPE may affect the cardiac NO signaling pathway. In the present study, the effects of KPE and sildenafil citrate on cyclic guanosine monophosphate (cGMP) level, modulation of cardiac function, and Ca transients in ventricular myocytes were investigated. In a rat model, cardiac cGMP level, cardiac function, and Ca transients were measured before and after treatment with KPE and sildenafil citrate. KPE significantly increased the cGMP level and decreased cardiac function and Ca transient. These effects were similar to those found in the sildenafil citrate-treated group. Furthermore, the nonspecific NOS inhibitor could abolish the effects of KPE and sildenafil citrate on Ca transient. KPE has positive effect on NO signaling in the heart, resulting in an increased cGMP level, similar to that of sildenafil citrate. This effect was found to influence the physiology of normal heart via the attenuation of cardiac function and the reduction of Ca transient in ventricular myocytes.

Histone H3K9 Demethylase JMJD2B Activates Adipogenesis by Regulating H3K9 Methylation on PPARγ and C/EBPα during Adipogenesis

PubMed Central

Jang, Min-Kyung; Kim, Ji-Hyun; Jung, Myeong Ho

2017-01-01

Previous studies have shown that tri- or di-methylation of histone H3 at lysine 9 (H3K9me3/me2) on the promoter of the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) contribute to the repression of PPARγ and C/EBPα and inhibition of adipogenesis in 3T3-L1 preadipocytes. The balance of histone methylation is regulated by histone methyltransferases and demethylases. However, it is poorly understood which demethylases are responsible for removing H3K9me3/me2 on the promoter of PPARγ and C/EBPα. JMJD2B is a H3K9me3/me2 demethylase that was previously shown to activate adipogenesis by promoting mitotic clonal expansion. Nevertheless, it remains unclear whether JMJD2B plays a role in the regulation of adipogenesis by removing H3K9me3/me2 on the promoter of PPARγ and C/EBPα and subsequently activating PPARγ and C/EBPα expression. Here, we showed that JMJD2B decreased H3K9me3/me2 on the promoter of PPARγ and C/EBPα, which in turn stimulated the expression of PPARγ and C/EBPα. JMJD2B knockdown using siRNA in 3T3-L1 preadipocytes repressed the expression of PPARγ and C/EBPα, resulting in inhibition of adipogenesis. This was accompanied by increased enrichment of H3K9me3/me2 on the promoter of PPARγ and C/EBPα. In contrast, overexpression of JMJD2B increased the expression of PPARγ and C/EBPα, which was accompanied by decreased enrichment of H3K9me3/me2 on the promoter and activated adipogenesis. Together, these results indicate that JMJD2B regulates PPARγ and C/EBPα during adipogenesis. PMID:28060835

Expression of androgen-binding protein (ABP) in human cardiac myocytes.

PubMed

Schock, H W; Herbert, Z; Sigusch, H; Figulla, H R; Jirikowski, G F; Lotze, U

2006-04-01

Cardiomyocytes are known to be androgen targets. Changing systemic steroid levels are thought to be linked to various cardiac ailments, including dilated cardiomyopathy (DCM). The mode of action of gonadal steroid hormones on the human heart is unknown to date. In the present study, we used high-resolution immunocytochemistry on semithin sections (1 microm thick), IN SITU hybridization, and mass spectrometry to investigate the expression of androgen-binding protein (ABP) in human myocardial biopsies taken from male patients with DCM. We observed distinct cytoplasmic ABP immunoreactivity in a fraction of the myocytes. IN SITU hybridization with synthetic oligonucleotide probes revealed specific hybridization signals in these cells. A portion of the ABP-positive cells contained immunostaining for androgen receptor. With SELDI TOF mass spectrometry of affinity purified tissue extracts of human myocardium, we confirmed the presence of a 50 kDa protein similar to ABP. Our observations provide evidence of an intrinsic expression of ABP in human heart. ABP may be secreted from myocytes in a paracrine manner perhaps to influence the bioavailabity of gonadal steroids in myocardium.

Phosphodiesterase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis. Essential role of nitric oxide signaling.

PubMed

Das, Anindita; Xi, Lei; Kukreja, Rakesh C

2005-04-01

We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 microM) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, L-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible

The role of spatial organization of Ca2+ release sites in the generation of arrhythmogenic diastolic Ca2+ release in myocytes from failing hearts

PubMed Central

Ho, Hsiang-Ting; Bonilla, Ingrid M.; Terentyeva, Radmila; Schober, Karsten E.; Terentyev, Dmitry; Carnes, Cynthia A.

2018-01-01

In heart failure (HF), dysregulated cardiac ryanodine receptors (RyR2) contribute to the generation of diastolic Ca2+ waves (DCWs), thereby predisposing adrenergically stressed failing hearts to life-threatening arrhythmias. However, the specific cellular, subcellular, and molecular defects that account for cardiac arrhythmia in HF remain to be elucidated. Patch-clamp techniques and confocal Ca2+ imaging were applied to study spatially defined Ca2+ handling in ventricular myocytes isolated from normal (control) and failing canine hearts. Based on their activation time upon electrical stimulation, Ca2+ release sites were categorized as coupled, located in close proximity to the sarcolemmal Ca2+ channels, and uncoupled, the Ca2+ channel-free non-junctional Ca2+ release units. In control myocytes, stimulation of β-adrenergic receptors with isoproterenol (Iso) resulted in a preferential increase in Ca2+ spark rate at uncoupled sites. This site-specific effect of Iso was eliminated by the phosphatase inhibitor okadaic acid, which caused similar facilitation of Ca2+ sparks at coupled and uncoupled sites. Iso-challenged HF myocytes exhibited increased predisposition to DCWs compared to control myocytes. In addition, the overall frequency of Ca2+ sparks was increased in HF cells due to preferential stimulation of coupled sites. Furthermore, coupled sites exhibited accelerated recovery from functional refractoriness in HF myocytes compared to control myocytes. Spatially resolved subcellular Ca2+ mapping revealed that DCWs predominantly originated from coupled sites. Inhibition of CaMK∏ suppressed DCWs and prevented preferential stimulation of coupled sites in Iso-challenged HF myocytes. These results suggest that CaMK∏-(and phosphatase)-dependent dysregulation of junctional Ca2+ release sites contributes to Ca2+-dependent arrhythmogenesis in HF. PMID:28612155

Alpha 1-acid glycoprotein reverses cocaine-induced sodium channel blockade in cardiac myocytes.

PubMed

Ma, Yu-Ling; Peters, Nicholas S; Henry, John A

2006-03-01

Alpha 1-acid glycoprotein (AAG) is an acute phase protein capable of binding basic drugs. This action explains its reversal of sodium channel blockade by drugs such as amitriptyline and quinidine. We report here the reversal of cocaine-induced sodium channel blockade by AAG. The sodium channel blocking property of cocaine is a major mechanism behind cocaine-induced sudden cardiac death, since sodium channels play a key role in the initiation and regulation of the heart beat. Voltage-gated sodium current (I(Na)) was recorded using whole-cell patch-clamp techniques. Guinea-pig cardiac ventricular myocytes were isolated and continuously perfused at room temperature with physiological solutions. At concentrations ranging from 5 to 320 microM cocaine showed a dose-dependent and reversible blockade of I(Na) with an IC50 of 45.9 microM. The addition of equimolar amounts of AAG to cocaine produced almost complete reversal of cocaine's effects, suggesting a single binding site for cocaine on the AAG molecule. With changes of peak I(Na) normalized against control as 1, cocaine at 20 and 40 microM reduced I(Na) to 0.62+/-0.042 (n = 6) and 0.57+/-0.052 (n = 9), respectively, and the addition of an equimolar concentration of AAG reversed I(Na) to 0.86+/-0.022 and 0.91+/-0.060, respectively. AAG reverses cocaine-induced sodium channel blockade in a dose-dependent manner, indicating a therapeutic potential to reverse acute cocaine cardiac toxicity.

AFos Dissociates Cardiac Myocyte Hypertrophy and Expression of the Pathological Gene Program

PubMed Central

Jeong, Mark Y.; Kinugawa, Koichiro; Vinson, Charles; Long, Carlin S.

2005-01-01

Background Although induction of activator protein-1 (AP-1) transcription factor activity has been observed in cardiac hypertrophy, a direct role for AP-1 in myocardial growth and gene expression remains obscure. Methods and Results Hypertrophy was induced in cultured neonatal rat cardiomyocytes with phenylephrine or overexpression of a constitutively active MAP3K, MKK6. In both treatment groups, induction of the pathological gene profile was observed, ie, expression of β-myosin heavy chain (βMHC), atrial/brain natriuretic peptides (ANP/BNP), and skeletal α-actin (sACT) was increased, whereas expression for α-myosin heavy chain (αMHC) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA) genes was repressed. The role of AP-1 in the hypertrophic phenotype was evaluated with the use of an adenoviral construct expressing a dominant negative mutant of the c-Fos proto-oncogene (AdAFos). Although AFos did not change the myocyte growth response, it abrogated the gene profile to both agonists, including the upregulation of both αMHC and SERCA expression. Conclusions Although c-Fos/AP-1 is necessary for induction of the pathological/fetal gene program, it does not appear to be critical for cardiomyocyte hypertrophy. PMID:15795322

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes.

PubMed

Allen, Bruce G; Phuong, Luu Lien; Farhat, Hala; Chevalier, Dominique

2003-02-01

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.

Pim-2 protects H9c2 cardiomyocytes from hypoxia/reoxygenation-induced apoptosis via downregulation of Bim expression.

PubMed

Xu, Yan; Xing, Yawei; Xu, Yanjie; Huang, Chahua; Bao, Huihui; Hong, Kui; Cheng, Xiaoshu

2016-12-01

We know that silencing Bim, a pro-apoptosis protein, significantly attenuates glucose and oxygen-deprived induced apoptosis in cardiomyocytes. However, the mechanisms underlying the regulation of the Bim activation in the heart have remained unknown. Pim-2 is one of three Pim serine/threonine kinase family members thought to be involved in cell survival and proliferation. H9c2 cardiomyocytes were subjected to a hypoxia/reoxygenation (H/R) condition in vitro, mimicking ischemic/reperfusion injury in vivo. H/R augmented the expression of Bim, Cyt C, and Pim-2 and induced H9c2 cell apoptosis. Overexpression of Pim-2 attenuated apoptosis which induced by H/R in H9c2 cells, via downregulation of Bim and Cyt C expression. Silencing of Pim-2 promoted H/R-induced apoptosis via upregulation of Bim and Cyt C expression. Co-IP revealed the interaction between Pim-2 and Bim protein, with Bim Ser 65 phosphorylated by Pim-2. Furthermore, blocking proteasome activity by MG132 prevented Bim degradation, and Bim S65A mutation could reverse the anti-apoptotic role of Pim-2 which induced by H/R. These data demonstrated that Pim-2 is a novel Bim-interacting protein, which negatively regulates Bim degradation and protects H9c2 cardiomyocytes from H/R-induced apoptosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of phloretin and phloridzin on Ca2+ handling, the action potential, and ion currents in rat ventricular myocytes.

PubMed

Olson, Marnie L; Kargacin, Margaret E; Ward, Christopher A; Kargacin, Gary J

2007-06-01

The effects of the phytoestrogens phloretin and phloridzin on Ca(2+) handling, cell shortening, the action potential, and Ca(2+) and K(+) currents in freshly isolated cardiac myocytes from rat ventricle were examined. Phloretin increased the amplitude and area and decreased the rate of decline of electrically evoked Ca(2+) transients in the myocytes. These effects were accompanied by an increase in the Ca(2+) load of the sarcoplasmic reticulum, as determined by the area of caffeine-evoked Ca(2+) transients. An increase in the extent of shortening of the myocytes in response to electrically evoked action potentials was also observed in the presence of phloretin. To further examine possible mechanisms contributing to the observed changes in Ca(2+) handling and contractility, the effects of phloretin on the cardiac action potential and plasma membrane Ca(2+) and K(+) currents were examined. Phloretin markedly increased the action potential duration in the myocytes, and it inhibited the Ca(2+)-independent transient outward K(+) current (I(to)). The inwardly rectifying K(+) current, the sustained outward delayed rectifier K(+) current, and L-type Ca(2+) currents were not significantly different in the presence and absence of phloretin, nor was there any evidence that the Na(+)/Ca(2+) exchanger was affected. The effects of phloretin on Ca(2+) handling in the myocytes are consistent with its effects on I(to). Phloridzin did not significantly alter the amplitude or area of electrically evoked Ca(2+) transients in the myocytes, nor did it have detectable effects on the sarcoplasmic reticulum Ca(2+) load, cell shortening, or the action potential.

Eliminating spiral waves pinned to an anatomical obstacle in cardiac myocytes by high-frequency stimuli.

PubMed

Isomura, Akihiro; Hörning, Marcel; Agladze, Konstantin; Yoshikawa, Kenichi

2008-12-01

The unpinning of spiral waves by the application of high-frequency wave trains was studied in cultured cardiac myocytes. Successful unpinning was observed when the frequency of the paced waves exceeded a critical level. The unpinning process was analyzed by a numerical simulation with a model of cardiac tissue. The mechanism of unpinning by high-frequency stimuli is discussed in terms of local entrainment failure, through a reduction of the two-dimensional spatial characteristics into one dimension.

Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.

PubMed Central

Kirshenbaum, L A; MacLellan, W R; Mazur, W; French, B A; Schneider, M D

1993-01-01

Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has, for cogent technical reasons, largely been undertaken to date in neonatal ventricular myocytes. To circumvent expected limitations of other methods, the present study was initiated to determine whether replication-deficient adenovirus would enable efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a cytomegalovirus immediate-early promoter-driven lacZ reporter gene and were assayed for the presence of beta-galactosidase 48 h after infection. The frequency of lacZ+ rod-shaped myocytes was half-maximal at 4 x 10(5) plaque-forming units (PFU) and approached 90% at 1 x 10(8) PFU. Uninfected cells and cells infected with lacZ- virus remained colorless. Beta-galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the exogenous lacZ gene. At 10(8) PFU/dish, cell number, morphology, and viability each were comparable to uninfected cells. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of putative regulators upon the endogenous genes and gene products of virally modified adult ventricular muscle cells. Images PMID:8326005

TRPM2 Channels Protect against Cardiac Ischemia-Reperfusion Injury

PubMed Central

Miller, Barbara A.; Hoffman, Nicholas E.; Merali, Salim; Zhang, Xue-Qian; Wang, JuFang; Rajan, Sudarsan; Shanmughapriya, Santhanam; Gao, Erhe; Barrero, Carlos A.; Mallilankaraman, Karthik; Song, Jianliang; Gu, Tongda; Hirschler-Laszkiewicz, Iwona; Koch, Walter J.; Feldman, Arthur M.; Madesh, Muniswamy; Cheung, Joseph Y.

2014-01-01

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca2+ uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca2+ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca2+ uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels. PMID:24492610

TRPV2 is critical for the maintenance of cardiac structure and function in mice

PubMed Central

Katanosaka, Yuki; Iwasaki, Keiichiro; Ujihara, Yoshihiro; Takatsu, Satomi; Nishitsuji, Koki; Kanagawa, Motoi; Sudo, Atsushi; Toda, Tatsushi; Katanosaka, Kimiaki; Mohri, Satoshi; Naruse, Keiji

2014-01-01

The heart has a dynamic compensatory mechanism for haemodynamic stress. However, the molecular details of how mechanical forces are transduced in the heart are unclear. Here we show that the transient receptor potential, vanilloid family type 2 (TRPV2) cation channel is critical for the maintenance of cardiac structure and function. Within 4 days of eliminating TRPV2 from hearts of the adult mice, cardiac function declines severely, with disorganization of the intercalated discs that support mechanical coupling with neighbouring myocytes and myocardial conduction defects. After 9 days, cell shortening and Ca2+ handling by single myocytes are impaired in TRPV2-deficient hearts. TRPV2-deficient neonatal cardiomyocytes form no intercalated discs and show no extracellular Ca2+-dependent intracellular Ca2+ increase and insulin-like growth factor (IGF-1) secretion in response to stretch stimulation. We further demonstrate that IGF-1 receptor/PI3K/Akt pathway signalling is significantly downregulated in TRPV2-deficient hearts, and that IGF-1 administration partially prevents chamber dilation and impairment in cardiac pump function in these hearts. Our results improve our understanding of the molecular processes underlying the maintenance of cardiac structure and function. PMID:24874017

TRPV2 is critical for the maintenance of cardiac structure and function in mice.

