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Background We reported 3 novel nonsynonymous single nucleotide variants of Bcl2-associated athanogene 3 (BAG3) in African Americans with heart failure (HF) that are associated with a 2-fold increase in cardiac events (HF hospitalization, heart transplantation, or death). Methods and Results We expressed BAG3 variants (P63A, P380S, and A479V) via adenovirus-mediated gene transfer in adult left ventricular myocytes isolated from either wild-type (WT) or cardiac-specific BAG3 haploinsufficient (cBAG3+/−) mice: the latter to simulate the clinical situation in which BAG3 variants are only found on 1 allele. Compared with WT myocytes, cBAG3+/− myocytes expressed approximately 50% of endogenous BAG3 levels and exhibited decreased [Ca2+]i and contraction amplitudes after isoproterenol owing to decreased L-type Ca2+ current. BAG3 repletion with WT BAG3 but not P380S, A479V, or P63A/P380S variants restored contraction amplitudes in cBAG3+/− myocytes to those measured in WT myocytes, suggesting excitation–contraction abnormalities partly account for HF in patients harboring these mutants. Because P63A is near the WW domain (residues 21–55) and A479V is in the BAG domain (residues 420–499), we expressed BAG3 deletion mutants (Δ1–61 and Δ421–575) in WT myocytes and demonstrated that the BAG but not the WW domain was involved in enhancement of excitation–contraction by isoproterenol. Conclusions The BAG3 variants contribute to HF in African American patients partly by decreasing myocyte excitation–contraction under stress, and that both the BAG and PXXP domains are involved in mediating β-adrenergic responsiveness in myocytes.

We have previously reported that methylene blue (MB) can counteract hydrogen sulfide (H2S) intoxication-induced circulatory failure. Because of the multifarious effects of high concentrations of H2S on cardiac function, as well as the numerous properties of MB, the nature of this interaction, if any, remains uncertain. The aim of this study was to clarify 1) the effects of MB on H2S-induced cardiac toxicity and 2) whether L-type Ca2+channels, one of the targets of H2S, could transduce some of the counteracting effects of MB. In sedated rats, H2S infused at a rate that would be lethal within 5 min (24 μM·kg−1·min−1), produced a rapid fall in left ventricle ejection fraction, determined by echocardiography, leading to a pulseless electrical activity. Blood concentrations of gaseous H2S reached 7.09 ± 3.53 μM when cardiac contractility started to decrease. Two to three injections of MB (4 mg/kg) transiently restored cardiac contractility, blood pressure, and V̇o2, allowing the animals to stay alive until the end of H2S infusion. MB also delayed PEA by several minutes following H2S-induced coma and shock in unsedated rats. Applying a solution containing lethal levels of H2S (100 μM) on isolated mouse cardiomyocytes significantly reduced cell contractility, intracellular calcium concentration ([Ca2+]i) transient amplitudes, and L-type Ca2+currents ( ICa) within 3 min of exposure. MB (20 mg/l) restored the cardiomyocyte function, ([Ca2+]i) transient, and ICa. The present results offer a new approach for counteracting H2S toxicity and potentially other conditions associated with acute inhibition of L-type Ca2+channels.

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.

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid protein that is found predominantly in the heart, skeletal muscle and many cancers. Deletions and truncations in BAG3 that result in haplo-insufficiency have been associated with the development of dilated cardiomyopathy. To study the cellular and molecular events attributable to BAG3 haplo-insufficiency we generated a mouse in which one allele of BAG3 was flanked by loxP recombination sites (BAG3(fl/+)). Mice were crossed with α-MHC-Cre mice in order to generate mice with cardiac-specific haplo-insufficiency (cBAG3(+/−)) and underwent bi-weekly echocardiography to assess their cardiac phenotype. By ten weeks of age, cBAG3(+/−) mice demonstrated increased heart size and diminished left ventricular ejection fraction when compared with non-transgenic littermates (Cre(−/−)BAG3(fl/+)). Contractility in adult myocytes isolated from cBAG3(+/−) mice were similar to those isolated from control mice at baseline, but showed a significantly decreased response to adrenergic stimulation. Intracellular calcium ([Ca(2+)](i)) transient amplitudes in myocytes isolated from cBAG3(+/−) mice were also similar to myocytes isolated from control mice at baseline but were significantly lower than myocytes from control mice in their response to isoproterenol. BAG3 haplo-insufficiency was also associated with decreased autophagy flux and increased apoptosis. Taken together, these results suggest that mice in which BAG3 has been deleted from a single allele provide a model that mirrors the biology seen in patients with heart failure and BAG3 haplo-insufficiency.

Since highly active antiretroviral therapy improved long-term survival of acquired immunodeficiency syndrome (AIDS) patients, AIDS cardiomyopathy has become an increasingly relevant clinical problem. We used human immunodeficiency virus (HIV)-1 transgenic (Tg26) mouse to explore molecular mechanisms of AIDS cardiomyopathy. Tg26 mice had significantly lower left ventricular (LV) mass and smaller end-diastolic and end-systolic LV volumes. Under basal conditions, cardiac contractility and relaxation and single myocyte contraction dynamics were not different between wild-type (WT) and Tg26 mice. Ten days after open heart surgery, contractility and relaxation remained significantly depressed in Tg26 hearts, suggesting that Tg26 mice did not tolerate surgical stress well. To simulate heart failure in which expression of Bcl2-associated athanogene 3 (BAG3) is reduced, we down-regulated BAG3 by small hairpin ribonucleic acid in WT and Tg26 hearts. BAG3 down-regulation significantly reduced contractility in Tg26 hearts. BAG3 overexpression rescued contractile abnormalities in myocytes expressing the HIV-1 protein Tat. We conclude: (i) Tg26 mice exhibit normal contractile function at baseline; (ii) Tg26 mice do not tolerate surgical stress well; (iii) BAG3 down-regulation exacerbated cardiac dysfunction in Tg26 mice; (iv) BAG3 overexpression rescued contractile abnormalities in myocytes expressing HIV-1 protein Tat; and (v) BAG3 may occupy a role in pathogenesis of AIDS cardiomyopathy.

Phospholemman (PLM), when phosphorylated at Ser68, inhibits cardiac Na+/Ca2+exchanger 1 (NCX1) and relieves its inhibition on Na+-K+-ATPase. We have engineered mice in which expression of the phosphomimetic PLM S68E mutant was induced when dietary doxycycline was removed at 5 wk. At 8–10 wk, compared with noninduced or wild-type hearts, S68E expression in induced hearts was ∼35–75% that of endogenous PLM, but protein levels of sarco(endo)plasmic reticulum Ca2+-ATPase, α1- and α2-subunits of Na+-K+-ATPase, α1c-subunit of L-type Ca2+channel, and phosphorylated ryanodine receptor were unchanged. The NCX1 protein level was increased by ∼47% but the NCX1 current was depressed by ∼34% in induced hearts. Isoproterenol had no effect on NCX1 currents but stimulated Na+-K+-ATPase currents equally in induced and noninduced myocytes. At baseline, systolic intracellular Ca2+concentrations ([Ca2+]i), sarcoplasmic reticulum Ca2+contents, and [Ca2+]itransient and contraction amplitudes were similar between induced and noninduced myocytes. Isoproterenol stimulation resulted in much higher systolic [Ca2+]i, sarcoplasmic reticulum Ca2+content, and [Ca2+]itransient and contraction amplitudes in induced myocytes. Echocardiography and in vivo close-chest catheterization demonstrated similar baseline myocardial function, but isoproterenol induced a significantly higher +dP/d t in induced compared with noninduced hearts. In contrast to the 50% mortality observed in mice constitutively overexpressing the S68E mutant, induced mice had similar survival as wild-type and noninduced mice. After ischemia-reperfusion, despite similar areas at risk and left ventricular infarct sizes, induced mice had significantly higher +dP/d t and −dP/d t and lower perioperative mortality compared with noninduced mice. We propose that phosphorylated PLM may be a novel therapeutic target in ischemic heart disease.