PubMed

Katanosaka, Yuki; Iwasaki, Keiichiro; Ujihara, Yoshihiro; Takatsu, Satomi; Nishitsuji, Koki; Kanagawa, Motoi; Sudo, Atsushi; Toda, Tatsushi; Katanosaka, Kimiaki; Mohri, Satoshi; Naruse, Keiji

2014-05-29

The heart has a dynamic compensatory mechanism for haemodynamic stress. However, the molecular details of how mechanical forces are transduced in the heart are unclear. Here we show that the transient receptor potential, vanilloid family type 2 (TRPV2) cation channel is critical for the maintenance of cardiac structure and function. Within 4 days of eliminating TRPV2 from hearts of the adult mice, cardiac function declines severely, with disorganization of the intercalated discs that support mechanical coupling with neighbouring myocytes and myocardial conduction defects. After 9 days, cell shortening and Ca(2+) handling by single myocytes are impaired in TRPV2-deficient hearts. TRPV2-deficient neonatal cardiomyocytes form no intercalated discs and show no extracellular Ca(2+)-dependent intracellular Ca(2+) increase and insulin-like growth factor (IGF-1) secretion in response to stretch stimulation. We further demonstrate that IGF-1 receptor/PI3K/Akt pathway signalling is significantly downregulated in TRPV2-deficient hearts, and that IGF-1 administration partially prevents chamber dilation and impairment in cardiac pump function in these hearts. Our results improve our understanding of the molecular processes underlying the maintenance of cardiac structure and function.

Antihistamines modulate the integrin signaling pathway in h9c2 rat cardiomyocytes: Possible association with cardiotoxicity.

PubMed

Yun, J S; Kim, S Y

2015-08-01

The identification of biomarkers for toxicity prediction is crucial for drug development and safety evaluation. The selective and specific biomarkers for antihistamines-induced cardiotoxicity is not well identified yet. In order to evaluate the mechanism of the life-threatening effects caused by antihistamines, we used DNA microarrays to analyze genomic profiles in H9C2 rat cardiomyocytes that were treated with antihistamines. The gene expression profiles from drug-treated cells revealed changes in the integrin signaling pathway, suggesting that cardiac arrhythmias induced by antihistamine treatment may be mediated by changes in integrin-mediated signaling. It has been reported that integrin plays a role in QT prolongation that may induce cardiac arrhythmia. These results indicate that the integrin-mediated signaling pathway induced by antihistamines is involved in various biological mechanisms that lead to cardiac QT prolongation. Therefore, we suggest that genomic profiling of antihistamine-treated cardiomyocytes has the potential to reveal the mechanism of adverse drug reactions, and this signal pathway is applicable to prediction of in vitro cardiotoxicity induced by antihistamines as a biomarker candidate. © The Author(s) 2014.

Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure

PubMed Central

Paudyal, Anju; Dewan, Sukriti; Ikie, Cindy; Whalley, Benjamin J; de Tombe, Pieter P.

2016-01-01

Key points The present study investigated the mechanism associated with impaired cardiac mechanosensing that leads to heart failure by examining the factors regulating muscle LIM protein subcellular distribution in myocytes.In myocytes, muscle LIM protein subcellular distribution is regulated by cell contractility rather than passive stretch via heme oxygenase‐1 and histone deacetylase signalling. The result of the present study provide new insights into mechanotransduction in cardiac myocytes.Myocyte mechanosensitivity, as indicated by the muscle LIM protein ratio, is also correlated with cardiac function in the transition to failure in a guinea‐pig model of disease. This shows that the loss mechanosensitivity plays an important role during the transition to failure in the heart.The present study provides the first indication that mechanosensing could be modified pharmacologically during the transition to heart failure. Abstract Impaired mechanosensing leads to heart failure and a decreased ratio of cytoplasmic to nuclear CSRP3/muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. In the present study, we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to a 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased by 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 μm blebbistatin resulted in an ∼3‐fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signalling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme

Enhanced effect of VEGF165 on L-type calcium currents in guinea-pig cardiac ventricular myocytes.

PubMed

Xing, Wenlu; Gao, Chuanyu; Qi, Datun; Zhang, You; Hao, Peiyuan; Dai, Guoyou; Yan, Ganxin

2017-01-01

The mechanisms of vascular endothelial growth factor 165 (VEGF165) on electrical properties of cardiomyocytes have not been fully elucidated. The aim of this study is to test the hypothesis that VEGF165, an angiogenesis-initiating factor, affects L-type calcium currents (I Ca,L ) and cell membrane potential in cardiac myocytes by acting on VEGF type-2 receptors (VEGFR2). I Ca,L and action potentials (AP) were recorded by the whole-cell patch clamp method in isolated guinea-pig ventricular myocytes treated with different concentrations of VEGF165 proteins. Using a VEGFR2 inhibitor, we also tested the receptor of VEGF165 in cardiomyocytes. We found that VEGF165 increased I Ca,L in a concentration-dependent manner. SU5416, a VEGFR2 inhibitor, almost completely eliminated VEGF165-induced I Ca,L increase. VEGF165 had no significant influence on action potential 90 (APD90) and other properties of AP. We conclude that in guinea-pig ventricular myocytes, I Ca,L can be increased by VEGF165 in a concentration-dependent manner through binding to VEGFR2 without causing any significant alteration to action potential duration. Results of this study may further expound the safety of VEGF165 when used in the intervention of heart diseases. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

YY1 Protects Cardiac Myocytes from Pathologic Hypertrophy by Interacting with HDAC5

PubMed Central

Dockstader, Karen; McKinsey, Timothy A.

2008-01-01

YY1 is a transcription factor that can repress or activate the transcription of a variety of genes. Here, we show that the function of YY1 as a repressor in cardiac myocytes is tightly dependent on its ability to interact with histone deacetylase 5 (HDAC5). YY1 interacts with HDAC5, and overexpression of YY1 prevents HDAC5 nuclear export in response to hypertrophic stimuli and the increase in cell size and re-expression of fetal genes that accompany pathological cardiac hypertrophy. Knockdown of YY1 results in up-regulation of all genes present during fetal development and increases the cell size of neonatal cardiac myocytes. Moreover, overexpression of a YY1 deletion construct that does not interact with HDAC5 results in transcription activation, suggesting that HDAC5 is necessary for YY1 function as a transcription repressor. In support of this relationship, we show that knockdown of HDAC5 results in transcription activation by YY1. Finally, we show that YY1 interaction with HDAC5 is dependent on the HDAC5 phosphorylation domain and that overexpression of YY1 reduces HDAC5 phosphorylation in response to hypertrophic stimuli. Our results strongly suggest that YY1 functions as an antihypertrophic factor by preventing HDAC5 nuclear export and that up-regulation of YY1 in human heart failure may be a protective mechanism against pathological hypertrophy. PMID:18632988

Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo.

PubMed Central

Kass-Eisler, A; Falck-Pedersen, E; Alvira, M; Rivera, J; Buttrick, P M; Wittenberg, B A; Cipriani, L; Leinwand, L A

1993-01-01

To optimize the use of modified adenoviruses as vectors for gene delivery to the myocardium, we have characterized infection of cultured fetal and adult rat cardiac myocytes in vitro and of adult cardiac myocytes in vivo by using a replication-defective adenovirus carrying the chloramphenicol acetyltransferase (CAT) reporter gene driven by the cytomegalovirus promoter (AdCMVCATgD). In vitro, virtually all fetal or adult cardiocytes express the CAT gene when infected with 1 plaque-forming unit of virus per cell. CAT enzymatic activity can be detected in these cells as early as 4 hr after infection, reaching near-maximal levels at 48 hr. In fetal cells, CAT expression was maintained without a loss in activity for at least 1 week. Using in vitro studies as a guide, we introduced the AdCMVCATgD virus directly into adult rat myocardium and compared the expression results obtained from virus injection with those obtained by direct injection of pAdCMVCATgD plasmid DNA. The amount of CAT activity resulting from adenovirus infection of the myocardium was orders of magnitude higher than that seen from DNA injection and was proportional to the amount of input virus. Immunostaining for CAT protein in cardiac tissue sections following adenovirus injection demonstrated large numbers of positive cells, reaching nearly 100% of the myocytes in many regions of the heart. Expression of genes introduced by adenovirus peaked at 5 days but was still detectable 55 days following infection. Adenoviruses are therefore a very useful tool for high-efficiency gene transfer into the cardiovascular system. Images Fig. 1 Fig. 5 PMID:8265580

Calcium release-dependent inactivation precedes formation of the tubular system in developing rat cardiac myocytes.

PubMed

Macková, Katarina; Zahradníková, Alexandra; Hoťka, Matej; Hoffmannová, Barbora; Zahradník, Ivan; Zahradníková, Alexandra

2017-12-01

Developing cardiac myocytes undergo substantial structural and functional changes transforming the mechanism of excitation-contraction coupling from the embryonic form, based on calcium influx through sarcolemmal DHPR calcium channels, to the adult form, relying on local calcium release through RYR calcium channels of sarcoplasmic reticulum stimulated by calcium influx. We characterized day-by-day the postnatal development of the structure of sarcolemma, using techniques of confocal fluorescence microscopy, and the development of the calcium current, measured by the whole-cell patch-clamp in isolated rat ventricular myocytes. We characterized the appearance and expansion of the t-tubule system and compared it with the appearance and progress of the calcium current inactivation induced by the release of calcium ions from sarcoplasmic reticulum as structural and functional measures of direct DHPR-RYR interaction. The release-dependent inactivation of calcium current preceded the development of the t-tubular system by several days, indicating formation of the first DHPR-RYR couplons at the surface sarcolemma and their later spreading close to contractile myofibrils with the growing t-tubules. Large variability of both of the measured parameters among individual myocytes indicates uneven maturation of myocytes within the growing myocardium.

Cardioprotective effects of calcitonin gene-related peptide in isolated rat heart and in H9c2 cells via redox signaling.

PubMed

Tullio, Francesca; Penna, Claudia; Cabiale, Karine; Femminò, Saveria; Galloni, Marco; Pagliaro, Pasquale

2017-06-01

The calcitonin-gene-related-peptide (CGRP) release is coupled to the signaling of Angeli's salt in determining vasodilator effects. However, it is unknown whether CGRP is involved in Angeli's salt cardioprotective effects and which are the mechanisms of protection. We aimed to determine whether CGRP is involved in myocardial protection induced by Angeli's salt. We also analyzed the intracellular signaling pathway activated by CGRP. Isolated rat hearts were pre-treated with Angeli's salt or Angeli's salt plus CGRP 8-37 , a specific CGRP-receptor antagonist, and subjected to ischemia (30-min) and reperfusion (120-min). Moreover, we studied CGRP-induced protection during oxidative stress (H 2 O 2 ) and hypoxia/reoxygenation protocols in H9c2 cardiomyocytes. Cell vitality and mitochondrial membrane potential (ΔYm, MMP) were measured using MTT and JC-1 dyes. Angeli's salt reduced infarct size and ameliorated post-ischemic cardiac function via a CGRP-receptor-dependent mechanism. Pre-treatment with CGRP increased H9c2 survival upon challenging with either H 2 O 2 (redox stress) or hypoxia/reoxygenation (H/R stress). Under these stress conditions, reduction in MMP and cell death were partly prevented by CGRP. These CGRP beneficial effects were blocked by CGRP 8-37. During H/R stress, pre-treatment with either CGRP-receptor, protein kinase C (PKC) or mitochondrial K ATP channel antagonists, and pre-treatment with an antioxidant (2-mercaptopropionylglycine) blocked the protection mediated by CGRP. In conclusion, CGRP is involved in the cardioprotective effects of Angeli's salt. In H9c2 cardiomyocytes, CGRP elicits PKC-dependent and mitochondrial-K ATP -redox-dependent mechanisms. Hence, CGRP is an important factor in the redox-sensible cardioprotective effects of Angeli's salt. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Urocortin2 prolongs action potential duration and modulates potassium currents in guinea pig myocytes and HEK293 cells.

PubMed

Yang, Li-Zhen; Zhu, Yi-Chun

2015-07-05

We previously reported that activation of corticotropin releasing factor receptor type 2 by urocortin2 up-regulates both L-type Ca(2+) channels and intracellular Ca(2+) concentration in ventricular myocytes and plays an important role in cardiac contractility and arrhythmogenesis. This study goal was to further test the hypothesis that urocortin2 may modulate action potentials as well as rapidly and slowly activating delayed rectifier potassium currents. With whole cell patch-clamp techniques, action potentials and slowly activating delayed rectifier potassium currents were recorded in isolated guinea pig ventricular myocytes, respectively. And rapidly activating delayed rectifier potassium currents were tested in hERG-HEK293 cells. Urocortin2 produced a time- and concentration-dependent prolongation of action potential duration. The EC50 values of action potential duration and action potential duration at 90% of repolarization were 14.73 and 24.3nM respectively. The prolongation of action potential duration of urocortin2 was almost completely or partly abolished by H-89 (protein kinase A inhibitor) or KB-R7943 (Na(+)/Ca(2+) exchange inhibitor) pretreatment respectively. And urocortin2 caused reduction of rapidly activating delayed rectifier potassium currents in hERG-HEK293 cells. In addition, urocortin2 slowed the rate of slowly activating delayed rectifier potassium channel activation, and rightward shifted the threshold of slowly activating delayed rectifier potassium currents to more positive potentials. Urocortin2 prolonged action potential duration via activation of protein kinase A and Na(+)/ Ca(2+) exchange in isolated guinea pig ventricular myocytes in a time- and concentration- dependent manner. In hERG-HEK293 cells, urocortin2 reduced rapidly activating delayed rectifier potassium current density which may contribute to action potential duration prolongation. Copyright © 2015 Elsevier B.V. All rights reserved.

Computer modeling of siRNA knockdown effects indicates an essential role of the Ca2+ channel alpha2delta-1 subunit in cardiac excitation-contraction coupling.

PubMed

Tuluc, Petronel; Kern, Georg; Obermair, Gerald J; Flucher, Bernhard E

2007-06-26

L-type Ca(2+) currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca(2+) signal, which regulates the contraction force. The auxiliary Ca(2+) channel subunits alpha(2)delta-1 and beta(2) are important regulators of membrane expression and current properties of the cardiac Ca(2+) channel (Ca(V)1.2). However, their role in cardiac excitation-contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the alpha(2)delta-1 subunit in a muscle expression system with simulation of APs and Ca(2+) transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with Ca(V)1.2 (GFP-alpha(1C)) recapitulates key properties of cardiac excitation-contraction coupling. Concomitant depletion of the alpha(2)delta-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-alpha(1C) subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, alpha(2)delta-1 depletion shifted the voltage dependence of Ca(2+) current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic Ca(2+) concentration during each contraction. Thus, the Ca(2+) channel alpha(2)delta-1 subunit is not essential for normal Ca(2+) channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of alpha(2)delta-1 function is predicted to severely perturb normal heart function.

Tetrodotoxin Blockade on Canine Cardiac L-Type Ca2+ Channels Depends on pH and Redox Potential

PubMed Central

Hegyi, Bence; Komáromi, István; Kistamás, Kornél; Ruzsnavszky, Ferenc; Váczi, Krisztina; Horváth, Balázs; Magyar, János; Bányász, Tamás; Nánási, Péter P.; Szentandrássy, Norbert

2013-01-01

Tetrodotoxin (TTX) is believed to be one of the most selective inhibitors of voltage-gated fast Na+ channels in excitable tissues. Recently, however, TTX has been shown to block L-type Ca2+ current (ICa) in canine cardiac cells. In the present study, the TTX-sensitivity of ICa was studied in isolated canine ventricular myocytes as a function of (1) channel phosphorylation, (2) extracellular pH and (3) the redox potential of the bathing medium using the whole cell voltage clamp technique. Fifty-five micromoles of TTX (IC50 value obtained under physiological conditions) caused 60% ± 2% inhibition of ICa in acidic (pH = 6.4), while only a 26% ± 2% block in alkaline (pH = 8.4) milieu. Similarly, the same concentration of TTX induced 62% ± 6% suppression of ICa in a reductant milieu (containing glutathione + ascorbic acid + dithiothreitol, 1 mM each), in contrast to the 31% ± 3% blockade obtained in the presence of a strong oxidant (100 μM H2O2). Phosphorylation of the channel protein (induced by 3 μM forskolin) failed to modify the inhibiting potency of TTX; an IC50 value of 50 ± 4 μM was found in forskolin. The results are in a good accordance with the predictions of our model, indicating that TTX binds, in fact, to the selectivity filter of cardiac L-type Ca channels. PMID:23771047

Simulation of action potentials from metabolically impaired cardiac myocytes. Role of ATP-sensitive K+ current.

PubMed

Ferrero, J M; Sáiz, J; Ferrero, J M; Thakor, N V

1996-08-01

The role of the ATP-sensitive K+ current (IK-ATP) and its contribution to electrophysiological changes that occur during metabolic impairment in cardiac ventricular myocytes is still being discussed. The aim of this work was to quantitatively study this issue by using computer modeling. A model of IK-ATP is formulated and incorporated into the Luo-Rudy ionic model of the ventricular action potential. Action potentials under different degrees of activation of IK-ATP are simulated. Our results show that in normal ionic concentrations, only approximately 0.6% of the KATP channels, when open, should account for a 50% reduction in action potential duration. However, increased levels of intracellular Mg2+ counteract this shortening. Under conditions of high [K+]0, such as those found in early ischemia, the activation of only approximately 0.4% of the KATP channels could account for a 50% reduction in action potential duration. Thus, our results suggest that opening of IK-ATP channels should play a significant role in action potential shortening during hypoxic/ischemic episodes, with the fraction of open channels involved being very low ( < 1%). However, the results of the model suggest that activation of IK-ATP alone does not quantitatively account for the observed K+ efflux in metabolically impaired cardiac myocytes. Mechanisms other than KATP channel activation should be responsible for a significant part of the K+ efflux measured in hypoxic/ischemic situations.

Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

PubMed

Lalli, M J; Yong, J; Prasad, V; Hashimoto, K; Plank, D; Babu, G J; Kirkpatrick, D; Walsh, R A; Sussman, M; Yatani, A; Marbán, E; Periasamy, M

2001-07-20

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

Cardiac complications associated with the influenza viruses A subtype H7N9 or pandemic H1N1 in critically ill patients under intensive care.

PubMed

Wang, Jiajia; Xu, Hua; Yang, Xinjing; Zhao, Daguo; Liu, Shenglan; Sun, Xue; Huang, Jian-An; Guo, Qiang

The clinical presentations and disease courses of patients hospitalized with either influenza A virus subtype H7N9 (H7N9) or 2009 pandemic H1N1 influenza virus were compared in a recent report, but associated cardiac complications remain unclear. The present retrospective study investigated whether cardiac complications in critically ill patients with H7N9 infections differed from those infected with the pandemic H1N1 influenza virus strain. Suspect cases were confirmed by reverse transcription polymerase chain reaction assays with specific confirmation of the pandemic H1N1 strain at the Centers for Disease Control and Prevention. Comparisons were conducted at the individual-level data of critically ill patients hospitalized with H7N9 (n=24) or pandemic H1N1 influenza virus (n=22) infections in Suzhou, China. Changes in cardiac biochemical markers, echocardiography, and electrocardiography during hospitalization in the intensive care unit were considered signs of cardiac complications. The following findings were more common among the H7N9 group relative to the pandemic H1N1 influenza virus group: greater tricuspid regurgitation pressure gradient, sinus tachycardia (heartbeat≥130bpm), ST segment depression, right ventricular dysfunction, and elevated cardiac biochemical markers. Pericardial effusion was more often found among pandemic H1N1 influenza virus patients than in the H7N9 group. In both groups, most of the cardiac complications were detected from day 6 to 14 after the onset of influenza symptoms. Those who developed cardiac complications were especially vulnerable during the first four days after initiation of mechanical ventilation. Cardiac complications were reversible in the vast majority of discharged H7N9 patients. Critically ill hospitalized H7N9 patients experienced a higher rate of cardiac complications than did patients with 2009 pandemic H1N1 influenza virus infections, with the exception of pericardial effusion. This study may help in the

Genetically engineered cardiac pacemaker: Stem cells transfected with HCN2 gene and myocytes—A model

NASA Astrophysics Data System (ADS)

Kanani, S.; Pumir, A.; Krinsky, V.

2008-01-01

One of the successfully tested methods to design genetically engineered cardiac pacemaker cells consists in transfecting a human mesenchymal stem cell (hMSC) with a HCN2 gene and connecting it to a myocyte. We develop and study a mathematical model, describing a myocyte connected to a hMSC transfected with a HCN2 gene. The cardiac action potential is described both with the simple Beeler Reuter model, as well as with the elaborate dynamic Luo Rudy model. The HCN2 channel is described by fitting electrophysiological records, in the spirit of Hodgkin Huxley. The model shows that oscillations can occur in a pair myocyte-stem cell, that was not observed in the experiments yet. The model predicted that: (1) HCN pacemaker channels can induce oscillations only if the number of expressed I channels is low enough. At too high an expression level of I channels, oscillations cannot be induced, no matter how many pacemaker channels are expressed. (2) At low expression levels of I channels, a large domain of values in the parameter space (n, N) exists, where oscillations should be observed. We denote N the number of expressed pacemaker channels in the stem cell, and n the number of gap junction channels coupling the stem cell and the myocyte. (3) The expression levels of I channels observed in ventricular myocytes, both in the Beeler Reuter and in the dynamic Luo Rudy models are too high to allow to observe oscillations. With expression levels below ˜1/4 of the original value, oscillations can be observed. The main consequence of this work is that in order to obtain oscillations in an experiment with a myocyte-stem cell pair, increasing the values of n, N is unlikely to be helpful, unless the expression level of I has been reduced enough. The model also allows us to explore levels of gene expression not yet achieved in experiments, and could be useful to plan new experiments, aimed at improving the robustness of the oscillations.

Tauroursodeoxycholic acid inhibits endoplasmic reticulum stress, blocks mitochondrial permeability transition pore opening, and suppresses reperfusion injury through GSK-3ß in cardiac H9c2 cells.

PubMed

Xie, Yuxi; He, Yonggui; Cai, Zhiliang; Cai, Jianhang; Xi, Mengyao; Zhang, Yidong; Xi, Jinkun

2016-01-01

This study investigates whether inhibition of endoplasmic reticulum (ER) stress prevents opening of the mitochondrial permeability transition pore (mPTP) and evaluates the corresponding signaling pathways involved in this process. Exposure of cardiac H9c2 cells to 800 µM H 2 O 2 for 20 min opened mPTP in response to oxidative stress, as demonstrated by quenching of tetramethylrhodamine ethyl ester (TMRE) fluorescence. Oxidative stress-induced mPTP opening was rescued by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) in a dose-dependent manner at low concentrations. The PI3K and PKG inhibitors LY294002 and KT5823 inhibited the effect of TUDCA on mPTP opening, suggesting the involvement of PI3K/Akt and PKG signaling pathways. TUDCA significantly increased glycogen synthase kinase 3 (GSK-3β) phosphorylation at Ser-9, with peak effect at 30 µM TUDCA. The level of GRP78 (ER chaperone) expression was significantly upregulated by 30 µM TUDCA. TUDCA-induced increases in Akt and GSK-3β phosphorylation were inhibited by LY294002, whereas KT5823 suppressed TUDCA-induced increases in VASP and GSK-3β phosphorylation. Oxidative stress severely affected cell morphology and ultrastructure. TUDCA prevented H 2 O 2 -induced ER swelling and mitochondrial damage. TUDCA boosted the viability of cells disrupted by ischemia/reperfusion (I/R), indicating that TUDCA eased reperfusion injury. However, TUDCA did not improve the viability of cells expressing the constitutively active GSK-3β mutant (GSK-3β-S9A-HA) that were subjected to I/R, suggesting an essential role of GSK-3β inactivation in TUDCA-mediated cardioprotection against reperfusion damage. These data indicate that ER stress inhibition prevents mPTP opening and attenuates reperfusion injury through GSK-3β inactivation. The PI3K/Akt and PKG pathways may mediate GSK-3β inactivation.

Tanshinone IIA protects H9c2 cells from oxidative stress-induced cell death via microRNA-133 upregulation and Akt activation.

PubMed

Gu, Yunfei; Liang, Zhuo; Wang, Haijun; Jin, Jun; Zhang, Shouyan; Xue, Shufeng; Chen, Jianfeng; He, Huijuan; Duan, Kadan; Wang, Jing; Chang, Xuewei; Qiu, Chunguang

2016-08-01

The aim of the present study was to investigate the cardioprotective effect of tanshinone IIA and the underlying molecular mechanisms. An in vitro model of oxidative stress injury was established in cardiac H9c2 cells, and the effects of tanshinone IIa were investigated using cell viability, reverse transcription-quantitative polymerase chain reaction and western blotting assays. The results demonstrated that tanshinone IIA protects H9c2 cells from H 2 O 2 -induced cell death in a concentration-dependent manner, via a mechanism involving microRNA-133 (miR-133), and that treatment with TIIA alone exerted no cytotoxic effects on H9c2. In order to further elucidate the mechanisms underlying the actions of TIIA, reverse transcription-quantitative polymease chain reaction and western blot analysis were performed. Reductions in miR-133 expression levels induced by increasing concentrations of H 2 O 2 were reversed by treatment with tanshinone IIA. In addition, the inhibition of miR-133 by transfection with an miR-133 inhibitor abolished the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Furthermore, western blot analysis demonstrated that tanshinone IIA activated Akt kinase via the phosphorylation of serine 473. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by pretreatment with the PI3K specific inhibitors wortmannin and LY294002 also eliminated the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Western blot analysis demonstrated that H 2 O 2 -induced reductions in B cell lymphoma 2 (Bcl-2) expression levels were reversed by tanshinone IIA. In addition, the effect of tanshinone IIA on Bcl-2 protein expression level in an oxidative environment was suppressed by a PI3K inhibitor, wortmannin, indicating that tanshinone IIA exerts cardioprotective effects against H 2 O 2 -induced cell death via the activation of the PI3K/Akt signal transduction pathway and the consequent

MYBL2 protects against H9c2 injury induced by hypoxia via AKT and NF‑κB pathways.

PubMed

Shao, Mingfeng; Ren, Zexiang; Zhang, Rongjun

2018-03-01

Cardiovascular diseases have become one of the major public health problems in many countries. The downregulation of MYBL2 was found in H9c2 and native cardiomyocytes cells after hypoxia treatment. The present study aimed to investigate the effects of MYB proto‑oncogene like 2 (MYBL2) on H9c2 injury induced by hypoxia. Reverse transcription‑quantitative polymerase chain reaction and western blot were performed on H9c2 cells to determine the mRNA and protein levels of MYBL2, respectively. Small interfering RNA (siRNA) was employed to downregulate MYBL2 expression in H9c2 cells to investigate changes in cell proliferation and apoptosis. Cell proliferation was assessed by a Cell Counting kit‑8 assay and the percentage of apoptotic cells was determined using an Annexin V‑fluorescein isothiocyanate/propidium iodide apoptosis detection kit. The nuclear factor‑κB (NF‑κB) and AKT signaling pathways in H9c2 cells were investigated by western blot analysis. The results demonstrated that the overexpression of MYBL2 promoted cell proliferation and suppressed apoptosis. Furthermore, overexpression of MYBL2 suppressed the expression of phosphorylated (p)‑AKT, p‑NF‑κB inhibitor α, p‑p65 and B‑cell CLL/lymphoma 3 (Bcl‑3). The results indicated that MYBL2 may improve cell viability and inhibit H9c2 apoptosis via the inhibition of AKT and NF‑κB pathways. Therefore, MYBL2 may be a potential therapeutic target for the treatment of myocardial infarction.

A cardiac pathway of cyclic GMP-independent signaling of guanylyl cyclase A, the receptor for atrial natriuretic peptide

PubMed Central

Klaiber, Michael; Dankworth, Beatrice; Kruse, Martin; Hartmann, Michael; Nikolaev, Viacheslav O.; Yang, Ruey-Bing; Völker, Katharina; Gaßner, Birgit; Oberwinkler, Heike; Feil, Robert; Freichel, Marc; Groschner, Klaus; Skryabin, Boris V.; Frantz, Stefan; Birnbaumer, Lutz; Pongs, Olaf; Kuhn, Michaela

2011-01-01

Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A, to exert its diverse functions. This process involves a cGMP-dependent signaling pathway preventing pathological [Ca2+]i increases in myocytes. In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization. Here we show that, in this situation, ANP binding to GC-A stimulates a unique cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca2+ levels. This pathway involves the activation of Ca2+‐permeable transient receptor potential canonical 3/6 (TRPC3/C6) cation channels by GC-A, which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting cation influx activates voltage-dependent L-type Ca2+ channels and ultimately increases myocyte Ca2+i levels. These observations reveal a dual role of the ANP/GC-A–signaling pathway in the regulation of cardiac myocyte Ca2+i homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca2+i-enhancing action of hypertrophic factors such as angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca2+]i might increase the propensity to cardiac hypertrophy and arrhythmias. PMID:22027011

Genetic Lineage Tracing of Non-Myocyte Population by Dual Recombinases.

PubMed

Li, Yan; He, Lingjuan; Huang, Xiuzhen; Issa Bhaloo, Shirin; Zhao, Huan; Zhang, Shaohua; Pu, Wenjuan; Tian, Xueying; Li, Yi; Liu, Qiaozhen; Yu, Wei; Zhang, Libo; Liu, Xiuxiu; Liu, Kuo; Tang, Juan; Zhang, Hui; Cai, Dongqing; Adams, Ralf H; Xu, Qingbo; Lui, Kathy O; Zhou, Bin

2018-04-26

Background -Whether the adult mammalian heart harbors cardiac stem cells (CSCs) for regeneration of cardiomyocytes is an important yet contentious topic in the field of cardiovascular regeneration. The putative myocyte stem cell populations recognized without specific cell markers such as the cardiosphere-derived cells or with markers such as Sca1 + , Bmi1 + , Isl1 + or Abcg2 + CSCs have been reported. Moreover, it remains unclear whether putative CSCs with unknown or unidentified markers exist and give rise to de novo cardiomyocytes in the adult heart. Methods -To address this question without relying on a particular stem cell marker, we developed a new genetic lineage tracing system to label all non-myocyte populations that contain putative CSCs. Using dual lineage tracing system, we assessed if non-myocytes generated any new myocytes during embryonic development, adult homeostasis and after myocardial infarction. Skeletal muscle was also examined after injury for internal control of new myocytes generation from non-myocytes. Results -By this stem cell marker-free and dual recombinases-mediated cell tracking approach, our fate mapping data show that new myocytes arise from non-myocytes in the embryonic heart, but not in the adult heart during homeostasis or after myocardial infarction. As positive control, our lineage tracing system detected new myocytes derived from non-myocytes in the skeletal muscle after injury. Conclusions -This study provides in vivo genetic evidence for non-myocyte to myocyte conversion in embryonic but not adult heart, arguing again the myogenic potential of putative stem cell populations for cardiac regeneration in the adult stage. This study also provides a new genetic strategy to identify endogenous stem cells, if any, in other organ systems for tissue repair and regeneration.