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.

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca2+ influx that was dependent on extracellular Ca2+, was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/d t between WT and KO hearts. At 2–3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/d t compared with WT hearts. Compared with WT I/R myocytes, expression of the Na+/Ca2+ exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na+-K+-ATPase and Na+ pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca2+ concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.

Phospholemman (PLM) regulates [Na+]i, [Ca2+]i and contractility through its interactions with Na+‐K+‐ATPase (NKA) and Na+/Ca2+ exchanger (NCX1) in the heart. Both expression and phosphorylation of PLM are altered after myocardial infarction (MI) and heart failure. We tested the hypothesis that absence of PLM regulation of NKA and NCX1 in PLM‐knockout (KO) mice is detrimental. Three weeks after MI, wild‐type (WT) and PLM‐KO hearts were similarly hypertrophied. PLM expression was lower but fractional phosphorylation was higher in WT‐MI compared to WT‐sham hearts. Left ventricular ejection fraction was severely depressed in WT‐MI but significantly less depressed in PLM‐KO‐MI hearts despite similar infarct sizes. Compared with WT‐sham myocytes, the abnormal [Ca2+], transient and contraction amplitudes observed in WT‐MI myocytes were ameliorated by genetic absence of PLM. In addition, NCX1 current was depressed in WT‐MI but not in PLM‐KO‐MI myocytes. Despite improved myocardial and myocyte performance, PLM‐KO mice demonstrated reduced survival after MI. Our findings indicate that alterations in PLM expression and phosphorylation are important adaptations post‐MI, and that complete absence of PLM regulation of NKA and NCX1 is detrimental in post‐MI animals. Clin Trans Sci 2012; Volume #: 1–8

Expression and activity of cardiac Na+/Ca2+ exchanger (NCX1) are altered in many disease states. We engineered mice in which the phosphomimetic phospholemman S68E mutant (inhibits NCX1 but not Na+-K+-ATPase) was constitutively overexpressed in a cardiac-specific manner (conS68E). At 4–6 wk, conS68E mice exhibited severe bradycardia, ventricular arrhythmias, increased left ventricular (LV) mass, decreased cardiac output (CO), and ∼50% mortality compared with wild-type (WT) littermates. Protein levels of NCX1, calsequestrin, ryanodine receptor, and α1- and α2-subunits of Na+-K+-ATPase were similar, but sarco(endo)plasmic reticulum Ca2+-ATPase was lower, whereas L-type Ca2+ channels were higher in conS68E hearts. Resting membrane potential and action potential amplitude were similar, but action potential duration was dramatically prolonged in conS68E myocytes. Diastolic intracellular Ca2+ ([Ca2+]i) was higher, [Ca2+]i transient and maximal contraction amplitudes were lower, and half-time of [Ca2+]i transient decline was longer in conS68E myocytes. Intracellular Na+ reached maximum within 3 min after isoproterenol addition, followed by decline in WT but not in conS68E myocytes. Na+/Ca2+ exchange, L-type Ca2+, Na+-K+-ATPase, and depolarization-activated K+ currents were decreased in conS68E myocytes. At 22 wk, bradycardia and increased LV mass persisted in conS68E survivors. Despite comparable baseline CO, conS68E survivors at 22 wk exhibited decreased chronotropic, inotropic, and lusitropic responses to isoproterenol. We conclude that constitutive overexpression of S68E mutant was detrimental, both in terms of depressed cardiac function and increased arrhythmogenesis.

Ornithine decarboxylase (ODC), the first enzyme of polyamine metabolism, is rapidly upregulated in response to agents that induce a pathological cardiac hypertrophy. Transgenic mice overexpressing ODC in the heart (MHC-ODC mice) experience a much more dramatic left ventricular hypertrophy in response to β-adrenergic stimulation with isoproterenol (ISO) compared to wild-type (WT) controls. ISO also induced arginase activity in transgenic hearts but not in controls. The current work studies the cooperation between the cardiac polyamines and L-arginine (L-Arg) availability in MHC-ODC mice. Although ISO-induced hypertrophy is well-compensated, MHC-ODC mice administered L-Arg along with ISO showed a rapid onset of systolic dysfunction and died within 48 h. Myocytes isolated from MHC-ODC mice administered L-Arg/ISO exhibited reduced contractility and altered calcium transients, suggesting an alteration in [Ca(2+)] homeostasis, and abbreviated action potential duration, which may contribute to arrhythmogenesis. The already elevated levels of spermidine and spermine were not further altered in MHC-ODC hearts by L-Arg/ISO treatment, suggesting alternative L-Arg utilization pathways lead to dysregulation of intracellular calcium. MHC-ODC mice administered an arginase inhibitor (Nor-NOHA) along with ISO died almost as rapidly as L-Arg/ISO-treated mice, while the iNOS inhibitor S-methyl-isothiourea (SMT) was strongly protective against L-Arg/ISO. These results point to the induction of arginase as a protective response to β-adrenergic stimulation in the setting of high polyamines. Further, NO generated by exogenously supplied L-Arg may contribute to the lethal consequences of L-Arg/ISO treatment. Since considerable variations in human cardiac polyamine and L-Arg content are likely, it is possible that alterations in these factors may influence myocyte contractility.

Phospholemman (PLM), a member of the FXYD family of regulators of ion transport, is a major sarcolemmal substrate for protein kinases A and C in cardiac and skeletal muscle. In the heart, PLM co-localizes and co-immunoprecipitates with Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and L-type Ca(2+) channel. Functionally, when phosphorylated at serine(68), PLM stimulates Na(+)-K(+)-ATPase but inhibits Na(+)/Ca(2+) exchanger in cardiac myocytes. In heterologous expression systems, PLM modulates the gating of cardiac L-type Ca(2+) channel. Therefore, PLM occupies a key modulatory role in intracellular Na(+) and Ca(2+) homeostasis and is intimately involved in regulation of excitation-contraction (EC) coupling. Genetic ablation of PLM results in a slight increase in baseline cardiac contractility and prolongation of action potential duration. When hearts are subjected to catecholamine stress, PLM minimizes the risks of arrhythmogenesis by reducing Na(+) overload and simultaneously preserves inotropy by inhibiting Na(+)/Ca(2+) exchanger. In heart failure, both expression and phosphorylation state of PLM are altered and may partly account for abnormalities in EC coupling. The unique role of PLM in regulation of Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and potentially L-type Ca(2+) channel in the heart, together with the changes in its expression and phosphorylation in heart failure, make PLM a rational and novel target for development of drugs in our armamentarium against heart failure. Clin Trans Sci 2010; Volume 3: 189-196.