Cardiac myocyte diversity and a fibroblast network in the junctional region of the zebrafish heart revealed by transmission and serial block-face scanning electron microscopy.

PubMed

Lafontant, Pascal J; Behzad, Ali R; Brown, Evelyn; Landry, Paul; Hu, Norman; Burns, Alan R

2013-01-01

The zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherentes junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart.

Modeling hypertrophic IP3 transients in the cardiac myocyte.

PubMed

Cooling, Michael; Hunter, Peter; Crampin, Edmund J

2007-11-15

Cardiac hypertrophy is a known risk factor for heart disease, and at the cellular level is caused by a complex interaction of signal transduction pathways. The IP3-calcineurin pathway plays an important role in stimulating the transcription factor NFAT which binds to DNA cooperatively with other hypertrophic transcription factors. Using available kinetic data, we construct a mathematical model of the IP3 signal production system after stimulation by a hypertrophic alpha-adrenergic agonist (endothelin-1) in the mouse atrial cardiac myocyte. We use a global sensitivity analysis to identify key controlling parameters with respect to the resultant IP3 transient, including the phosphorylation of cell-membrane receptors, the ligand strength and binding kinetics to precoupled (with G(alpha)GDP) receptor, and the kinetics associated with precoupling the receptors. We show that the kinetics associated with the receptor system contribute to the behavior of the system to a great extent, with precoupled receptors driving the response to extracellular ligand. Finally, by reparameterizing for a second hypertrophic alpha-adrenergic agonist, angiotensin-II, we show that differences in key receptor kinetic and membrane density parameters are sufficient to explain different observed IP3 transients in essentially the same pathway.

Polyphenol-rich apple (Malus domestica L.) peel extract attenuates arsenic trioxide induced cardiotoxicity in H9c2 cells via its antioxidant activity.

PubMed

Vineetha, Vadavanath Prabhakaran; Girija, Seetharaman; Soumya, Rema Sreenivasan; Raghu, Kozhiparambil Gopalan

2014-03-01

Evidences suggest that apple peel has a wide range of polyphenols having antioxidant activity and its consumption has been linked with improved health benefits. Arsenic trioxide (ATO) is a very effective drug for the treatment of acute promyelocytic leukemia (APL) but it leads to cardiotoxicity mediated through alterations in various cardiac ion channels and by increasing the intracellular calcium level and reactive oxygen species (ROS). The aim of the present investigation was to study the effect of methanolic extract of apple peel (APME) and aqueous extract of apple peel (APAE) on ATO (5 μM) induced toxicity in the H9c2 cardiac myoblast cell line. We estimated the cellular status of innate antioxidant enzymes, level of ROS, mitochondrial superoxide, glutathione and intracellular calcium with ATO and apple peel extracts. Prior to the cell line based study, we had evaluated the antioxidant potential of apple peel extract by 1,1-diphenyl-2-picrylhydrazyl (DPPH), total reducing power (TRP), superoxide anion and hydroxyl radical scavenging activity, in addition to quantifying total phenolic and flavonoid content. Both the extracts showed considerable antioxidant activity in cell-free chemical assays. In addition, both APME and APAE prevented the alteration in antioxidant status induced by ATO in H9c2 cells. Significant differential alterations had been observed in the activity of lactate dehydrogenase, superoxide dismutase, catalase, glutathione, glutathione peroxidase, thioredoxin reductase, xanthine oxidase, calcium overload and caspase 3 activity with ATO. The overall result revealed the protective property of polyphenol-rich apple peel extract against ATO induced cardiac toxicity via its antioxidant activity.

Toward an Integrative Computational Model of the Guinea Pig Cardiac Myocyte

PubMed Central

Gauthier, Laura Doyle; Greenstein, Joseph L.; Winslow, Raimond L.

2012-01-01

The local control theory of excitation-contraction (EC) coupling asserts that regulation of calcium (Ca2+) release occurs at the nanodomain level, where openings of single L-type Ca2+ channels (LCCs) trigger openings of small clusters of ryanodine receptors (RyRs) co-localized within the dyad. A consequence of local control is that the whole-cell Ca2+ transient is a smooth continuous function of influx of Ca2+ through LCCs. While this so-called graded release property has been known for some time, its functional importance to the integrated behavior of the cardiac ventricular myocyte has not been fully appreciated. We previously formulated a biophysically based model, in which LCCs and RyRs interact via a coarse-grained representation of the dyadic space. The model captures key features of local control using a low-dimensional system of ordinary differential equations. Voltage-dependent gain and graded Ca2+ release are emergent properties of this model by virtue of the fact that model formulation is closely based on the sub-cellular basis of local control. In this current work, we have incorporated this graded release model into a prior model of guinea pig ventricular myocyte electrophysiology, metabolism, and isometric force production. The resulting integrative model predicts the experimentally observed causal relationship between action potential (AP) shape and timing of Ca2+ and force transients, a relationship that is not explained by models lacking the graded release property. Model results suggest that even relatively subtle changes in AP morphology that may result, for example, from remodeling of membrane transporter expression in disease or spatial variation in cell properties, may have major impact on the temporal waveform of Ca2+ transients, thus influencing tissue level electromechanical function. PMID:22783206

Toward an integrative computational model of the Guinea pig cardiac myocyte.

PubMed

Gauthier, Laura Doyle; Greenstein, Joseph L; Winslow, Raimond L

2012-01-01

The local control theory of excitation-contraction (EC) coupling asserts that regulation of calcium (Ca(2+)) release occurs at the nanodomain level, where openings of single L-type Ca(2+) channels (LCCs) trigger openings of small clusters of ryanodine receptors (RyRs) co-localized within the dyad. A consequence of local control is that the whole-cell Ca(2+) transient is a smooth continuous function of influx of Ca(2+) through LCCs. While this so-called graded release property has been known for some time, its functional importance to the integrated behavior of the cardiac ventricular myocyte has not been fully appreciated. We previously formulated a biophysically based model, in which LCCs and RyRs interact via a coarse-grained representation of the dyadic space. The model captures key features of local control using a low-dimensional system of ordinary differential equations. Voltage-dependent gain and graded Ca(2+) release are emergent properties of this model by virtue of the fact that model formulation is closely based on the sub-cellular basis of local control. In this current work, we have incorporated this graded release model into a prior model of guinea pig ventricular myocyte electrophysiology, metabolism, and isometric force production. The resulting integrative model predicts the experimentally observed causal relationship between action potential (AP) shape and timing of Ca(2+) and force transients, a relationship that is not explained by models lacking the graded release property. Model results suggest that even relatively subtle changes in AP morphology that may result, for example, from remodeling of membrane transporter expression in disease or spatial variation in cell properties, may have major impact on the temporal waveform of Ca(2+) transients, thus influencing tissue level electromechanical function.

Adiponectin downregulation is associated with volume overload-induced myocyte dysfunction in rats

PubMed Central

Wang, Li-li; Miller, Dori; Wanders, Desiree; Nanayakkara, Gayani; Amin, Rajesh; Judd, Robert; Morrison, Edward E; Zhong, Ju-ming

2016-01-01

Aim: Adiponectin has been reported to exert protective effects during pathological ventricular remodeling, but the role of adiponectin in volume overload-induced heart failure remains unclear. In this study we investigated the effect of adiponectin on cardiac myocyte contractile dysfunction following volume overload in rats. Methods: Volume overload was surgically induced in rats by infrarenal aorta-vena cava fistula. The rats were intravenously administered adenoviral adiponectin at 2-, 6- and 9-weeks following fistula. The protein expression of adiponectin, adiponectin receptors (AdipoR1/R2 and T-cadherin) and AMPK activity were measured using Western blot analyses. Isolated ventricular myocytes were prepared at 12 weeks post-fistula to examine the contractile performance of myocytes and intracellular Ca2+ transient. Results: A-V fistula resulted in significant reductions in serum and myocardial adiponectin levels, myocardial adiponectin receptor (AdipoR1/R2 and T-cadherin) levels, as well as myocardial AMPK activity. Consistent with these changes, the isolated myocytes exhibited significant depression in cell shortening and intracellular Ca2+ transient. Administration of adenoviral adiponectin significantly increased serum adiponectin levels and prevented myocyte contractile dysfunction in fistula rats. Furthermore, pretreatment of isolated myocytes with recombinant adiponectin (2.5 μg/mL) significantly improved their contractile performance in fistula rats, but had no effects in control or adenoviral adiponectin-administered rats. Conclusion: These results demonstrate a positive correlation between adiponectin downregulation and volume overload-induced ventricular remodeling. Adiponectin plays a protective role in volume overload-induced heart failure. PMID:26616727

Acute heat tolerance of cardiac excitation in the brown trout (Salmo trutta fario).

PubMed

Vornanen, Matti; Haverinen, Jaakko; Egginton, Stuart

2014-01-15

The upper thermal tolerance and mechanisms of heat-induced cardiac failure in the brown trout (Salmo trutta fario) was examined. The point above which ion channel function and sinoatrial contractility in vitro, and electrocardiogram (ECG) in vivo, started to fail (break point temperature, BPT) was determined by acute temperature increases. In general, electrical excitation of the heart was most sensitive to heat in the intact animal (electrocardiogram, ECG) and least sensitive in isolated cardiac myocytes (ion currents). BPTs of Ca(2+) and K(+) currents of cardiac myocytes were much higher (>28°C) than BPT of in vivo heart rate (23.5 ± 0.6°C) (P<0.05). A striking exception among sarcolemmal ion conductances was the Na(+) current (INa), which was the most heat-sensitive molecular function, with a BPT of 20.9 ± 0.5°C. The low heat tolerance of INa was reflected as a low BPT for the rate of action potential upstroke in vitro (21.7 ± 1.2°C) and the velocity of impulse transmission in vivo (21.9 ± 2.2°C). These findings from different levels of biological organization strongly suggest that heat-dependent deterioration of Na(+) channel function disturbs normal spread of electrical excitation over the heart, leading to progressive variability of cardiac rhythmicity (missed beats, bursts of fast beating), reduction of heart rate and finally cessation of the normal heartbeat. Among the cardiac ion currents INa is 'the weakest link' and possibly a limiting factor for upper thermal tolerance of electrical excitation in the brown trout heart. Heat sensitivity of INa may result from functional requirements for very high flux rates and fast gating kinetics of the Na(+) channels, i.e. a trade-off between high catalytic activity and thermal stability.

Early NADPH oxidase-2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells.

PubMed

Hahn, Nynke E; Musters, René J P; Fritz, Jan M; Pagano, Patrick J; Vonk, Alexander B A; Paulus, Walter J; van Rossum, Albert C; Meischl, Christof; Niessen, Hans W M; Krijnen, Paul A J

2014-09-01

Reactive oxygen species (ROS) produced by different NADPH oxidases (NOX) play a role in cardiomyocyte hypertrophy induced by different stimuli, such as angiotensin II and pressure overload. However, the role of the specific NOX isoforms in phenylephrine (PE)-induced cardiomyocyte hypertrophy is unknown. Therefore we aimed to determine the involvement of the NOX isoforms NOX1, NOX2 and NOX4 in PE-induced cardiomyocyte hypertrophy. Hereto rat neonatal cardiomyoblasts (H9c2 cells) were incubated with 100 μM PE to induce hypertrophy after 24 and 48h as determined via cell and nuclear size measurements using digital imaging microscopy, electron microscopy and an automated cell counter. Digital-imaging microscopy further revealed that in contrast to NOX1 and NOX4, NOX2 expression increased significantly up to 4h after PE stimulation, coinciding and co-localizing with ROS production in the cytoplasm as well as the nucleus. Furthermore, inhibition of NOX-mediated ROS production with apocynin, diphenylene iodonium (DPI) or NOX2 docking sequence (Nox2ds)-tat peptide during these first 4h of PE stimulation significantly inhibited PE-induced hypertrophy of H9c2 cells, both after 24 and 48h of PE stimulation. These data show that early NOX2-mediated ROS production is crucial in PE-induced hypertrophy of H9c2 cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Vitamin C prevents zidovudine-induced NAD(P)H oxidase activation and hypertension in the rat.

PubMed

Papparella, Italia; Ceolotto, Giulio; Berto, Laura; Cavalli, Maurizio; Bova, Sergio; Cargnelli, Gabriella; Ruga, Ezia; Milanesi, Ornella; Franco, Lorenzo; Mazzoni, Martina; Petrelli, Lucia; Nussdorfer, Gastone G; Semplicini, Andrea

2007-01-15

Cardiovascular risk is increased among HIV-infected patients receiving antiretroviral therapy due to the development of hypertension and metabolic abnormalities. In this study, we investigated the effects of long-term treatment with zidovudine (AZT) and vitamin C, alone and in combination, on blood pressure and on the chain of events linking oxidative stress to cardiac damage in the rat. Six adult Wistar Kyoto rats received AZT (1 mg/ml) in the drinking water for 8 months, six vitamin C (10 g/kg of food) and AZT, six vitamin C alone, and six served as controls. AZT increased systolic blood pressure, expression of gp91(phox) and p47(phox) subunits of NAD(P)H oxidase, and protein kinase C (PKC) delta activation and reduced antioxidant power of plasma and cardiac homogenates. AZT also caused morphological alterations in cardiac myocyte mitochondria, indicative of functional damage. All of these effects were prevented by vitamin C. Chronic AZT administration increases blood pressure and promotes cardiovascular damage through a NAD(P)H oxidase-dependent mechanism that involves PKC delta. Vitamin C antagonizes these adverse effects of AZT in the cardiovascular system.

The protective effect of lipid emulsion in preventing bupivacaine-induced mitochondrial injury and apoptosis of H9C2 cardiomyocytes.

PubMed

Chen, Zhe; Jin, Zhousheng; Xia, Yun; Zhao, Shishi; Xu, Xuzhong; Papadimos, Thomas J; Wang, Quanguang

2017-11-01

Lipid emulsion (LE) has been shown to be effective in the resuscitation of bupivacaine-induced cardiac arrest, but the precise mechanism of this action has not been fully elucidated. Pursuant to this lack of information on the mechanism in which LE protects the myocardium during bupivacaine-induced toxicity, we explored mitochondrial function and cell apoptosis. H9C2 cardiomyocytes were used in study. Cells were randomly divided in different groups and were cultivated 6 h, 12 h, and 24 h. The mitochondria were extracted and mitochondrial ATP content was measured, as was mitochondrial membrane potential, the concentration of calcium ion (Ca2+), and the activity of Ca2+-ATP enzyme (Ca2+-ATPase). Cells from groups Bup1000, LE group, and Bup1000LE were collected to determine cell viability, cell apoptosis, and electron microscopy scanning of mitochondrial ultrastructure (after 24 h). We found that LE can reverse the inhibition of the mitochondrial function induced by bupivacaine, regulate the concentration of calcium ion in mitochondria, resulting in the protection of myocardial cells from toxicity induced by bupivacaine.

Noncontact minimally invasive technique for the assessment of mechanical properties of single cardiac myocyte via magnetic field loading

NASA Astrophysics Data System (ADS)

Yin, Shizhuo; Zhang, Xueqian; Cheung, Joseph; Wu, Juntao; Zhan, Chun; Xue, Jinchao

2004-07-01

In this paper, a unique non-contact, minimum invasive technique for the assessment of mechanical properties of single cardiac myocyte is presented. The assessment process includes following major steps: (1) attach a micro magnetic bead to the cell to be measured, (2) measure the contractile performance of the cell under the different magnetic field loading, (3) calculate mechanical loading force, and (4) derive the contractile force from the measured contraction data under different magnetic field loading.