We have produced mice in which expression of the rat cardiac Na+/Ca2+ exchanger (NCX1) transgene was switched on when doxycycline was removed from the feed at 5 wk. At 8 to 10 wk, NCX1 expression in induced (Ind) mouse hearts was 2.5-fold higher but protein levels of sarco(endo)plasmic reticulum Ca2+-ATPase, α1- and α2-subunits of Na+-K+-ATPase, phospholamban, ryanodine receptor, calsequestrin, and unphosphorylated and phosphorylated phospholemman were unchanged compared with wild-type (WT) or noninduced (non-Ind) hearts. There was no cellular hypertrophy since WT, non-Ind, and Ind myocytes had similar whole cell membrane capacitance. In Ind myocytes, NCX1 current amplitude was ∼42% higher, L-type Ca2+ current amplitude was unchanged, and action potential duration was prolonged compared with WT or non-Ind myocytes. Contraction and intracellular Ca2+ concentration ([Ca2+]i) transient amplitudes in Ind myocytes were lower at 0.6, not different at 1.8, and higher at 5.0 mM extracellular Ca2+ concentration ([Ca2+]o) compared with WT or non-Ind myocytes. Despite similar Ca2+ current amplitude and sarcoplasmic reticulum (SR) Ca2+ uptake, SR Ca2+ content at 5.0 mM [Ca2+]o was significantly higher in Ind compared with non-Ind myocytes, indicating that NCX1 directly contributed to SR Ca2+ loading. Echocardiography demonstrated that heart rate, left ventricular mass, ejection fraction, stroke volume, and cardiac output were similar among the three groups of animals. In vivo close-chest catheterization demonstrated similar contractility and relaxation among the three groups of mice, both at baseline and after stimulation with isoproterenol. We conclude that induced expression of NCX1 transgene resulted in altered [Ca2+]i homeostasis, myocyte contractility, and action potential morphology. In addition, heart failure did not occur 3 to 5 wk after NCX1 transgene was induced to be expressed at levels found in diseased hearts.

Phospholemman (PLM) regulates cardiac Na+/Ca2+ exchanger (NCX1) and Na+-K+-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser68, disinhibits Na+-K+-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na+-K+-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca2+-ATPase levels, and retained near normal contractility, but α1-subunit of Na+-K+-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca2+ concentration ([Ca2+]i) transients reverted back to those observed in cultured WT myocytes. Both Na+-K+-ATPase current ( Ipump) and Na+/Ca2+ exchange current ( INaCa) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of INaCa but had no effect on Ipump. Contractility, [Ca2+]i transient amplitudes, and sarcoplasmic reticulum Ca2+ contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on INaCa but decreased Ipump. Contractility, [Ca2+]i transient amplitudes, and sarcoplasmic reticulum Ca2+ contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca2+]i homeostasis primarily by its direct inhibitory effects on Na+/Ca2+ exchange.

Previous studies in adult myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated abnormal contractility and intracellular Ca2+ concentration ([Ca2+]i) homeostasis and decreased sarcoplasmic reticulum Ca2+-ATPase (SERCA2) expression and activity, but sarcoplasmic reticulum Ca2+ leak was unchanged. In the present study, we investigated whether SERCA2 overexpression in MI myocytes would restore contraction and [Ca2+]i transients to normal. Compared with sham-operated hearts, 3-wk MI hearts exhibited significantly higher left ventricular end-diastolic and end-systolic volumes but lower fractional shortening and ejection fraction, as measured by M-mode echocardiography. Seventy-two hours after adenovirus-mediated gene transfer, SERCA2 overexpression in 3-wk MI myocytes did not affect Na+-Ca2+ exchanger expression but restored the depressed SERCA2 levels toward those measured in sham myocytes. In addition, the reduced sarcoplasmic reticulum Ca2+ uptake in MI myocytes was improved to normal levels by SERCA2 overexpression. At extracellular Ca2+ concentration of 5 mM, the subnormal contraction and [Ca2+]i transient amplitudes in MI myocytes (compared with sham myocytes) were restored to normal by SERCA2 overexpression. However, at 0.6 mM extracellular Ca2+ concentration, the supernormal contraction and [Ca2+]i transient amplitudes in MI myocytes (compared with sham myocytes) were exacerbated by SERCA2 overexpression. We conclude that SERCA2 overexpression was only partially effective in ameliorating contraction and [Ca2+]i transient abnormalities in our rat model of ischemic cardiomyopathy. We suggest that other Ca2+ transport pathways, e.g., Na+-Ca2+ exchanger, may also play an important role in contractile and [Ca2+]i homeostatic abnormalities in MI myocytes.

Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+concentration ([Ca2+]i) homeostasis and inhibited Na+/Ca2+exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca2+]itransients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca2+-ATPase, Na+-K+-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, and Thr69) were deleted, a partial COOH-terminal deletion mutant in which Ser68and Thr69were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68or Thr69in mediating PLM's effect on cardiac contractility. Focusing on Ser68, the Ser68to Glu mutant was fully effective, the Ser63to Ala (leaving Ser68intact) mutant was partially effective, and the Ser68to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]itransients, and NCX1 currents. Both the Ser63to Ala and Ser68to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser68in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca2+]itransients, and NCX1 activity by PLM is critically dependent on Ser68. We suggest that PLM phosphorylation at Ser68may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.

Phospholemman (PLM) expression was increased in rat hearts after myocardial infarction (MI). Overexpression of PLM in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+concentration ([Ca2+]i) homeostasis in a manner similar to that observed in post-MI myocytes. In this study, we tested whether PLM downregulation in normal adult rat myocytes resulted in contractility and [Ca2+]itransient changes opposite to those observed in post-MI myocytes. Compared with control myocytes infected with adenovirus (Adv) expressing green fluorescent protein (GFP) alone, myocytes infected with Adv expressing both GFP and rat antisense PLM (rASPLM) had 23% less PLM protein ( P < 0.012) at 3 days, but no differences were found in sarcoplasmic reticulum (SR) Ca2+-ATPase, Na+/Ca2+exchanger (NCX1), Na+-K+-ATPase, and calsequestrin levels. SR Ca2+uptake and whole cell capacitance were not affected by rASPLM treatment. Relaxation from caffeine-induced contracture was faster, and NCX1 current amplitudes were higher in rASPLM myocytes, indicating that PLM downregulation enhanced NCX1 activity. In native rat cardiac myocytes, coimmunoprecipitation experiments indicated an association of PLM with NCX1. At 0.6 mM [Ca2+]o, rASPLM myocytes had significantly ( P < 0.003) lower contraction and [Ca2+]itransient amplitudes than control GFP myocytes. At 5 mM [Ca2+]o, both contraction and [Ca2+]itransient amplitudes were higher in rASPLM myocytes. This pattern of contractile and [Ca2+]itransient behavior in rASPLM myocytes was opposite to that observed in post-MI rat myocytes. We conclude that downregulation of PLM in normal rat cardiac myocytes enhanced NCX1 function and affected [Ca2+]itransient and contraction amplitudes. We suggest that PLM downregulation offers a potential therapeutic strategy for ameliorating contractile abnormalities in MI myocytes.

Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca2+homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na+/Ca2+exchanger (NCX1) activity. PLM was overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. After 72 h, the half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was prolonged 1.8-fold ( P < 0.003) in myocytes overexpressing PLM compared with control myocytes overexpressing green fluorescent protein alone. Reverse NCX1 current (3 Na+out:1 Ca2+in) was significantly ( P < 0.0001) lower in PLM myocytes, especially at more positive voltages. Immunofluorescence demonstrated colocalization of PLM and NCX1 to the plasma membrane and t-tubules. Resting membrane potential, action potential amplitude and duration, myocyte size, and NCX1 and calsequestrin protein levels were not affected by PLM overexpression. At 5 mM extracellular [Ca2+] ([Ca2+]o), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca2+]o, the supranormal contraction amplitudes in PLM myocytes were reduced by cooverexpression with NCX1. We conclude that PLM modulated myocyte contractility partly by inhibiting Na+/Ca2+exchange.