Validation of an in vitro contractility assay using canine ventricular myocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harmer, A.R., E-mail: alex.harmer@astrazeneca.com; Abi-Gerges, N.; Morton, M.J.

Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose. Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscopemore » at ∼ 36 °C. Myocytes were stimulated to contract at a pacing frequency of 1 Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration–effect curves were constructed for each compound in 4–30 myocytes taken from 1 or 2 dog hearts. The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%. With a throughput of 6–8 compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds. -- Highlights: ► Cardiac contractility is an important physiological function of the heart. ► Assessment of contractility is a logical part of pre-clinical drug safety testing. ► There are limited validated assays that predict effects of compounds on contractility. ► Using dog myocytes, we have developed an in vitro cardiac contractility assay. ► The assay predicted the in vivo contractility with a good level of

Increased Response to β2-Adrenoreceptor Stimulation Augments Inhibition of IKr in Heart Failure Ventricular Myocytes

PubMed Central

Wang, Hegui; Chen, Yanhong; Zhu, Hongjun; Wang, Sen; Zhang, Xiwen; Xu, Dongjie; Cao, Kejiang; Zou, Jiangang

2012-01-01

Background Increasing evidence indicates that the rapid component of delayed rectifier potassium current (IKr) is modulated by α- and β-adrenergic stimulation. However, the role and mechanism regulating IKr through β2-adrenoreceptor (β-AR) stimulation in heart failure (HF) are unclear. Methodology/Principal Findings In the present study, we investigated the effects of fenoterol, a highly selective β2-AR agonist, on IKr in left ventricular myocytes obtained from control and guinea pigs with HF induced by descending aortic banding. IKr was measured by using whole cell patch clamp technique. In control myocytes, superfusion of fenoterol (10 µM) caused a 17% decrease in IKr. In HF myocytes, the same concentration of fenoterol produced a significantly greater decrease (33%) in IKr. These effects were not modified by the incubation of myocytes with CGP-20712A, a β1-AR antagonist, but were abolished by pretreatment of myocytes with ICI-118551, a β2-AR antagonist. An inhibitory cAMP analog, Rp-cAMPS and PKA inhibitor significantly attenuated fenoterol-induced inhibition of IKr in HF myocytes. Moreover, fenoterol markedly prolonged action potential durations at 90% (APD90) repolarization in HF ventricular myocytes. Conclusions The results indicate that inhibition of IKr induced by β2-AR stimulation is increased in HF. The inhibitory effect is likely to be mediated through a cAMP/PKA pathway in HF ventricular myocytes. PMID:23029432

Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes*

PubMed Central

Markandeya, Yogananda S.; Phelan, Laura J.; Woon, Marites T.; Keefe, Alexis M.; Reynolds, Courtney R.; August, Benjamin K.; Hacker, Timothy A.; Roth, David M.; Patel, Hemal H.; Balijepalli, Ravi C.

2015-01-01

Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca2+ cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca2+ signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca2+ current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy. PMID:26170457

β1-adrenergic regulation of rapid component of delayed rectifier K+ currents in guinea-pig cardiac myocytes.

PubMed

Wang, Sen; Xu, Di; Wu, Ting-Ting; Guo, Yan; Chen, Yan-Hong; Zou, Jian-Gang

2014-05-01

Human ether-à-go-go-related gene (hERG) potassium channels conduct the rapid component of the delayed rectifier potassium current (IKr), which is crucial for repolarization of cardiac action potential. Patients with hERG‑associated long QT syndrome usually develop tachyarrhythmias during physical and/or emotional stress, both known to stimulate adrenergic receptors. The present study aimed to investigate a putative functional link between β1-adrenergic stimulation and IKr in guinea-pig left ventricular myocytes and to analyze how IKr is regulated following activation of the β1-adrenergic signaling pathway. The IKr current was measured using a whole-cell patch-clamp technique. A selective β1-adrenergic receptor agonist, xamoterol, at concentrations of 0.01-100 µM decreased IKr in a concentration-dependent manner. The 10 µM xamoterol-induced inhibition of IKr was attenuated by the protein kinase A (PKA) inhibitor KT5720, the protein kinase C (PKC) inhibitor chelerythrine, and the phospholipase (PLC) inhibitor U73122, indicating involvement of PKA, PKC and PLC in β1-adrenergic inhibition of IKr. The results of the present study indicate an association between IKr and the β1-adrenergic receptor in arrhythmogenesis, involving the activation of PKA, PKC and PLC.

Differing Effects of Younger and Older Human Plasma on C2C12 Myocytes in Vitro.

PubMed

Kalampouka, Ifigeneia; van Bekhoven, Angel; Elliott, Bradley T

2018-01-01

Ageing is associated with a general reduction of physiological function and a reduction of muscle mass and strength. Endocrine factors such as myostatin, activin A, growth and differentiation factor 11 (GDF-11) and their inhibitory peptides influence muscle mass in health and disease. We hypothesised that myocytes cultured in plasma from older and younger individuals would show an ageing effect, with reduced proliferation and differentiation in older environments. C2C12 myoblasts were grown as standard and stimulated with media conditioned with 5% plasma from healthy male participants that were either younger ( n = 6, 18-35 years of age) or older ( n = 6, >57 years of age). Concentration of plasma myostatin (total and free), follistatin-like binding protein (FLRG), GDF-11 and activin A were quantified by ELISA. Both FLRG and activin A were elevated in older individuals (109.6 and 35.1% increase, respectively), whilst myostatin (free and total) and GDF-11 were not. Results indicated that plasma activin A and FLRG were increased in older vs. younger participants, GDF11 and myostatin did not differ. Myoblasts in vitro showed no difference in proliferation rate between ages, however scratch closure was greater in younger vs. older plasma stimulated myoblasts (78.2 vs. 87.2% of baseline scratch diameter, respectively). Myotube diameters were larger in cells stimulated with younger plasma than with older at 24 and 48 h, but not at 2 h. A significant negative correlation was noted between in vivo plasma FLRG concentration and in vitro myotube diameter 48 h following plasma stimulation ( r 2 = 0.392, p = 0.030). Here we show that myoblasts and myotubes cultured in media conditioned with plasma from younger or older individuals show an ageing effect, and further this effect moderately correlates with circulating FLRG concentration in vivo . The effect of ageing on muscle function may not be innate to the tissue, but involve a general cellular environment change. Further

Cathepsin K knockout alleviates aging-induced cardiac dysfunction

PubMed Central

Hua, Yinan; Robinson, Timothy J; Cao, Yongtao; Shi, Guo-Ping; Ren, Jun; Nair, Sreejayan

2015-01-01

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis. PMID:25692548

Protection of Luteolin-7-O-glucoside against apoptosis induced by hypoxia/reoxygenation through the MAPK pathways in H9c2 cells.

PubMed

Chen, Shenjie; Yang, Bingsheng; Xu, Yifei; Rong, Yiqing; Qiu, Yuangang

2018-05-01

Myocardial hypertrophy is often associated with myocardial infarction. Luteolin-7-O-glucoside (LUTG) has the prosperity of preventing cardiomyocyte injury. The current study aimed to explore the potential protective effect of LUTG and its relevant mechanisms in the heart. To establish the cardiac hypertrophy model in vitro, Angiotensin II (Ang II) was used to stimuli H9c2 cells in this study. The CCK‑8 assay showed that LUTG pretreatment improved cell viability of cardiomyocytes co‑treated with Ang II and ischemia/reperfusion. LUTG decreased the reactive oxygen species levels. Furthermore, it was demonstrated LUTG could reduce the release amount of lactate dehydrogenase and recover the catalase activity according to the flow cytometry analysis, and activity detection, respectively in Ang II‑H/R‑treated H9c2 cells. In addition, the flow cytometry analysis showed that the pretreatment of LUTG mitigated cell apoptosis induced by hypoxia/reoxygenation in the cardiac hypertrophy model. Meanwhile, reverse transcription‑quantitative polymerase chain reaction and western blot assays showed that the apoptosis‑related genes, including poly (ADP‑ribose) polymerase, Fas, Fasl and Caspase‑3 were downregulated at the transcriptional and translational levels. Notably, the protien expression of phosphorylated (p)‑extracellular signal‑regulated kinas (ERK) 1/2, p‑janus kinase and p‑P38 were reduced, while the expression of p‑ERK5 was elevated in the LUTG pretreatment groups compared with the hypoxia/reoxygenation treatment group. Based on these results, it was suggested that the anti‑apoptosis effect of LUTG may be associated with regulating the activation of mitogen‑activated protein kinases signaling pathways.

Association of cardiac myosin binding protein-C with the ryanodine receptor channel: putative retrograde regulation?

PubMed

Stanczyk, Paulina J; Seidel, Monika; White, Judith; Viero, Cedric; George, Christopher H; Zissimopoulos, Spyros; Lai, F Anthony

2018-06-21

The cardiac muscle ryanodine receptor-Ca 2+ release channel (RyR2) constitutes the sarcoplasmic reticulum (SR) Ca 2+ efflux mechanism that initiates myocyte contraction, while cardiac myosin binding protein-C (cMyBP-C) mediates regulation of acto-myosin cross-bridge cycling. In this report, we provide the first evidence for the presence of direct interaction between these two proteins, forming a RyR2:cMyBP-C complex. The C-terminus of cMyBP-C binds with the RyR2 N-terminus in mammalian cells and is not mediated by a fibronectin-like domain. Notably, we detected complex formation between both recombinant cMyBP-C and RyR2, as well as with the native proteins in cardiac tissue. Cellular Ca 2+ dynamics in HEK293 cells is altered upon co-expression of cMyBP-C and RyR2, with lowered frequency of RyR2-mediated spontaneous Ca 2+ oscillations, suggesting cMyBP-C exerts a potential inhibitory effect on RyR2-dependent Ca 2+ release. Discovery of a functional RyR2 association with cMyBP-C provides direct evidence for a putative mechanistic link between cytosolic soluble cMyBP-C and SR-mediated Ca 2+ release, via RyR2. Importantly, this interaction may have clinical relevance to the observed cMyBP-C and RyR2 dysfunction in cardiac pathologies, such as hypertrophic cardiomyopathy. © 2018. Published by The Company of Biologists Ltd.

Long Term Ablation of Protein Kinase A (PKA)-mediated Cardiac Troponin I Phosphorylation Leads to Excitation-Contraction Uncoupling and Diastolic Dysfunction in a Knock-in Mouse Model of Hypertrophic Cardiomyopathy*

PubMed Central

Dweck, David; Sanchez-Gonzalez, Marcos A.; Chang, Audrey N.; Dulce, Raul A.; Badger, Crystal-Dawn; Koutnik, Andrew P.; Ruiz, Edda L.; Griffin, Brittany; Liang, Jingsheng; Kabbaj, Mohamed; Fincham, Frank D.; Hare, Joshua M.; Overton, J. Michael; Pinto, Jose R.

2014-01-01

The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca2+ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca2+-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca2+ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca2+ content. This abnormal Ca2+ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na+-Ca2+ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy. PMID:24973218

Role of transglutaminase 2 in A1 adenosine receptor- and β2-adrenoceptor-mediated pharmacological pre- and post-conditioning against hypoxia-reoxygenation-induced cell death in H9c2 cells.

PubMed

Vyas, Falguni S; Nelson, Carl P; Dickenson, John M

2018-01-15

Pharmacologically-induced pre- and post-conditioning represent attractive therapeutic strategies to reduce ischaemia/reperfusion injury during cardiac surgery and following myocardial infarction. We have previously reported that transglutaminase 2 (TG2) activity is modulated by the A 1 adenosine receptor and β 2 -adrenoceptor in H9c2 cardiomyoblasts. The primary aim of this study was to determine the role of TG2 in A 1 adenosine receptor and β 2 -adrenoceptor-induced pharmacological pre- and post-conditioning in the H9c2 cells. H9c2 cells were exposed to 8h hypoxia (1% O 2 ) followed by 18h reoxygenation, after which cell viability was assessed by monitoring mitochondrial reduction of MTT, lactate dehydrogenase release and caspase-3 activation. N 6 -cyclopentyladenosine (CPA; A 1 adenosine receptor agonist), formoterol (β 2 -adrenoceptor agonist) or isoprenaline (non-selective β-adrenoceptor agonist) were added before hypoxia/reoxygenation (pre-conditioning) or at the start of reoxygenation following hypoxia (post-conditioning). Pharmacological pre- and post-conditioning with CPA and isoprenaline significantly reduced hypoxia/reoxygenation-induced cell death. In contrast, formoterol did not elicit protection. Pre-treatment with pertussis toxin (G i/o -protein inhibitor), DPCPX (A 1 adenosine receptor antagonist) or TG2 inhibitors (Z-DON and R283) attenuated the A 1 adenosine receptor-induced pharmacological pre- and post-conditioning. Similarly, pertussis toxin, ICI 118,551 (β 2 -adrenoceptor antagonist) or TG2 inhibition attenuated the isoprenaline-induced cell survival. Knockdown of TG2 using small interfering RNA (siRNA) attenuated CPA and isoprenaline-induced pharmacological pre- and post-conditioning. Finally, proteomic analysis following isoprenaline treatment identified known (e.g. protein S100-A6) and novel (e.g. adenine phosphoribosyltransferase) protein substrates for TG2. These results have shown that A 1 adenosine receptor and β 2 -adrenoceptor

Phospholipase C/protein kinase C pathway mediates angiotensin II-dependent apoptosis in neonatal rat cardiac fibroblasts expressing AT1 receptor.

PubMed

Vivar, Raul; Soto, Cristian; Copaja, Miguel; Mateluna, Francisca; Aranguiz, Pablo; Muñoz, Juan Pablo; Chiong, Mario; Garcia, Lorena; Letelier, Alan; Thomas, Walter G; Lavandero, Sergio; Díaz-Araya, Guillermo

2008-08-01

Cardiac fibroblasts are the major non-myocyte cell constituent in the myocardium, and they are involved in heart remodeling. Angiotensin II type 1 receptor (AT1R) mediates the established actions of angiotensin II (Ang II), and changes in its expression have been reported in cardiac fibroblasts after myocardial infarction. However, the AT1R-dependent signaling pathways involved in cardiac fibroblast death remain unknown. Using adenovirus, we ectopically expressed AT1R in cultured neonatal rat cardiac fibroblasts and investigated the role of the phospholipase (PLC)/protein kinase C (PKC) pathway on Ang II-dependent death. Ang II induced cardiac fibroblast death characterized by an early loss of mitochondrial membrane potential, increased Bax/Bcl-2 ratio, caspase-3 activation, and DNA fragmentation. All these effects were prevented by the AT1R antagonist losartan, PLC inhibitor U73122, and PKC inhibitor Gö6976. We conclude that Ang II stimulates the intrinsic apoptotic pathway in cultured cardiac fibroblasts by the AT1R/PLC/PKC signaling pathway.

Can optical recordings of membrane potential be used to screen for drug-induced action potential prolongation in single cardiac myocytes?