The effects of 6–8 wk of high-intensity sprint training (HIST) on rat myocyte contractility and intracellular Ca2+concentration ([Ca2+]i) transients were investigated. Compared with sedentary (Sed) myocytes, HIST induced a modest (5%) but significant ( P < 0.0005) increase in cell length with no changes in cell width. In addition, the percentage of myosin heavy chain α-isoenzyme increased significantly ( P < 0.02) from 0.566 ± 0.077% in Sed rats to 0.871 ± 0.006% in HIST rats. At all three (0.6, 1.8, and 5 mM) extracellular Ca2+concentrations ([Ca2+]o) examined, maximal shortening amplitudes and maximal shortening velocities were significantly ( P < 0.0001) lower and half-times of relaxation were significantly ( P < 0.005) longer in HIST myocytes. HIST myocytes had significantly ( P < 0.0001) higher diastolic [Ca2+]ilevels. Compared with Sed myocytes, systolic [Ca2+]ilevels in HIST myocytes were higher at 0.6 mM [Ca2+]o, similar at 1.8 mM [Ca2+]o, and lower at 5 mM [Ca2+]o. The amplitudes of [Ca2+]itransients were significantly ( P < 0.0001) lower in HIST myocytes. Half-times of [Ca2+]itransient decline, an estimate of sarcoplasmic reticulum (SR) Ca2+uptake activity, were not different between Sed and HIST myocytes. Compared with Sed hearts, Western blots demonstrated a significant ( P < 0.03) threefold decrease in Na+/Ca2+exchanger, but SR Ca2+-ATPase and calsequestrin protein levels were unchanged in HIST hearts. We conclude that HIST effected diminished myocyte contractile function and [Ca2+]itransient amplitudes under the conditions studied. We speculate that downregulation of Na+/Ca2+exchanger may partly account for the decreased contractility in HIST myocytes.

Postmyocardial infarction (MI) rat myocytes demonstrated depressed Na+/Ca2+exchange (NCX1) activity, altered contractility, and intracellular Ca2+ concentration ([Ca2+]i) transients. We investigated whether NCX1 downregulation in normal myocytes resulted in contractility changes observed in MI myocytes. Myocytes infected with adenovirus expressing antisense (AS) oligonucleotides to NCX1 had 30% less NCX1 at 3 days and 66% less NCX1 at 6 days. The half-time of relaxation from caffeine-induced contracture was twice as long in ASNCX1 myocytes. Sarcoplasmic reticulum (SR) Ca2+-ATPase abundance, SR Ca2+uptake, resting membrane potential, action potential amplitude and duration, L-type Ca2+ current density and cell size were not affected by ASNCX1 treatment. At extracellular Ca2+ concentration ([Ca2+]o) of 5 mM, ASNCX1 myocytes had significantly lower contraction and [Ca2+]i transient amplitudes and SR Ca2+ contents than control myocytes. At 0.6 mM [Ca2+]o, contraction and [Ca2+]i transient amplitudes and SR Ca2+ contents were significantly higher in ASNCX1 myocytes. At 1.8 mM [Ca2+]o, contraction and [Ca2+]i transient amplitudes were not different between control and ASNCX1 myocytes. This pattern of contractile and [Ca2+]i transient abnormalities in ASNCX1 myocytes mimics that observed in rat MI myocytes. We conclude that downregulation of NCX1 in adult rat myocytes resulted in decreases in both Ca2+ influx and efflux during a twitch. We suggest that depressed NCX1 activity may partly account for the contractile abnormalities after MI.

Previous studies showed increased phospholemman (PLM) mRNA after myocardial infarction (MI) in rats (Sehl PD, Tai JTN, Hillan KJ, Brown LA, Goddard A, Yang R, Jin H, and Lowe DG. Circulation 101: 1990–1999, 2000). We tested the hypothesis that, in normal adult rat cardiac myocytes, PLM overexpression alters contractile function and cytosolic Ca2+concentration ([Ca2+]i) homeostasis in a manner similar to that observed in post-MI myocytes. Compared with myocytes infected by control adenovirus expressing green fluorescent protein (GFP) alone, Western blots indicated a 41% increase in PLM expression after 72 h ( P < 0.001) but no changes in Na+/Ca2+exchanger, SERCA2, and calsequestrin levels in myocytes infected by adenovirus expressing GFP and PLM. At 5 mM extracellular [Ca2+] ([Ca2+]o), maximal contraction amplitudes in PLM-overexpressed myocytes were 24% ( P < 0.005) and [Ca2+]itransient amplitudes were 18% ( P < 0.05) lower than control myocytes. At 0.6 mM [Ca2+]o, however, contraction and [Ca2+]itransient amplitudes were significantly ( P < 0.05) higher in PLM-overexpressed than control myocytes (18% and 42%, respectively); at 1.8 mM [Ca2+]o, the differences in contraction and [Ca2+]itransient amplitudes were narrowed. This pattern of contractile and [Ca2+]itransient abnormalities in PLM-overexpressed myocytes mimics that observed in post-MI rat myocytes. We suggest that PLM overexpression observed in post-MI myocytes may partly account for contractile abnormalities by perturbing Ca2+fluxes during excitation-contraction.

The functional consequences of overexpression of rat heart Na+/Ca2+exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]iwhen compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+content depended on extracellular Ca2+concentration ([Ca2+]o), with a decrease at low [Ca2+]oand an increase at high [Ca2+]o.The half-times for [Ca2+]itransient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant ( P ≤ 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]oresulted in enhanced Ca2+influx via reverse NCX1 function, as evidenced by greater SR Ca2+content, larger twitch, and [Ca2+]itransient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.

Previous studies have shown that myocytes isolated from sedentary (Sed) rat hearts 3 wk after myocardial infarction (MI) undergo hypertrophy, exhibit altered intracellular Ca2+concentration ([Ca2+]i) dynamics and abnormal contraction, and impaired sarcoplasmic reticulum (SR) function manifested as prolonged half-time of [Ca2+]idecline. Because exercise training elicits positive adaptations in cardiac contractile function and myocardial Ca2+regulation, the present study examined whether 6–8 wk of high-intensity sprint training (HIST) would restore [Ca2+]idynamics and SR function in MI myocytes toward normal. In MI rats, HIST ameliorated myocyte hypertrophy as indicated by significant ( P ≤ 0.05) decreases in whole cell capacitances [Sham-Sed 179 ±12 ( n = 20); MI-Sed 226 ± 7 ( n = 20); MI-HIST 183 ± 11 pF ( n = 19)]. HIST significantly ( P < 0.0001) restored both systolic [Ca2+]i[Sham-Sed 421 ± 9 ( n = 79); MI-Sed 350 ± 6 ( n = 70); MI-HIST 399 ± 9 nM ( n = 70)] and half-time of [Ca2+]idecline (Sham-Sed 0.197 ± 0.005; MI-Sed 0.247 ± 0.006; MI-HIST 0.195 ± 0.006 s) toward normal. Compared with Sham-Sed myocytes, SR Ca2+-ATPase expression significantly ( P < 0.001) decreased by 44% in MI-Sed myocytes. Surprisingly, expression of SR Ca2+-ATPase was further reduced in MI-HIST myocytes to 26% of that measured in Sham-Sed myocytes. There were no differences in calsequestrin expression among the three groups. Expression of phospholamban was not different between Sham-Sed and MI-Sed myocytes but was significantly ( P < 0.01) reduced in MI-HIST myocytes by 25%. Our results indicate that HIST instituted shortly after MI improves [Ca2+]idynamics in surviving myocytes. Improvement in SR function by HIST is mediated not by increased SR Ca2+-ATPase expression, but by modulating phospholamban regulation of SR Ca2+-ATPase activity.

Erythropoietin (Epo) induces a dose-dependent increase in intracellular free Ca2+ ([Ca2+]i ) in human erythroblasts, which is dependent on extracellular Ca2+ and blocked by high doses of nifedipine or Ni2+. In addition, pretreatment of human erythroblasts with mouse antihuman erythropoietin receptor antibody but not mouse immunopure IgG blocked the Epo-induced [Ca2+]i increase, indicating the specificity of the Ca2+ response to Epo stimulation. In this study, the erythropoietin-regulated calcium channel was identified by single channel recordings. Use of conventional whole cell patch-clamp failed to detect Epo-induced whole cell Ca2+ current. To minimize washout of cytosolic constituents, we next used nystatin perforated patch, but did not find any Epo-induced whole cell Ca2+ current. Using Ba2+ (30 mmol/L) as charge carrier in cell-attached patches, we detected single channels with unitary conductance of 3.2 pS, reversal potential of +72 mV, and whose unitary current (at +10 mV) increased monotonically with increasing Ba2+ concentrations. Channel open probability did not appreciably change over the voltage range (−50 to +30 mV) tested. Epo (2 U/mL) increased both mean open time (from 4.27 ± 0.75 to 11.15 ± 1.80 ms) and open probability (from 0.26 ± 0.06 to 2.56 ± 0.59%) of this Ba2+-permeable channel. Our data strongly support the conclusion that the Epo-induced [Ca2+]i increase in human erythroblasts is mediated via Ca2+ entry through a voltage-independent Ca2+ channel.

Myocytes isolated from rat hearts that have suffered 35% myocardial infarction (MI) 3 wk prior have lower peak cytosolic Ca2+ concentration ([Ca2+]i) during contraction compared with Sham myocytes, a difference that is amplified by isoproterenol or high extracellular Ca2+ concentration ([Ca2+]o). To evaluate whether reduced [Ca2+]i in MI myocytes is due to decreased Ca2+ entry, we measured [3H]PN-200-110 [dihydropyridine (DHP)] binding and whole cell Ca2+ current (ICa). DHP binding decreased in both sarcolemmal vesicles and intact myocytes from hearts 3 wk after MI. In contrast, ICa was not different between Sham and MI myocytes incubated at 1.8 mM [Ca2+]o. At 5.0 mM [Ca2+]o, ICa increased similarly in Sham and MI myocytes. Steady-state voltage dependence of activation and inactivation were similar between Sham and MI myocytes, as were the fast- and slow-inactivation time constants. Isoproterenol (1 microM) significantly increased ICa in Sham but not in MI myocytes. Forskolin (10 microM) dibutyryl adenosine 3',5'-cyclic monophosphate (5 mM) significantly increased ICa in MI myocytes; the magnitude of ICa increase was similar to that observed in Sham myocytes. We conclude that 1) decreased systolic [Ca2+]i in MI myocytes was not due to reduced Ca2+ entry via L-type Ca2+ channels; 2) discrepancy between DHP binding (decrease) and ICa (no change) results may be explained by higher channel availability and/or increased long-opening modes (mode 2) in MI myocytes; 3) reduction in isoproterenol-induced [Ca2+]i increase in MI myocytes was partly due to decreased ICa, resulting in less Ca2+ release from sarcoplasmic reticulum; 4) the adenylate cyclase-protein kinase A signal-transduction pathway functioned normally in MI myocytes; and 5) decreased beta-adrenergic responsiveness in MI myocytes was likely due to altered coupling by G proteins. d

Changes in intracellular calcium concentration ([Ca2+]i) in paced fura 2-loaded myocytes isolated from Sham, renovascular hypertensive (Hyp), and myocardial-infarcted (MI) rats were examined. Compared with controls, Hyp myocytes paced at physiological rates had similar systolic but elevated diastolic [Ca2+]i. By contrast, systolic [Ca2+]i was significantly lower and diastolic [Ca2+]i higher in MI myocytes. The different patterns of alterations in [Ca2+]i dynamics in Hyp and MI myocytes may partly explain predominantly diastolic dysfunction in hypertensive hearts and systolic dysfunction in hearts surviving MI. In the presence of 1 microM isoproterenol, both Hyp and MI myocytes had much lower systolic [Ca2+]i when compared with their respective controls. Isoproterenol restored the elevated diastolic [Ca2+]i in Hyp myocytes toward normal but had no effect on the intrinsic differences in diastolic [Ca2+]i between Sham and MI myocytes. The observation that isoproterenol lowers diastolic [Ca2+]i in Hyp myocytes toward normal may provide a cellular mechanism for the lack of efficacy of beta-adrenergic blockers to improve diastolic compliance in patients with hypertensive hypertrophic cardiomyopathy.

Alterations in expression and activity of cardiac Na(+)/Ca(2+) exchanger (NCX1) have been implicated in the pathogenesis of heart failure.Using transgenic mice in which expression of rat NCX1 was induced at 5 weeks of age, we performed transverse aortic constriction (TAC) at 8 weeks and examined cardiac and myocyte function at 15-18 weeks after TAC (age 23-26 weeks). TAC induced left ventricular (LV) and myocyte hypertrophy and increased myocardial fibrosis in both wild-type (WT) and NCX1-overexpressed mice. NCX1 and phosphorylated ryanodine receptor expression was increased by TAC, whereas sarco(endo)plasmic reticulum Ca(2+)-ATPase levels were decreased by TAC. Action potential duration was prolonged by TAC, but to a greater extent in NCX1 myocytes. Na(+)/Ca(2+) exchange current was similar between WT-TAC and WT-sham myocytes, but was higher in NCX1-TAC myocytes. Both myocyte contraction and [Ca(2+)](i) transient amplitudes were reduced in WT-TAC myocytes, but restored to WT-sham levels in NCX1-TAC myocytes. Despite improvement in single myocyte contractility and Ca(2+) dynamics, induced NCX1 overexpression in TAC animals did not ameliorate LV hypertrophy, increase ejection fraction, or enhance inotropic (maximal first derivative of LV pressure rise, +dP/dt) responses to isoproterenol.In pressure-overload hypertrophy, induced overexpression of NCX1 corrected myocyte contractile and [Ca(2+)](i) transient abnormalities but did not aggravate or improve myocardial dysfunction.

Background— [Arg8]-vasopressin (AVP) activates 3 G-protein–coupled receptors: V1A, V2, and V1B. The AVP-V1A receptor is the primary AVP receptor in the heart; however, its role in cardiac homeostasis is controversial. To better understand AVP-mediated signaling in the heart, we created a transgenic mouse with controlled overexpression of the V1A receptor. Methods and Results— The V1A receptor transgene was placed under the control of the tetracycline-regulated, cardiac-specific α-myosin heavy chain promoter (V1A-TG). V1A-TG mice had a normal cardiac function phenotype at 10 weeks of age; however, by 24 weeks of age, tetracycline-transactivating factor/V1A-TG mouse hearts had reduced cardiac function, cardiac hypertrophy, and dilatation of the ventricular cavity. Contractile dysfunction was also observed in isolated adult cardiac myocytes. When V1A receptor transgene was induced to be expressed in adult mice (V1A-TG Ind ), left ventricular dysfunction and dilatation were also seen, albeit at a later time point. Because the V1A receptor mediates cell signaling through Gα q protein, we blocked Gα q signaling by crossing tetracycline-transactivating factor/V1A mice with transgenic mice that expressed a small inhibitory peptide against Gα q . Gα q blockade abrogated the development of the heart failure phenotype in tetracycline-transactivating factor/V1A-TG mice. The heart failure phenotype could be reversed by administration of doxycycline. Conclusions— Our results demonstrate a role for V1A-mediated signaling in the development of heart failure and support a role for V1A blockade in the treatment of patients with elevated levels of vasopressin.