PubMed

Hardy, M E L; Lawrence, C L; Standen, N B; Rodrigo, G C

2006-01-01

Potential-sensitive dyes have primarily been used to optically record action potentials (APs) in whole heart tissue. Using these dyes to record drug-induced changes in AP morphology of isolated cardiac myocytes could provide an opportunity to develop medium throughout assays for the pharmaceutical industry. Ideally, this requires that the dye has a consistent and rapid response to membrane potential, is insensitive to movement, and does not itself affect AP morphology. We recorded the AP from isolated adult guinea-pig ventricular myocytes optically using di-8-ANEPPS in a single-excitation dual-emission ratiometric system, either separately in electrically field stimulated myocytes, or simultaneously with an electrical AP recorded with a patch electrode in the whole-cell bridge mode. The ratio of di-8-ANEPPS fluorescence signal was calibrated against membrane potential using a switch-clamp to voltage clamp the myocyte. Our data show that the ratio of the optical signals emitted at 560/620 nm is linearly related to voltage over the voltage range of an AP, producing a change in ratio of 7.5% per 100 mV, is unaffected by cell movement and is identical to the AP recorded simultaneously with a patch electrode. However, the APD90 recorded optically in myocytes loaded with di-8-ANEPPS was significantly longer than in unloaded myocytes recorded with a patch electrode (355.6+/-13.5 vs. 296.2+/-16.2 ms; p<0.01). Despite this effect, the apparent IC50 for cisapride, which prolongs the AP by blocking IKr, was not significantly different whether determined optically or with a patch electrode (91+/-46 vs. 81+/-20 nM). These data show that the optical AP recorded ratiometrically using di-8-ANEPPS from a single ventricular myocyte accurately follows the action potential morphology. This technique can be used to estimate the AP prolonging effects of a compound, although di-8-ANEPPS itself prolongs APD90. Optical dyes require less technical skills and are less invasive than

Resveratrol prevents doxorubicin-induced cardiotoxicity in H9c2 cells through the inhibition of endoplasmic reticulum stress and the activation of the Sirt1 pathway.

PubMed

Lou, Yu; Wang, Zhen; Xu, Yi; Zhou, Ping; Cao, Junxian; Li, Yuanshi; Chen, Yeping; Sun, Junfeng; Fu, Lu

2015-09-01

Treatment with doxorubicin (DOX) is one of the major causes of chemotherapy-induced cardiotoxicity and is therefore, the principal limiting factor in the effectiveness of chemotherapy for cancer patients. DOX‑induced heart failure is thought to result from endoplasmic reticulum (ER) stress and cardiomyocyte apoptosis. Resveratrol (RV), a polyphenol antioxidant found in red wine, has been shown to play a cardioprotective role. The aim of the present study was to examine the effects of RV on DOX‑induced cardiotoxicity in H9c2 cells. We hypothesized that RV would protect H9c2 cells against DOX‑induced ER stress and subsequent cell death through the activation of the Sirt1 pathway. Our results demonstrated that the decrease observed in the viability of the H9c2 cells following exposure to DOX was accompanied by a significant increase in the expression of the ER stress‑related proteins, glucose‑regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP). However, we found that RV downregulated the expression of ER stress marker protein in the presence of DOX and restored the viability of the H9c2 cells. Exposure to RV or DOX alone only slightly increased the protein expression of Sirt1, whereas a significant increase in Sirt1 protein levels was observed in the cells treated with both RV and DOX. The Sirt1 inhibitor, nicotinamide (NIC), partially neutralized the effects of RV on the expression of Sirt1 in the DOX‑treated cells and completely abolished the effects of RV on the expression of GRP78 and CHOP. The findings of our study suggest that RV protects H9c2 cells against DOX‑induced ER stress through ER stabilization, and more specifically through the activation of the Sirt1 pathway, thereby leading to cardiac cell survival.

Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes.

PubMed

Zimmermann, W H; Fink, C; Kralisch, D; Remmers, U; Weil, J; Eschenhagen, T

2000-04-05

A technique is presented that allows neonatal rat cardiac myocytes to form spontaneously and coherently beating 3-dimensional engineered heart tissue (EHT) in vitro, either as a plane biconcaval matrix anchored at both sides on Velcro-coated silicone tubes or as a ring. Contractile activity was monitored in standard organ baths or continuously in a CO(2) incubator for up to 18 days (=26 days after casting). Long-term measurements showed an increase in force between days 8 and 18 after casting and stable forces thereafter. At day 10, the twitch amplitude (TA) of electrically paced EHTs (average length x width x thickness, 11 x 6 x 0.4 mm) was 0.51 mN at length of maximal force development (L(max)) and a maximally effective calcium concentration. EHTs showed typical features of neonatal rat heart: a positive force-length and a negative force-frequency relation, high sensitivity to calcium (EC(50) 0.24 mM), modest positive inotropic (increase in TA by 46%) and pronounced positive lusitropic effect of isoprenaline (decrease in twitch duration by 21%). Both effects of isoprenaline were sensitive to the muscarinic receptor agonist carbachol in a pertussis toxin-sensitive manner. Adenovirus-mediated gene transfer of beta-galactosidase into EHTs reached 100% efficiency. In summary, EHTs retain many of the physiological characteristics of rat cardiac tissue and allow efficient gene transfer with subsequent force measurement. Copyright 2000 John Wiley & Sons, Inc.

ERβ targets ZAK and attenuates cellular hypertrophy via SUMO-1 modification in H9c2 cells.

PubMed

Pai, Peiying; Shibu, Marthandam Asokan; Chang, Ruey-Lin; Yang, Jaw-Ji; Su, Chia-Chi; Lai, Chao-Hung; Liao, Hung-En; Viswanadha, Vijaya Padma; Kuo, Wei-Wen; Huang, Chih-Yang

2018-06-22

Aberrant expression of leucine zipper- and sterile ɑ motif-containing kinase (ZAK) observed in pathological human myocardial tissue is associated with the progression and elevation of hypertrophy. Our previous reports have correlated high levels of estrogen (E2) and abundant estrogen receptor (ER) α with a low incidence of pathological cardiac-hypertrophy and heart failure in the premenopause female population. However, the effect of elevated ERβ expression is not well known yet. Therefore, in this study, we have analyzed the cardioprotective effects and mechanisms of E2 and/or ERβ against ZAK overexpression-induced cellular hypertrophy. We have used transient transfection to overexpress ERβ into the ZAK tet-on H9c2 cells that harbor the doxycycline-inducible ZAK plasmid. The results show that ZAK overexpression in H9c2 cells resulted in hypertrophic effects, which was correlated with the upregulation of p-JNK and p-p38 MAPKs and their downstream transcription factors c-Jun and GATA-4. However, ERβ and E2 with ERβ overexpressions totally suppressed the effects of ZAK overexpression and inhibited the levels of p-JNK, p-p38, c-Jun, and GATA-4 effectively. Our results further reveal that ERβ directly binds with ZAK under normal conditions; however, ZAK overexpression reduced the association of ZAK-ERβ. Interestingly, increase in ERβ and E2 along with ERβ overexpression both enhanced the binding strengths of ERβ and ZAK and reduced the ZAK protein level. ERβ overexpression also suppressed the E3 ligase-casitas B-lineage lymphoma (CBL) and attenuated CBL-phosphoinositide 3-kinase (PI3K) protein association to prevent PI3K protein degradation. Moreover, ERβ and/or E2 blocked ZAK nuclear translocation via the inhibition of small ubiquitin-like modifier (SUMO)-1 modification. Taken together, our results further suggest that ERβ overexpression strongly suppresses ZAK-induced cellular hypertrophy and myocardial damage. © 2018 Wiley Periodicals, Inc.

Myocardial deletion of transcription factor CHF1/Hey2 results in altered myocyte action potential and mild conduction system expansion but does not alter conduction system function or promote spontaneous arrhythmias.

PubMed

Hartman, Matthew E; Liu, Yonggang; Zhu, Wei-Zhong; Chien, Wei-Ming; Weldy, Chad S; Fishman, Glenn I; Laflamme, Michael A; Chin, Michael T

2014-07-01

CHF1/Hey2 is a Notch-responsive basic helix-loop-helix transcription factor involved in cardiac development. Common variants in Hey2 are associated with Brugada syndrome. We hypothesized that absence of CHF1/Hey2 would result in abnormal cellular electrical activity, altered cardiac conduction system (CCS) development, and increased arrhythmogenesis. We isolated neonatal CHF/Hey2-knockout (KO) cardiac myocytes and measured action potentials and ion channel subunit gene expression. We also crossed myocardial-specific CHF1/Hey2-KO mice with cardiac conduction system LacZ reporter mice and stained for conduction system tissue. We also performed ambulatory ECG monitoring for arrhythmias and heart rate variability. Neonatal cardiomyocytes from CHF1/Hey2-KO mice demonstrate a 50% reduction in action potential dV/dT, a 50-75% reduction in SCN5A, KCNJ2, and CACNA1C ion channel subunit gene expression, and an increase in delayed afterdepolarizations from 0/min to 12/min. CHF1/Hey2 cKO CCS-lacZ mice have a ∼3-fold increase in amount of CCS tissue. Ambulatory ECG monitoring showed no difference in cardiac conduction, arrhythmias, or heart rate variability. Wild-type cells or animals were used in all experiments. CHF1/Hey2 may contribute to Brugada syndrome by influencing the expression of SCN5A and formation of the cardiac conduction system, but its absence does not cause baseline conduction defects or arrhythmias in the adult mouse.-Hartman, M. E., Liu, Y., Zhu, W.-Z., Chien, W.-M., Weldy, C. S., Fishman, G. I., Laflamme, M. A., Chin, M. T. Myocardial deletion of transcription factor CHF1/Hey2 results in altered myocyte action potential and mild conduction system expansion but does not alter conduction system function or promote spontaneous arrhythmias. © FASEB.

Thyroxine-induced cardiac hypertrophy: influence of adrenergic nervous system versus renin-angiotensin system on myocyte remodeling.

PubMed

Hu, L W; Benvenuti, L A; Liberti, E A; Carneiro-Ramos, M S; Barreto-Chaves, M L M

2003-12-01

The present study assessed the possible involvement of the renin-angiotensin system (RAS) and the sympathetic nervous system (SNS) in thyroxine (T4)-induced cardiac hypertrophy. Hemodynamic parameters, heart weight (HW), ratio of HW to body weight (HW/BW), and myocyte width were evaluated in absence of thyroid hormone (hypothyroidism) and after T4 administration. Male Wistar rats were used. Some were subjected to thyroidectomies, whereas hyperthyroidism was induced in others via daily intraperitoneal injection of T4 (25 or 100 microg x 100 g BW(-1) x day(-1)) for 7 days. In some cases, T4 administration was combined with the angiotensin I-converting enzyme inhibitor enalapril (Ena), with the angiotensin type 1 (AT1) receptor blocker losartan (Los) or with the beta-adrenergic blocker propanolol (Prop). Hemodynamics and morphology were then evaluated. Systolic blood pressure (SBP) was not altered by administration of either T4 alone or T4 in combination with the specific inhibitors. However, SBP decreased significantly in hypothyroid rats. An increased heart rate was seen after administration of either T4 alone or T4 in combination with either Los or Ena. Although the higher dose of T4 significantly increased HW, HW/BW increased in both T4-treated groups. Ena and Prop inhibited the increase in HW or HW/BW in hyperthyroid rats. Morphologically, both T4 dose levels significantly increased myocyte width, an occurrence prevented by RAS or SNS blockers. There was a good correlation between changes in HW/BW and myocyte width. These results indicate that T4-induced cardiac hypertrophy is associated with both the SNS and the RAS.

Adrenergic Receptors in Individual Ventricular Myocytes: The Beta-1 and Alpha-1B Are in All Cells, the Alpha-1A Is in a Subpopulation, and the Beta-2 and Beta-3 Are Mostly Absent.

PubMed

Myagmar, Bat-Erdene; Flynn, James M; Cowley, Patrick M; Swigart, Philip M; Montgomery, Megan D; Thai, Kevin; Nair, Divya; Gupta, Rumita; Deng, David X; Hosoda, Chihiro; Melov, Simon; Baker, Anthony J; Simpson, Paul C

2017-03-31

It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), β1, β2, β3, α1A, and α1B. The β1 and β2 are thought to be the dominant myocyte ARs. Quantify the 5 cardiac ARs in individual ventricular myocytes. We studied ventricular myocytes from wild-type mice, mice with α1A and α1B knockin reporters, and β1 and β2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal-regulated kinase and phospholamban), and contraction. We found that the β1 and α1B were present in all myocytes. The α1A was present in 60%, with high levels in 20%. The β2 and β3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total β-ARs were β2 and 20% were β3, both mainly in nonmyocytes. The dominant ventricular myocyte ARs present in all cells are the β1 and α1B. The β2 and β3 are mostly absent in myocytes but are abundant in nonmyocytes. The α1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with β1 and α1B only; 60% that also have the α1A; and 5% each that also have the β2 or β3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms. © 2017 American Heart Association, Inc.

Vagus nerve stimulation mitigates intrinsic cardiac neuronal and adverse myocyte remodeling postmyocardial infarction

PubMed Central

Beaumont, Eric; Southerland, Elizabeth M.; Hardwick, Jean C.; Wright, Gary L.; Ryan, Shannon; Li, Ying; KenKnight, Bruce H.; Armour, J. Andrew

2015-01-01

This paper aims to determine whether chronic vagus nerve stimulation (VNS) mitigates myocardial infarction (MI)-induced remodeling of the intrinsic cardiac nervous system (ICNS), along with the cardiac tissue it regulates. Guinea pigs underwent VNS implantation on the right cervical vagus. Two weeks later, MI was produced by ligating the ventral descending coronary artery. VNS stimulation started 7 days post-MI (20 Hz, 0.9 ± 0.2 mA, 14 s on, 48 s off; VNS-MI, n = 7) and was compared with time-matched MI animals with sham VNS (MI n = 7) vs. untreated controls (n = 8). Echocardiograms were performed before and at 90 days post-MI. At termination, IC neuronal intracellular voltage recordings were obtained from whole-mount neuronal plexuses. MI increased left ventricular end systolic volume (LVESV) 30% (P = 0.027) and reduced LV ejection fraction (LVEF) 6.5% (P < 0.001) at 90 days post-MI compared with baseline. In the VNS-MI group, LVESV and LVEF did not differ from baseline. IC neurons showed depolarization of resting membrane potentials and increased input resistance in MI compared with VNS-MI and sham controls (P < 0.05). Neuronal excitability and sensitivity to norepinephrine increased in MI and VNS-MI groups compared with controls (P < 0.05). Synaptic efficacy, as determined by evoked responses to stimulating input axons, was reduced in VNS-MI compared with MI or controls (P < 0.05). VNS induced changes in myocytes, consistent with enhanced glycogenolysis, and blunted the MI-induced increase in the proapoptotic Bcl-2-associated X protein (P < 0.05). VNS mitigates MI-induced remodeling of the ICNS, correspondingly preserving ventricular function via both neural and cardiomyocyte-dependent actions. PMID:26276818

Vagus nerve stimulation mitigates intrinsic cardiac neuronal and adverse myocyte remodeling postmyocardial infarction.

PubMed

Beaumont, Eric; Southerland, Elizabeth M; Hardwick, Jean C; Wright, Gary L; Ryan, Shannon; Li, Ying; KenKnight, Bruce H; Armour, J Andrew; Ardell, Jeffrey L

2015-10-01

This paper aims to determine whether chronic vagus nerve stimulation (VNS) mitigates myocardial infarction (MI)-induced remodeling of the intrinsic cardiac nervous system (ICNS), along with the cardiac tissue it regulates. Guinea pigs underwent VNS implantation on the right cervical vagus. Two weeks later, MI was produced by ligating the ventral descending coronary artery. VNS stimulation started 7 days post-MI (20 Hz, 0.9 ± 0.2 mA, 14 s on, 48 s off; VNS-MI, n = 7) and was compared with time-matched MI animals with sham VNS (MI n = 7) vs. untreated controls (n = 8). Echocardiograms were performed before and at 90 days post-MI. At termination, IC neuronal intracellular voltage recordings were obtained from whole-mount neuronal plexuses. MI increased left ventricular end systolic volume (LVESV) 30% (P = 0.027) and reduced LV ejection fraction (LVEF) 6.5% (P < 0.001) at 90 days post-MI compared with baseline. In the VNS-MI group, LVESV and LVEF did not differ from baseline. IC neurons showed depolarization of resting membrane potentials and increased input resistance in MI compared with VNS-MI and sham controls (P < 0.05). Neuronal excitability and sensitivity to norepinephrine increased in MI and VNS-MI groups compared with controls (P < 0.05). Synaptic efficacy, as determined by evoked responses to stimulating input axons, was reduced in VNS-MI compared with MI or controls (P < 0.05). VNS induced changes in myocytes, consistent with enhanced glycogenolysis, and blunted the MI-induced increase in the proapoptotic Bcl-2-associated X protein (P < 0.05). VNS mitigates MI-induced remodeling of the ICNS, correspondingly preserving ventricular function via both neural and cardiomyocyte-dependent actions. Copyright © 2015 the American Physiological Society.