Phospholemman (PLM), when phosphorylated at serine 68, relieves its inhibition on Na+-K+-ATPase but inhibits Na+/Ca2+exchanger 1 (NCX1) in cardiac myocytes. Under stress when catecholamine levels are high, enhanced Na+-K+-ATPase activity by phosphorylated PLM attenuates intracellular Na+concentration ([Na+]i) overload. To evaluate the effects of PLM on NCX1 on in vivo cardiac contractility, we injected recombinant adeno-associated virus (serotype 9) expressing either the phosphomimetic PLM S68E mutant or green fluorescent protein (GFP) directly into left ventricles (LVs) of PLM-knockout (KO) mice. Five weeks after virus injection, ∼40% of isolated LV myocytes exhibited GFP fluorescence. Expression of S68E mutant was confirmed with PLM antibody. There were no differences in protein levels of α1- and α2-subunits of Na+-K+-ATPase, NCX1, and sarco(endo)plasmic reticulum Ca2+-ATPase between KO-GFP and KO-S68E LV homogenates. Compared with KO-GFP myocytes, Na+/Ca2+exchange current was suppressed, but resting [Na+]i, Na+-K+-ATPase current, and action potential amplitudes were similar in KO-S68E myocytes. Resting membrane potential was slightly lower and action potential duration at 90% repolarization (APD90) was shortened in KO-S68E myocytes. Isoproterenol (Iso; 1 μM) increased APD90in both groups of myocytes. After Iso, [Na+]iincreased monotonically in paced (2 Hz) KO-GFP but reached a plateau in KO-S68E myocytes. Both systolic and diastolic [Ca2+]iwere higher in Iso-stimulated KO-S68E myocytes paced at 2 Hz. Echocardiography demonstrated similar resting heart rate, ejection fraction, and LV mass between KO-GFP and KO-S68E mice. In vivo closed-chest catheterization demonstrated enhanced contractility in KO-S68E compared with KO-GFP hearts stimulated with Iso. We conclude that under catecholamine stress when [Na+]iis high, PLM minimizes [Na+]ioverload by relieving its inhibition of Na+-K+-ATPase and preserves inotropy by simultaneously inhibiting Na+/Ca2+exchanger.

Phosphorylation at serine 68 of phospholemman (PLM) in response to β-adrenergic stimulation results in simultaneous inhibition of cardiac Na+/Ca2+exchanger NCX1 and relief of inhibition of Na+-K+-ATPase. The role of PLM in mediating β-adrenergic effects on in vivo cardiac function was investigated with congenic PLM-knockout (KO) mice. Echocardiography showed similar ejection fraction between wild-type (WT) and PLM-KO hearts. Cardiac catheterization demonstrated higher baseline contractility (+dP/d t) but similar relaxation (−dP/d t) in PLM-KO mice. In response to isoproterenol (Iso), maximal +dP/d t was similar but maximal −dP/d t was reduced in PLM-KO mice. Dose-response curves to Iso (0.5–25 ng) for WT and PLM-KO hearts were superimposable. Maximal +dP/d t was reached 1–2 min after Iso addition and declined with time in WT but not PLM-KO hearts. In isolated myocytes paced at 2 Hz. contraction and intracellular Ca2+concentration ([Ca2+]i) transient amplitudes and [Na+]ireached maximum 2–4 min after Iso addition, followed by decline in WT but not PLM-KO myocytes. Reducing pacing frequency to 0.5 Hz resulted in much smaller increases in [Na+]iand no decline in contraction and [Ca2+]itransient amplitudes with time in Iso-stimulated WT and PLM-KO myocytes. Although baseline Na+-K+-ATPase current was 41% higher in PLM-KO myocytes because of increased α1- but not α2-subunit activity, resting [Na+]iwas similar between quiescent WT and PLM-KO myocytes. Iso increased α1-subunit current ( Iα1) by 73% in WT but had no effect in PLM-KO myocytes. Iso did not affect α2-subunit current ( Iα2) in WT and PLM-KO myocytes. In both WT and NCX1-KO hearts, PLM coimmunoprecipitated with Na+-K+-ATPase α1- and α2-subunits, indicating that association of PLM with Na+-K+-ATPase did not require NCX1. We conclude that under stressful conditions in which [Na+]iwas high, β-adrenergic agonist-mediated phosphorylation of PLM resulted in time-dependent reduction in inotropy due to relief of inhibition of Na+-K+-ATPase.

In the heart, adenosine binds to pharmacologically distinct G protein-coupled receptors (R) located on the cardiomyocyte (A1-R, A2A-R and A3-R). While the role of the A1- and A3-Rs in the heart has been clarified by selective overexpression or ablation in murine hearts, the effects of genetically manipulating the A2A-R has not been defined. Thus, we created mice overexpressing a cardiac-restricted A2A-R transgene. Mice with both low (Lo) and high (Hi) levels of A2A-R overexpression demonstrated a marked increase in cardiac contractility at 12 weeks-of-age. These changes were associated with a significantly higher systolic but not diastolic [Ca2+]i, higher maximal contraction amplitudes, maximal shortening and re-lengthening velocities, and a significantly enhanced sarcoplasmic reticulum Ca2+ uptake activity. The alterations in Ca2+ handling were associated with an increase in the level of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2). At 20 weeks-of-age, the effects of A2A-R overexpression on cardiac contractility diminished. The positive effects elicited by A2A-R overexpression differ from the heart failure phenotype we observed with A1-R overexpresson. Interestingly, co-expression of A2A-R TGHi, but not A2A-R TGLo, enhanced survival, prevented the development of left ventricular dysfunction and heart failure and improved Ca2+ handling in mice overexpressing the A1-R. These results suggest that adenosine-mediated signaling in the heart requires a balance between A1- and A2A-Rs – a finding that may have important implications for the ongoing clinical evaluation of adenosine receptor subtype-specific agonists and antagonists for the treatment of cardiovascular diseases.

Phospholemman (PLM) regulates contractility and Ca2+homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice backbred to a pure congenic C57BL/6 background. Cell length, cell width, and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared with wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na+/Ca2+exchanger, sarcoplasmic reticulum (SR) Ca2+-ATPase, α1-subunit of Na+-K+-ATPase, and calsequestrin levels in PLM-null myocytes. At 5 mM extracellular Ca2+concentration ([Ca2+]o), contraction and cytosolic [Ca2+] ([Ca2+]i) transient amplitudes and SR Ca2+contents in PLM-null myocytes were significantly ( P < 0.0004) higher than wild-type myocytes, whereas the converse was true at 0.6 mM [Ca2+]o. This pattern of contractile and [Ca2+]itransient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na+/Ca2+exchanger. Indeed, we have previously reported that Na+/Ca2+exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged ( P < 0.04) in PLM-null myocytes. Whole cell Ca2+current densities were similar between wild-type and PLM-null myocytes, as were the fast- and slow-inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca2+fluxes, likely by modulating Na+/Ca2+exchange activity.

Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postmyocardial infarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na+-K+-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by two- and fourfold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine68of PLM. Na+-K+-ATPase activity was measured as ouabain-sensitive Na+-K+pump current (Ip). Compared with control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly ( P < 0.0001) lower at similar membrane voltages, pipette Na+([Na+]pip) and extracellular K+([K+]o) concentrations. From −70 to +60 mV, neither [Na+]pipnor [K+]orequired to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased Vmaxwithout appreciable changes in Kmfor Na+and K+in PLM-overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na+-K+-ATPase expression because there were no changes in either protein or messenger RNA levels of either α1- or α2-isoforms of Na+-K+-ATPase. In native rat cardiac myocytes, PLM coimmunoprecipitated with α-subunits of Na+-K+-ATPase. Inhibition of Na+-K+-ATPase by PLM overexpression, in addition to previously reported decrease in Na+-K+-ATPase expression, may explain altered Vmaxbut not Kmof Na+-K+-ATPase in postinfarction rat myocytes.

Exercise training instituted after myocardial infarction improves many steps involved in cardiac excitation-contraction coupling. Focusing on Na/Caexchange, current controversies regarding whether it mediates Cainflux during an action potential, whether it is increased or decreased in disease models, whether protein kinase A alters its activity, and whether exercise training affects its function are reviewed. Finally, a novel target for exercise training in the heart is suggested.

Previous studies in adult myocytes isolated from rat hearts 3–9 wk after myocardial infarction (MI) demonstrated abnormal contractility and decreased Na+/Ca2+ exchanger (NCX1) activity. In addition, a program of high-intensity sprint training (HIST) instituted shortly after MI restored both contractility and NCX1 activity toward normal. The present study examined the hypotheses that reduced NCX1 activity caused abnormal contractility in myocytes isolated from sedentary (Sed) rat hearts 9–11 wk after coronary artery ligation and that HIST ameliorated contractile dysfunction in post-MI myocytes by increasing NCX1 activity. The approach was to upregulate NCX1 in MI-sedentary (MISed) myocytes and downregulate NCX1 in MI-exercised (MIHIST) myocytes by adenovirus-mediated gene transfer. Overexpression of NCX1 in MISed myocytes did not affect sarco(endo)plasmic reticulum Ca2+-ATPase and calsequestrin levels but rescued contractile abnormalities observed in MISed myocytes. That is, at 5 mM extracellular Ca2+ concentration, the subnormal contraction amplitude in MISed myocytes (compared with Sham myocytes) was increased toward normal by NCX1 overexpression, whereas at 0.6 mM extracellular Ca2+ concentration the supernormal contraction amplitude in MISed myocytes was lowered. Conversely, NCX1 downregulation by antisense in MIHIST myocytes abolished the beneficial effects of HIST on contraction amplitudes in MI myocytes. We suggest that decreased NCX1 activity may play an important role in contractile abnormalities in post-MI myocytes and that HIST ameliorated contractile dysfunction in post-MI myocytes partly by enhancing NCX1 activity.

Previous studies on myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated increased cell length, reduced Na+/Ca2+ exchange (NCX1) activity, altered contractility, and intracellular Ca2+ concentration ([Ca2+]i) transients. In the present study, we investigated whether NCX1 overexpression in MI myocytes would restore contraction and [Ca2+]i transients to normal. When myocytes were placed in culture under continued electrical-field stimulation conditions, differences in contraction amplitudes and cell lengths between sham and MI myocytes were preserved for at least 48 h. Infection of both sham and MI myocytes by adenovirus expressing green fluorescent protein resulted in >95% infection, as evidenced by green fluorescent protein fluorescence, but contraction amplitudes at 6-, 24-, and 48-h postinfection were not affected. NCX1 overexpression in MI myocytes resulted in lower diastolic [Ca2+]i levels at all extracellular Ca2+ concentrations ([Ca2+]o) examined, suggesting enhanced forward NCX1 activity. At 5 mM [Ca2+]o, subnormal contraction and [Ca2+]i transient amplitudes in MI myocytes (compared with sham myocytes) were restored toward normal levels by overexpressing NCX1. At 0.6 mM [Ca2+]o, supranormal contraction and [Ca2+]i transient amplitudes in MI myocytes (compared with sham myocytes) were lowered by NCX1 overexpression. We conclude that overexpression of NCX1 in MI myocytes was effective in improving contractile dysfunction, most likely because of enhancement of both Ca2+ efflux and influx during a cardiac cycle. We suggest that decreased NCX1 activity may play an important role in contractile abnormalities in postinfarction myocytes.

Two electrophysiological manifestations of myocardial infarction (MI)-induced myocyte hypertrophy are prolongation of action potential duration (APD) and reduction of transient outward current ( I to) density. Because high-intensity sprint training (HIST) ameliorated myocyte hypertrophy and improved myocyte Ca2+ homeostasis and contractility after MI, the present study evaluated whether 6–8 wk of HIST would shorten the prolonged APD and improve the depressed I to in post-MI myocytes. There were no differences in resting membrane potential and action potential amplitude (APA) measured in myocytes isolated from sham-sedentary (Sed), MI-Sed, and MI-HIST groups. Times required for repolarization to 50 and 90% APA were significantly ( P < 0.001) prolonged in MI-Sed myocytes. HIST reduced times required for repolarization to 50 and 90% APA to values observed in Sham-Sed myocytes. The fast and slow components of I towere significantly ( P < 0.0001) reduced in MI-Sed myocytes. HIST significantly ( P < 0.001) enhanced the fast and slow components of I to in MI myocytes, although not to levels observed in Sham-Sed myocytes. There were no significant differences in steady-state I toinactivation and activation parameters among Sham-Sed, MI-Sed, and MI-HIST myocytes. Likewise, recovery from time-dependent inactivation was also similar among the three groups. We suggest that normalization of APD after MI by HIST may be mediated by restoration of I to toward normal levels.

The significance of 6–8 wk of high-intensity sprint training (HIST) on contractile abnormalities of myocytes isolated from rat hearts with prior myocardial infarction (MI) was investigated. Compared with the sedentary (Sed) condition, HIST attenuated myocyte hypertrophy observed post-MI primarily by reducing cell lengths but not cell widths. At high extracellular Ca2+ concentration (5 mM) and low pacing frequency (0.1 Hz), conditions that preferentially favored Ca2+ influx over efflux, MI-Sed myocytes shortened less than Sham-Sed myocytes did. HIST significantly improved contraction amplitudes in MI myocytes. Under conditions that favored Ca2+ efflux, i.e., low extracellular Ca2+ concentration (0.6 mM) and high pacing frequency (2 Hz), MI-Sed myocytes contracted more than Sham-Sed myocytes. HIST did not appreciably affect contraction amplitudes of MI myocytes under these conditions. Compared with MI-Sed myocytes, HIST myocytes showed significant improvement in time required to reach one-half maximal contraction amplitude shortening, maximal myocyte shortening and relengthening velocities, and half time of relaxation. Our results indicate that HIST instituted shortly after MI improved cellular contraction in surviving myocytes. Because our previous studies demonstrated that, in post-MI myocytes, HIST improved intracellular Ca2+ dynamics, enhanced sarcoplasmic reticulum Ca2+ uptake and Ca2+ content, and restored Na+/Ca2+ exchange current toward normal, we hypothesized that improvement in MI myocyte contractile function by HIST was likely mediated by normalization of cellular Ca2+ homeostatic mechanisms.

Previous studies have shown lower systolic intracellular Ca2+concentrations ([Ca2+]i) and reduced sarcoplasmic reticulum (SR)-releasable Ca2+contents in myocytes isolated from rat hearts 3 wk after moderate myocardial infarction (MI). Ca2+entry via L-type Ca2+channels was normal, but that via reverse Na+/Ca2+exchange was depressed in 3-wk MI myocytes. To elucidate mechanisms of reduced SR Ca2+contents in MI myocytes, we measured SR Ca2+uptake and SR Ca2+leak in situ, i.e., in intact cardiac myocytes. For sham and MI myocytes, we first demonstrated that caffeine application to release SR Ca2+and inhibit SR Ca2+uptake resulted in a 10-fold prolongation of half-time ( t½) of [Ca2+]itransient decline compared with that measured during a normal twitch. These observations indicate that early decline of the [Ca2+]itransient during a twitch in rat myocytes was primarily mediated by SR Ca2+-ATPase and that the t½of [Ca2+]idecline is a measure of SR Ca2+uptake in situ. At 5.0 mM extracellular Ca2+, systolic [Ca2+]iwas significantly ( P ≤ 0.05) lower (337 ± 11 and 416 ± 18 nM in MI and sham, respectively) and t½of [Ca2+]idecline was significantly longer (0.306 ± 0.014 and 0.258 ± 0.014 s in MI and sham, respectively) in MI myocytes. The 19% prolongation of t½of [Ca2+]idecline was associated with a 23% reduction in SR Ca2+-ATPase expression (detected by immunoblotting) in MI myocytes. SR Ca2+leak was measured by a novel electrophysiological technique that did not require assigning empirical constants for intracellular Ca2+buffering. SR Ca2+leak rate was not different between sham and MI myocytes: the time constants of SR Ca2+loss after thapsigargin were 290 and 268 s, respectively. We conclude that, independent of decreased SR filling by Ca2+influx, the lower SR Ca2+content in MI myocytes was due to reduced SR Ca2+uptake and SR Ca2+-ATPase expression, but not to enhanced SR Ca2+leak.