Inhibition of gap junction intercellular communication is involved in silica nanoparticles-induced H9c2 cardiomyocytes apoptosis via the mitochondrial pathway.

PubMed

Du, Zhong-Jun; Cui, Guan-Qun; Zhang, Juan; Liu, Xiao-Mei; Zhang, Zhi-Hu; Jia, Qiang; Ng, Jack C; Peng, Cheng; Bo, Cun-Xiang; Shao, Hua

2017-01-01

Gap junction intercellular communication (GJIC) between cardiomyocytes is essential for synchronous heart contraction and relies on connexin-containing channels. Connexin 43 (Cx43) is a major component involved in GJIC in heart tissue, and its abnormal expression is closely associated with various cardiac diseases. Silica nanoparticles (SNPs) are known to induce cardiovascular toxicity. However, the mechanisms through which GJIC plays a role in cardiomyocytes apoptosis induced by SNPs remain unknown. The aim of the present study is to determine whether SNPs-decreased GJIC promotes apoptosis in rat cardiomyocytes cell line (H9c2 cells) via the mitochondrial pathway using CCK-8 Kit, scrape-loading dye transfer technique, Annexin V/PI double-staining assays, and Western blot analysis. The results showed that SNPs elicited cytotoxicity in H9c2 cells in a time- and concentration-dependent manner. SNPs also reduced GJIC in H9c2 cells in a concentration-dependent manner through downregulation of Cx43 and upregulation of P-Cx43. Inhibition of gap junctions by gap junction blocker carbenoxolone disodium resulted in decreased survival and increased apoptosis, whereas enhancement of the gap junctions by retinoic acid led to enhanced survival but decreased apoptosis. Furthermore, SNPs-induced apoptosis through the disrupted functional gap junction was correlated with abnormal expressions of the proteins involved in the mitochondrial pathway-related apoptosis such as Bcl-2/Bax, cytochrome C, Caspase-9, and Caspase-3. Taken together, our results provide the first evidence that SNPs-decreased GJIC promotes apoptosis in cardiomyocytes via the mitochondrial pathway. In addition, downregulation of GJIC by SNPs in cardiomyocytes is mediated through downregulation of Cx43 and upregulation of P-Cx43. These results suggest that in rat cardiomyocytes cell line, GJIC plays a protective role in SNPs-induced apoptosis and that GJIC may be one of the targets for SNPs-induced biological

Cardiomyocyte H9c2 cells present a valuable alternative to fish lethal testing for azoxystrobin.

PubMed

Rodrigues, Elsa T; Pardal, Miguel Â; Laizé, Vincent; Cancela, M Leonor; Oliveira, Paulo J; Serafim, Teresa L

2015-11-01

The present study aims at identifying, among six mammalian and fish cell lines, a sensitive cell line whose in vitro median inhibitory concentration (IC50) better matches the in vivo short-term Sparus aurata median lethal concentration (LC50). IC50s and LC50 were assessed after exposure to the widely used fungicide azoxystrobin (AZX). Statistical results were relevant for most cell lines after 48 h of AZX exposure, being H9c2 the most sensitive cells, as well as the ones which provided the best prediction of fish toxicity, with a LC50,96h/IC50,48h = 0.581. H9c2 cell proliferation upon 72 h of AZX exposure revealed a LC50,96h/IC50,72h = 0.998. Therefore, identical absolute sensitivities were attained for both in vitro and in vivo assays. To conclude, the H9c2 cell-based assay is reliable and represents a suitable ethical alternative to conventional fish assays for AZX, and could be used to get valuable insights into the toxic effects of other pesticides. Copyright © 2015 Elsevier Ltd. All rights reserved.

BAG3 regulates contractility and Ca(2+) homeostasis in adult mouse ventricular myocytes.

PubMed

Feldman, Arthur M; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D; Tilley, Douglas G; Gao, Erhe; Hoffman, Nicholas E; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y

2016-03-01

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na(+)-K(+)-ATPase and L-type Ca(2+) channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca(2+) channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca(2+)]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca(2+) current (ICa) and sarcoplasmic reticulum (SR) Ca(2+) content but not Na(+)/Ca(2+) exchange current (INaCa) or SR Ca(2+) uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca(2+) entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca(2+) channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. Copyright © 2016 Elsevier Ltd. All rights

BAG3 regulates contractility and Ca2+ homeostasis in adult mouse ventricular myocytes

PubMed Central

Feldman, Arthur M.; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D.; Tilley, Douglas G.; Gao, Erhe; Hoffman, Nicholas E.; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J.; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y.

2016-01-01

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na+-K+-ATPase and L-type Ca2+ channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca2+ channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca2+]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ content but not Na+/Ca2+ exchange current (INaCa) or SR Ca2+ uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyrl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca2+ entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca2+ channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. PMID:26796036

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes

PubMed Central

Santhosh, KT; Elkhateeb, O; Nolette, N; Outbih, O; Halayko, AJ; Dakshinamurti, S

2011-01-01

BACKGROUND AND PURPOSE Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor–mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. EXPERIMENTAL APPROACH We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca2+ response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. KEY RESULTS Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. CONCLUSIONS AND IMPLICATIONS TP receptor sensitivity and EC50 for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca2+ mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. PMID:21385177

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes.

PubMed

Santhosh, K T; Elkhateeb, O; Nolette, N; Outbih, O; Halayko, A J; Dakshinamurti, S

2011-07-01

Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Phase Equilibria and Transport Properties in the Systems AgNO3/RCN/H2O. R = CH3, C2H5, C3H7, C4H,, C6H5, and C6H5CH2

NASA Astrophysics Data System (ADS)

Das, Surjya P.; Wittekopf, Burghard; Weil, Konrad G.

1988-11-01

Silver nitrate can form homogeneous liquid phases with some organic nitriles and water, even when there is no miscibility between the pure liquid components. We determined the shapes of the single phase regions in the ternary phase diagram for the following systems: silver nitrate /RCN /H2O with R =CH3, C3H7, C6H5, and C6H5CH2 at room temperature and for R =C6H5 also at 60 °C and O °C. Furthermore we studied kinematic viscosities, electrical conductivities, and densities of mixtures containing silver nitrate, RCN, and water with the mole ratios X /4 /1 (0.2≦ X ≦S 3.4). In these cases also R = C2H5 and C4H9 were studied. The organic nitriles show different dependences of viscosity and conductivity on the silver nitrate content from the aliphatic ones.

STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis.

PubMed

Chen, Lei; Zhao, Lin; Samanta, Anweshan; Mahmoudi, Seyed Morteza; Buehler, Tanner; Cantilena, Amy; Vincent, Robert J; Girgis, Magdy; Breeden, Joshua; Asante, Samuel; Xuan, Yu-Ting; Dawn, Buddhadeb

2017-01-01

Signal transducers and activators of transcription 3 (STAT3) is known to participate in various cardiovascular signal transduction pathways, including those responsible for cardiac hypertrophy and cytoprotection. However, the role of STAT3 signaling in cardiomyocyte autophagy remains unclear. We tested the hypothesis that Angiotensin II (Ang II)-induced cardiomyocyte hypertrophy is effected, at least in part, through STAT3-mediated inhibition of cellular autophagy. In H9c2 cells, Ang II treatment resulted in STAT3 activation and cellular hypertrophy in a dose-dependent manner. Ang II enhanced autophagy, albeit without impacting AMPKα/mTOR signaling or cellular ADP/ATP ratio. Pharmacologic inhibition of STAT3 with WP1066 suppressed Ang II-induced myocyte hypertrophy and mRNA expression of hypertrophy-related genes ANP and β-MHC. These molecular events were recapitulated in cells with STAT3 knockdown. Genetic or pharmacologic inhibition of STAT3 significantly increased myocyte ADP/ATP ratio and enhanced autophagy through AMPKα/mTOR signaling. Pharmacologic activation and inhibition of AMPKα attenuated and exaggerated, respectively, the effects of Ang II on ANP and β-MHC gene expression, while concomitant inhibition of STAT3 accentuated the inhibition of hypertrophy. Together, these data indicate that novel nongenomic effects of STAT3 influence myocyte energy status and modulate AMPKα/mTOR signaling and autophagy to balance the transcriptional hypertrophic response to Ang II stimulation. These findings may have significant relevance for various cardiovascular pathological processes mediated by Ang II signaling.

STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis

PubMed Central

Samanta, Anweshan; Mahmoudi, Seyed Morteza; Buehler, Tanner; Cantilena, Amy; Vincent, Robert J.; Girgis, Magdy; Breeden, Joshua; Asante, Samuel; Xuan, Yu-Ting

2017-01-01

Signal transducers and activators of transcription 3 (STAT3) is known to participate in various cardiovascular signal transduction pathways, including those responsible for cardiac hypertrophy and cytoprotection. However, the role of STAT3 signaling in cardiomyocyte autophagy remains unclear. We tested the hypothesis that Angiotensin II (Ang II)-induced cardiomyocyte hypertrophy is effected, at least in part, through STAT3-mediated inhibition of cellular autophagy. In H9c2 cells, Ang II treatment resulted in STAT3 activation and cellular hypertrophy in a dose-dependent manner. Ang II enhanced autophagy, albeit without impacting AMPKα/mTOR signaling or cellular ADP/ATP ratio. Pharmacologic inhibition of STAT3 with WP1066 suppressed Ang II-induced myocyte hypertrophy and mRNA expression of hypertrophy-related genes ANP and β-MHC. These molecular events were recapitulated in cells with STAT3 knockdown. Genetic or pharmacologic inhibition of STAT3 significantly increased myocyte ADP/ATP ratio and enhanced autophagy through AMPKα/mTOR signaling. Pharmacologic activation and inhibition of AMPKα attenuated and exaggerated, respectively, the effects of Ang II on ANP and β-MHC gene expression, while concomitant inhibition of STAT3 accentuated the inhibition of hypertrophy. Together, these data indicate that novel nongenomic effects of STAT3 influence myocyte energy status and modulate AMPKα/mTOR signaling and autophagy to balance the transcriptional hypertrophic response to Ang II stimulation. These findings may have significant relevance for various cardiovascular pathological processes mediated by Ang II signaling. PMID:28686615

Direct toxic effects of aqueous extract of cigarette smoke on cardiac myocytes at clinically relevant concentrations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamada, Shigeyuki; Zhang Xiuquan; Kadono, Toshie

Aims: Our goal was to determine if clinically relevant concentrations of aqueous extract of cigarette smoke (CSE) have direct deleterious effects on ventricular myocytes during simulated ischemia, and to investigate the mechanisms involved. Methods: CSE was prepared with a smoking chamber. Ischemia was simulated by metabolic inhibition (MI) with cyanide (CN) and 0 glucose. Adult rabbit and mouse ventricular myocyte [Ca{sup 2+}]{sub i} was measured by flow cytometry using fluo-3. Mitochondrial [Ca{sup 2+}] was measured with confocal microscopy, and Rhod-2 fluorescence. The mitochondrial permeability transition (MPT) was detected by TMRM fluorescence and myocyte contracture. Myocyte oxidative stress was quantified bymore » dichlorofluorescein (DCF) fluorescence with confocal microscopy. Results: CSE 0.1% increased myocyte contracture caused by MI. The nicotine concentration (HPLC) in 0.1% CSE was 15 ng/ml, similar to that in humans after smoking cigarettes. CSE 0.1% increased mitochondrial Ca{sup 2+} uptake, and increased the susceptibility of mitochondria to the MPT. CSE 0.1% increased DCF fluorescence in isolated myocytes, and increased [Ca{sup 2+}]{sub i} in paced myocytes exposed to 2.0 mM CN, 0 glucose (P-MI). These effects were inhibited by the superoxide scavenger Tiron. The effect of CSE on [Ca{sup 2+}]{sub i} during P-MI was also prevented by ranolazine. Conclusions: CSE in clinically relevant concentrations increases myocyte [Ca{sup 2+}]{sub i} during simulated ischemia, and increases myocyte susceptibility to the MPT. These effects appear to be mediated at least in part by oxidative radicals in CSE, and likely contribute to the effects of cigarette smoke to increase myocardial infarct size, and to decrease angina threshold.« less

Novel mechanisms for caspase inhibition protecting cardiac function with chronic pressure overload

PubMed Central

Vatner, Stephen F.; Yan, Lin; Gao, Shumin; Yoon, Seunghun; Lee, Grace Jung Ah; Xie, Lai-Hua; Kitsis, Richard N.; Vatner, Dorothy E.

2013-01-01

Myocyte apoptosis is considered a major mechanism in the pathogenesis of heart failure. Accordingly, manipulations that inhibit apoptosis are assumed to preserve cardiac function by maintaining myocyte numbers. We tested this assumption by examining the effects of caspase inhibition (CI) on cardiac structure and function in C57BL/6 mouse with pressure overload model induced by transverse aortic constriction (TAC). CI preserved left ventricular (LV) function following TAC compared with the vehicle. TAC increased apoptosis in non-myocytes more than in myocytes and these increases were blunted more in non-myocytes by CI. Total myocyte number, however, did not differ significantly among control and TAC groups and there was no correlation between myocyte number and apoptosis, but there was a strong correlation between myocyte number and an index of myocyte proliferation, Ki67-positive myocytes. Despite comparable pressure gradients, LV hypertrophy was less in the CI group, likely attributable to decreased wall stress. Since changes in myocyte numbers did not account for protection from TAC, several other CI-mediated mechanisms were identified including: (a) lessening of TAC-induced fibrosis, (b) augmentation of isolated myocyte contractility, and (c) increased angiogenesis and Ki67-positive myocytes, which were due almost entirely to the non-myocyte apoptosis, but not myocyte apoptosis, with CI. CI maintained LV function following TAC not by protecting against myocyte loss, but rather by augmenting myocyte contractile function, myocyte proliferation, and angiogenesis resulting in reduced LV wall stress, hypertrophy, and fibrosis. PMID:23277091

B-type natriuretic peptide attenuates endoplasmic reticulum stress in H9c2 cardiomyocytes underwent hypoxia/reoxygenation injury under high glucose/high fat conditions.

PubMed

Chang, Pan; Zhang, Mingyang; Zhang, Xiaomeng; Li, Guohua; Hu, Haiyan; Wu, Juan; Wang, Xihui; Yang, Zihua; Zhang, Jing; Chen, Weiguo; Ren, Minggang; Li, Xin; Zhu, Miaozhang; Chen, Baoying; Yu, Jun

2018-04-22

Exogenously administered B-type natriuretic peptide (BNP) has been shown to provide cardioprotection against various heart diseases. However, the underlying mechanisms remain elusive. This study explores whether BNP exerts its cardioprotection against hypoxia/reoxygenation (H/R) injury under high glucose/high fat (HG/HF) conditions in cardiac H9c2 cells and uncovers the underlying mechanisms. Our data revealed that BNP significantly increased the cell viability and decreased the release of lactate dehydrogenase (LDH) and creatine kinase (CK), with a maximal effect at the BNP concentration of 10 -7  mol/L. In addition, by analyzing the activation of cleaved caspase-3 and by Annexin V-FITC/PI staining, we showed that BNP attenuated H/R-induced cell apoptosis in HG/HF conditions. Western blot analysis showed enhanced phosphorylation of protein kinase RNA (PKR)-like endoplastmic reticulum (ER) kinase (PERK) and eukaryotic initiation factor 2α (eIF2α)(one of the three main signaling pathways in endoplastmic reticulum (ER) stress), and increased expression of GRP78 and CHOP proteins (ER stress-related proteins) in H9c2 cells which underwent H/R in HG/HF conditions. Treatment with BNP or 8-Br-cGMP (an analog of cGMP) reversed this activation. However, this effect was significantly weakened by KT-5823, a selective cGMP-dependent protein kinase G (PKG) inhibitor. In addition, similar to BNP, treatment with a specific inhibitor of ER stress tauroursodeoxycholic acid (TUDCA) protected the cells against H/R injury exposed to HG/HF conditions. In conclusion, these findings demonstrated that BNP effectively protected cells against H/R injury under HG/HF conditions by inhibiting the ER stress via activation of the cGMP-PKG signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

MiR-130a inhibition protects rat cardiac myocytes from hypoxia-triggered apoptosis by targeting Smad4.

PubMed

Li, Yuanshi; Du, Yingrong; Cao, Junxian; Gao, Qianping; Li, Hongjuan; Chen, Yangjun; Lu, Nihong

2018-02-05

Cardiomyocyte death facilitates the pathological process underlying ischemic heart diseases, such as myocardial infarction. Emerging evidence suggests that microRNAs play a critical role in the pathological process underlying myocardial infarction by regulating cardiomyocyte apoptosis. However, the relevance of miR-130a in regulating cardiomyocyte apoptosis and the mechanism of regulation is still uncertain. This study aimed to explore the regulatory effect of miR-130a on hypoxic cardiomyocyte apoptosis. The expression of miR-130a was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell survival was determined by the MTT assay. The lactate dehydrogenase (LDH) assay was performed to determine the severity of hypoxia-induced cell injury. Apoptosis was assessed via caspase-3 analysis. Protein expression level was determined by Western blotting. The genes targeted by miR-130a were predicted using bioinformatics and were validated via the dual-luciferase reporter assay. We found that miR-130a expression was greatly increased in hypoxic cardiac myocytes, and that the downregulation of miR-130a effectively shielded cardiac myocytes from hypoxia-triggered apoptosis. The results of our bioinformatic analysis predicted the Smad4 gene to be the target of miR-130a. This finding was validated through the Western blot assay, dual-luciferase reporter gene assay, and qRT-PCR. MiR-130a inhibition significantly promoted the activation of Smad4 in hypoxic cardiomyocytes. Interestingly, knockdown of Smad4 markedly reversed the protective effects induced by miR-130a inhibition. Moreover, we found that the inhibition of miR-130a promoted the activation of TGF-β signaling. Blocking Smad4 signaling significantly abrogated the protective effects of miR-130a inhibition. Overall, these findings indicate that inhibition of miR-130a, which targets the Smad4 gene, shields cardiac myocytes from hypoxic apoptosis. This study offers a novel perspective of the

5-AIQ inhibits H2O2-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3β signaling pathway in H9c2 cardiomyocytes.

PubMed

Park, Eun-Seok; Kang, Jun Chul; Kang, Do-Hyun; Jang, Yong Chang; Yi, Kyu Yang; Chung, Hun-Jong; Park, Jong Seok; Kim, Bokyung; Feng, Zhong-Ping; Shin, Hwa-Sup

2013-04-01

Poly(adenosine 5'-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H2O2-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxide dismutase and catalase accompanied the protective effect of 5-AIQ on H2O2-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H2O2-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3β (GSK-3β), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3β activation. In addition, inhibiting the Akt/GSK-3β pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H2O2-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3β pathway. Copyright © 2013 Elsevier Inc. All rights reserved.

The involvement of protein kinase C-ε in isoflurane induced preconditioning of human embryonic stem cell--derived Nkx2.5(+) cardiac progenitor cells.

PubMed

Song, In-Ae; Oh, Ah-Young; Kim, Jin-Hee; Choi, Young-Min; Jeon, Young-Tae; Ryu, Jung-Hee; Hwang, Jung-Won

2016-02-20

Anesthetic preconditioning can improve survival of cardiac progenitor cells exposed to oxidative stress. We investigated the role of protein kinase C and isoform protein kinase C-ε in isoflurane-induced preconditioning of cardiac progenitor cells exposed to oxidative stress. Cardiac progenitor cells were obtained from undifferentiated human embryonic stem cells. Immunostaining with anti-Nkx2.5 was used to confirm the differentiated cardiac progenitor cells. Oxidative stress was induced by H2O2 and FeSO4. For anesthetic preconditioning, cardiac progenitor cells were exposed to 0.25, 0.5, and 1.0 mM of isoflurane. PMA and chelerythrine were used for protein kinase C activation and inhibition, while εψRACK and εV1-2 were used for protein kinase C -ε activation and inhibition, respectively. Isoflurane-preconditioning decreased the death rate of Cardiac progenitor cells exposed to oxidative stress (death rates isoflurane 0.5 mM 12.7 ± 9.3%, 1.0 mM 12.0 ± 7.7% vs. control 31.4 ± 10.2%). Inhibitors of both protein kinase C and protein kinase C -ε abolished the preconditioning effect of isoflurane 0.5 mM (death rates 27.6 ± 13.5% and 25.9 ± 8.7% respectively), and activators of both protein kinase C and protein kinase C - ε had protective effects from oxidative stress (death rates 16.0 ± 3.2% and 10.6 ± 3.8% respectively). Both PKC and PKC-ε are involved in isoflurane-induced preconditioning of human embryonic stem cells -derived Nkx2.5(+) Cardiac progenitor cells under oxidative stress.

The pH dependence of cocaine interaction with cardiac sodium channels.

PubMed

Crumb, W J; Clarkson, C W

1995-09-01

Previous in vitro and in vivo studies have provided evidence implicating cocaine block of cardiac sodium channels as a putative mechanism for cocaine-induced arrhythmias and sudden death. Cocaine also has been shown to cause seizures which can result in respiratory and/or metabolic acidosis. In this study we investigated how changes in both internal pH (pHi) and external pH (pHo) over the range of 6.6 to 9.2 modify the sodium channel blocking properties of cocaine in isolated guinea pig ventricular myocytes by using the whole-cell variant of the patch clamp technique. Use-dependent block produced by a train of 1-sec pulses to -20 mV was not affected by changes in pHi, but both the amplitude and time constant for approaching steady-state block were significantly affected by changes in pHo. Characterization of the time course of cocaine binding during a depolarizing pulse indicated that the kinetics of drug interaction with inactivated channels were independent of pHi, but were significantly affected by changes in pHo. The rate of recovery from channel block at a holding potential of -140 mV also was independent of pHi, but strongly dependent on pHo, with the unblocking time constant decreasing exponentially as pHo was increased. The results of this study indicate that cocaine's effect on cardiac sodium channels can be modulated significantly by changes in pHo, and provide further support for previously poorly tested assumptions of the modulated receptor hypothesis.

5-AIQ inhibits H{sub 2}O{sub 2}-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3β signaling pathway in H9c2 cardiomyocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Eun-Seok; Kang, Jun Chul; Kang, Do-Hyun

2013-04-01

Poly(adenosine 5′-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H{sub 2}O{sub 2}-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxidemore » dismutase and catalase accompanied the protective effect of 5-AIQ on H{sub 2}O{sub 2}-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H{sub 2}O{sub 2}-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3β (GSK-3β), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3β activation. In addition, inhibiting the Akt/GSK-3β pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H{sub 2}O{sub 2}-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3β pathway. - Highlights: ► 5-AIQ, a PARP inhibitor, decreased H{sub 2}O{sub 2}-induced H9c2 cell death and apoptosis. ► 5-AIQ upregulated antioxidant Mn-SOD and catalase, while decreasing ROS production. ► 5-AIQ decreased H{sub 2}O{sub 2}-induced increase in cleaved caspase-3 and Bax and decrease in Bcl2. ► 5-AIQ activated Akt and

Combined use of UV-labile calcium chelators and calcium-sensitive dyes in a microscope with two light sources influencing different regions in a group of coordinated contracting cardiac myocytes

NASA Astrophysics Data System (ADS)

Pilarczyk, Goetz; Greulich, Karl-Otto

1997-12-01

The coordination of excitation in a biological system of cells such as cardiac myocytes in heart tissue has crucial influence on the function of the entire organ. This coordinated behavior can be visualized in a small group of embryonic cardiac myocytes derived from the hearts of unborn chicken. Loaded with a calcium sensitive dye the excitation can be imaged via the occurring transient rise in cytosolic calcium concentration. It can be shown that in regions with physiological or morphological restrictions the transient rise in cytosolic calcium occurs with a temporal delay compared to the ordinary array of coupled myocytes. The height of the transient rise of cytosolic calcium is related to the ability of the individual cell to participate in the coordinated contraction. The free cytosolic calcium concentration is decreased with the UV-labile calcium, chelator diazo-2. Our setup allows to decrease the free cytosolic calcium in a single cell of the contracting array of cells. This allows us to introduce mismatches in selected regions of the coordinated contraction and to visualize the effects simultaneously.

Na/K pump inactivation, subsarcolemmal Na measurements, and cytoplasmic ion turnover kinetics contradict restricted Na spaces in murine cardiac myocytes

PubMed Central

Lu, Fang-Min

2017-01-01

Decades ago, it was proposed that Na transport in cardiac myocytes is modulated by large changes in cytoplasmic Na concentration within restricted subsarcolemmal spaces. Here, we probe this hypothesis for Na/K pumps by generating constitutive transsarcolemmal Na flux with the Na channel opener veratridine in whole-cell patch-clamp recordings. Using 25 mM Na in the patch pipette, pump currents decay strongly during continuous activation by extracellular K (τ, ∼2 s). In contradiction to depletion hypotheses, the decay becomes stronger when pump currents are decreased by hyperpolarization. Na channel currents are nearly unchanged by pump activity in these conditions, and conversely, continuous Na currents up to 0.5 nA in magnitude have negligible effects on pump currents. These outcomes are even more pronounced using 50 mM Li as a cytoplasmic Na congener. Thus, the Na/K pump current decay reflects mostly an inactivation mechanism that immobilizes Na/K pump charge movements, not cytoplasmic Na depletion. When channel currents are increased beyond 1 nA, models with unrestricted subsarcolemmal diffusion accurately predict current decay (τ ∼15 s) and reversal potential shifts observed for Na, Li, and K currents through Na channels opened by veratridine, as well as for Na, K, Cs, Li, and Cl currents recorded in nystatin-permeabilized myocytes. Ion concentrations in the pipette tip (i.e., access conductance) track without appreciable delay the current changes caused by sarcolemmal ion flux. Importantly, cytoplasmic mixing volumes, calculated from current decay kinetics, increase and decrease as expected with osmolarity changes (τ >30 s). Na/K pump current run-down over 20 min reflects a failure of pumps to recover from inactivation. Simulations reveal that pump inactivation coupled with Na-activated recovery enhances the rapidity and effectivity of Na homeostasis in cardiac myocytes. In conclusion, an autoregulatory mechanism enhances cardiac Na/K pump activity when

Acute desensitization of acetylcholine and endothelin-1 activated inward rectifier K+ current in myocytes from the cardiac atrioventricular node.

PubMed

Choisy, Stéphanie C M; James, Andrew F; Hancox, Jules C

2012-07-06

The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits 'fade' or desensitization in the AVN, as established for the heart's primary pacemaker--the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37 °C. 0.1-1 μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300 nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1 μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0 s and τs 76.9 s at -120 mV; 1 μM ACh). 10 nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6 s at -120 mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization. Copyright © 2012 Elsevier Inc. All rights reserved.

The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca2+ leak in ventricular myocytes.

PubMed

Bovo, Elisa; Huke, Sabine; Blatter, Lothar A; Zima, Aleksey V

2017-03-01

Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca 2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca 2+ ] and intra-SR [Ca 2+ ] SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10μM) increased Ca 2+ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca 2+ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10μM) abolished the cAMP-dependent increase of SR Ca 2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca 2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca 2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca 2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca 2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca 2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca 2+ leak. Copyright © 2017 Elsevier

Statistical Metamodeling and Sequential Design of Computer Experiments to Model Glyco-Altered Gating of Sodium Channels in Cardiac Myocytes.

PubMed

Du, Dongping; Yang, Hui; Ednie, Andrew R; Bennett, Eric S

2016-09-01

Glycan structures account for up to 35% of the mass of cardiac sodium ( Nav ) channels. To question whether and how reduced sialylation affects Nav activity and cardiac electrical signaling, we conducted a series of in vitro experiments on ventricular apex myocytes under two different glycosylation conditions, reduced protein sialylation (ST3Gal4(-/-)) and full glycosylation (control). Although aberrant electrical signaling is observed in reduced sialylation, realizing a better understanding of mechanistic details of pathological variations in INa and AP is difficult without performing in silico studies. However, computer model of Nav channels and cardiac myocytes involves greater levels of complexity, e.g., high-dimensional parameter space, nonlinear and nonconvex equations. Traditional linear and nonlinear optimization methods have encountered many difficulties for model calibration. This paper presents a new statistical metamodeling approach for efficient computer experiments and optimization of Nav models. First, we utilize a fractional factorial design to identify control variables from the large set of model parameters, thereby reducing the dimensionality of parametric space. Further, we develop the Gaussian process model as a surrogate of expensive and time-consuming computer models and then identify the next best design point that yields the maximal probability of improvement. This process iterates until convergence, and the performance is evaluated and validated with real-world experimental data. Experimental results show the proposed algorithm achieves superior performance in modeling the kinetics of Nav channels under a variety of glycosylation conditions. As a result, in silico models provide a better understanding of glyco-altered mechanistic details in state transitions and distributions of Nav channels. Notably, ST3Gal4(-/-) myocytes are shown to have higher probabilities accumulated in intermediate inactivation during the repolarization and yield a

Difference in propagation of Ca2+ release in atrial and ventricular myocytes.

PubMed

Tanaami, Takeo; Ishida, Hideyuki; Seguchi, Hidetaka; Hirota, Yuki; Kadono, Toshie; Genka, Chokoh; Nakazawa, Hiroe; Barry, William H

2005-04-01

Intracellular [Ca2+] ([Ca2+]i) was imaged in atrial and ventricular rat myocytes by means of a high-speed Nipkow confocal microscope. Atrial myocytes with an absent t-tubule system on 8-di- ANEPPS staining showed an initial rise in Ca2+ at the periphery of the cell, which propagated to the interior of the cell. Ventricular myocytes showed a uniform rise in [Ca2+]i after electrical stimulation, consistent with a prominent t-tubular network. In atrial myocytes, there was a much shorter time between the peak of the [Ca2+]i transient and the peak contraction as compared to ventricular myocytes. A regional release of Ca2+ induced by an exposure of one end of the myocyte to caffeine with a rapid solution switcher resulted in a uniform propagation of Ca2+ down the length of the cell in atrial myocytes, but we found no propagation in ventricular myocytes. A staining with rhodamine 123 indicated a much greater density of mitochondria in ventricular myocytes than in atrial myocytes. Thus the atrial myocytes display a lack of "local control" of Ca2+ release, with propagation after the Ca2+ release at the periphery induced by stimulation or at one end of the cell induced by exposure to caffeine. Ventricular myocytes showed the presence of local control, as indicated by an absence of the propagation of a local caffeine-induced Ca2+ transient. We suggest that this finding, as well as a reduced delay between the peak of the [Ca2+]i transient and the peak shortening in atrial myocytes, could be due in part to reduced Ca2+ buffering provided by mitochondria in atrial myocytes as opposed to ventricular myocytes.