The significance of altered Ca2+ influx and efflux pathways on contractile abnormalities of myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) was investigated by varying extracellular Ca2+concentration ([Ca2+]o, 0.6–5.0 mM) and pacing frequency (0.1–5.0 Hz). Myocytes isolated from 3-wk MI hearts were significantly longer than those from sham-treated (Sham) hearts (125 ± 1 vs. 114 ± 1 μm, P < 0.0001). At high [Ca2+]oand low pacing frequency, conditions that preferentially favored Ca2+ influx over efflux, Sham myocytes shortened to a greater extent than 3-wk MI myocytes. Conversely, under conditions that favored Ca2+ efflux (low [Ca2+]oand high pacing frequency), MI myocytes shortened more than Sham myocytes. At intermediate [Ca2+]oand pacing frequencies, differences in steady-state contraction amplitudes between Sham and MI myocytes were no longer significant. Collectively, the interpretation of these data was that Ca2+ influx and efflux pathways were subnormal in MI myocytes and that they contributed to abnormal cellular contractile behavior. Because Na+/Ca2+exchange activity, but not whole cell Ca2+ current, was depressed in 3-wk MI rat myocytes, our results on steady-state contraction are consistent with, but not proof of, the hypothesis that depressed Na+/Ca2+exchange accounted for abnormal contractility in MI myocytes. The effects of depressed Na+/Ca2+exchange on MI myocyte mechanical activity were further evaluated in relaxation from caffeine-induced contractures. Because Ca2+ uptake by sarcoplasmic reticulum was inhibited by caffeine and with the assumption that intracellular Na+ and membrane potential were similar between Sham and MI myocytes, myocyte relaxation from caffeine-induced contracture can be taken as an estimate of Ca2+ extrusion by Na+/Ca2+exchange. In MI myocytes, in which Na+/Ca2+exchange activity was depressed, the half time of relaxation (1.54 ± 0.14 s) was significantly ( P < 0.02) prolonged compared with that measured in Sham myocytes (1.10 ± 0.10 s).

TRPM2 channels are expressed in the heart and vasculature. To elucidate the function of TRPM2, we generated a global TRPM2-KO mouse. To evaluate whether TRPM2 channels were functional in the heart, we activated TRPM2 channels with H2O2 and demonstrated that Ca2+ influx was dependent on extracellular Ca2+ and significantly higher in WT compared to TRPM2-KO myocytes. We then examined the effects of TRPM2 channels on cardiac function, both at rest and after ischemia-reperfusion (I/R) injury. At rest, there were no differences in LV mass, heart rate, fractional shortening (FS) and +dP/dt between WT and TRPM2-KO hearts. At 2-3 days post-I/R injury, despite similar areas-at-risk and infarct sizes, TRPM2-KO hearts had lower FS and +dP/dt when compared to WT hearts. Compared to WT I/R myocytes, expression of Na+/Ca2+ exchanger (NCX1) and NCX1 current were increased, and action potential duration was prolonged in TRPM2-KO I/R myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation to simulate I/R in vitro, levels of reactive oxygen species (ROS) were significantly higher in TRPM2-KO when compared to WT myocytes. Oxygen radical scavenging enzymes (SOD1 and SOD2) and their upstream regulators (FoxO1, FoxO3a and HIF-1α) were lower while NADPH oxidase (Nox2) was higher in TRPM2-KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by enhancing expression of oxygen radical scavenging enzymes, thereby reducing oxidative stress associated with ischemia-reperfusion.

Myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) had lower peak cytosolic free Ca2+ concentration ([Ca2+]i) and reduced maximal extent of cell shortening during contraction, but Ca2+ entry via L-type Ca2+ channels was normal. In the current study using whole cell patch-clamp technique, reverse Na+/Ca2+ exchange current (INa/Ca; 3 Na+ out:1 Ca2+ in) was measured in myocytes in which Na+, K+, and Ca2+ currents were blocked or minimized. Steady-state outward currents measured under these conditions increased with depolarization or with elevation of extracellular Ca2+ concentration ([Ca2+]o) from 1.8 to 5.0 mM, but were inhibited by 5 mM Ni2+ or by reduction of [Ca2+]i to near zero. In addition, reduction of cytosolic free Na+ concentration or of [Ca2+]i also decreased the amplitude of the outward current. These characteristics indicate the outward current was INa/Ca operating in reverse mode. Reverse INa/Ca was significantly lower in MI myocytes, especially at more positive voltages. In addition, sarcoplasmic reticulum (SR)-releasable Ca2+ content as estimated by integrating forward INa/Ca during caffeine-induced SR Ca2+ release was also significantly lower in MI myocytes. Depressed Na+/Ca2+ exchange activity may contribute to abnormal [Ca2+]i dynamics in MI myocytes.

Adenosine binds to three G protein-coupled receptors (R) located on the cardiomyocyte (A(1)-R, A(2A)-R and A(3)-R) and provides cardiac protection during both ischemic and load-induced stress. While the role of adenosine receptor-subtypes has been well defined in the setting of ischemia-reperfusion, far less is known regarding their roles in protecting the heart during other forms of cardiac stress. Because of its ability to increase cardiac contractility and heart rate, we hypothesized that enhanced signaling through A(2A)-R would protect the heart during the stress of transverse aortic constriction (TAC). Using a cardiac-specific and inducible promoter, we selectively over-expressed A(2A)-R in FVB mice. Echocardiograms were obtained at baseline, 2, 4, 8, 12, 14 weeks and hearts were harvested at 14 weeks, when WT mice developed a significant decrease in cardiac function, an increase in end systolic and diastolic dimensions, a higher heart weight to body weight ratio (HW/BW), and marked fibrosis when compared with sham-operated WT. More importantly, these changes were significantly attenuated by over expression of the A(2A)-R. Furthermore, WT mice also demonstrated marked increases in the hypertrophic genes β-myosin heavy chain (β-MHC), and atrial natriuretic factor (ANF)--changes that are mediated by activation of the transcription factor GATA-4. Levels of the mRNAs encoding β-MHC, ANP, and GATA-4 were significantly lower in myocardium from A(2A)-R TG mice after TAC when compared with WT and sham-operated controls. In addition, three inflammatory factors genes encoding cysteine dioxygenase, complement component 3, and serine peptidase inhibitor, member 3N, were enhanced in WT TAC mice, but their expression was suppressed in A(2A)-R TG mice. A(2A)-R over-expression is protective against pressure-induced heart failure secondary to TAC. These cardioprotective effects are associated with attenuation of GATA-4 expression and inflammatory factors. The A(2A)-R may provide a novel new target for pharmacologic therapy in patients with cardiovascular disease.