Steve Lancel

Nitroxyl Activates SERCA in Cardiac Myocytes via Glutathiolation of Cysteine 674

Nitroxyl (HNO) exerts inotropic and lusitropic effects in myocardium, in part via activation of SERCA (sarcoplasmic reticulum calcium ATPase). To elucidate the molecular mechanism, adult rat ventricular myocytes were exposed to HNO derived from Angeli’s salt. HNO increased the maximal rate of thapsigargin-sensitive Ca2+ uptake mediated by SERCA in sarcoplasmic vesicles and caused reversible oxidative modification of SERCA thiols. HNO increased the S-glutathiolation of SERCA, and adenoviral overexpression of glutaredoxin-1 prevented both the HNO-stimulated oxidative modification of SERCA and its activation, as did overexpression of a mutated SERCA in which cysteine 674 was replaced with serine. Thus, HNO increases the maximal activation of SERCA via S-glutathiolation at cysteine 674.

Redox-mediated reciprocal regulation of SERCA and Na+-Ca2+ exchanger contributes to sarcoplasmic reticulum Ca2+ depletion in cardiac myocytes.

Myocardial failure is associated with increased oxidative stress and abnormal excitation-contraction coupling characterized by depletion of sarcoplasmic reticulum (SR) Ca(2+) stores and a reduction in Ca(2+)-transient amplitude. Little is known about the mechanisms whereby oxidative stress affects Ca(2+) handling and contractile function; however, reactive thiols may be involved. We used an in vitro cardiomyocyte system to test the hypothesis that short-term oxidative stress induces SR Ca(2+) depletion via redox-mediated regulation of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) and the sodium-Ca(2+) exchanger (NCX) and that this is associated with thiol oxidation. Adult rat ventricular myocytes paced at 5 Hz were superfused with H(2)O(2) (100 microM, 15 min). H(2)O(2) caused a progressive decrease in cell shortening followed by diastolic arrest, which was associated with decreases in SR Ca(2+) content, systolic [Ca(2+)](i), and Ca(2+)-transient amplitude, but no change in diastolic [Ca(2+)](i). H(2)O(2) caused reciprocal effects on the activities of SERCA (decreased) and NCX (increased). Pretreatment with the NCX inhibitor KB-R7943 before H(2)O(2) increased diastolic [Ca(2+)](i) and mimicked the effect of SERCA inhibition with thapsigargin. These functional effects were associated with oxidative modification of thiols on both SERCA and NCX. In conclusion, redox-mediated SR Ca(2+) depletion involves reciprocal regulation of SERCA and NCX, possibly via direct oxidative modification of both proteins.

Oxidative Posttranslational Modifications Mediate Decreased SERCA Activity and Myocyte Dysfunction in Gαq-Overexpressing Mice

Background: Myocyte contractile dysfunction occurs in pathological remodeling in association with abnormalities in calcium regulation. Mice with cardiac myocyte–specific overexpression of G&agr;q develop progressive left ventricular failure associated with myocyte contractile dysfunction and calcium dysregulation. Objective: We tested the hypothesis that myocyte contractile dysfunction in the G&agr;q mouse heart is mediated by reactive oxygen species, and in particular, oxidative posttranslational modifications, which impair the function of sarcoplasmic reticulum Ca2+-ATPase (SERCA). Methods and Results: Freshly isolated ventricular myocytes from G&agr;q mice had marked abnormalities of myocyte contractile function and calcium transients. In G&agr;q myocardium, SERCA protein was not altered in quantity but displayed evidence of oxidative cysteine modifications reflected by decreased biotinylated iodoacetamide labeling and evidence of specific irreversible oxidative modifications consisting of sulfonylation at cysteine 674 and nitration at tyrosines 294/295. Maximal calcium-stimulated SERCA activity was decreased 47% in G&agr;q myocardium. Cross-breeding G&agr;q mice with transgenic mice that have cardiac myocyte-specific overexpression of catalase (a) decreased SERCA oxidative cysteine modifications, (b) decreased SERCA cysteine 674 sulfonylation and tyrosine 294/295 nitration, (c) restored SERCA activity, and (d) improved myocyte calcium transients and contractile function. Conclusions: In G&agr;q-induced cardiomyopathy, myocyte contractile dysfunction is mediated, at least in part, by 1 or more oxidative posttranslational modifications of SERCA. Protein oxidative posttranslational modifications contribute to the pathophysiology of myocardial dysfunction and thus may provide a target for therapeutic intervention.

Cardiac-Specific Overexpression of Catalase Identifies Hydrogen Peroxide-Dependent and -Independent Phases of Myocardial Remodeling and Prevents the Progression to Overt Heart Failure in Gαq-Overexpressing Transgenic Mice

Background—Although it seems that reactive oxygen species contribute to chronic myocardial remodeling, questions remain about (1) the specific types of reactive oxygen species involved, (2) the role of reactive oxygen species in mediating specific cellular events, and (3) the cause-and-effect relationship between myocardial reactive oxygen species and the progression to heart failure. Transgenic mice with myocyte-specific overexpression of G&agr;q develop a dilated cardiomyopathy that progresses to heart failure. We used this model to examine the role of H2O2 in mediating myocardial remodeling and the progression to failure. Methods and Results—In G&agr;q myocardium, markers of oxidative stress were increased at 4 weeks and increased further at 20 weeks. G&agr;q mice were crossbred with transgenic mice having myocyte-specific overexpression of catalase. At 4 weeks of age, left ventricular end-diastolic dimension was increased and left ventricular fractional shortening decreased in G&agr;q mice and deteriorated further through 20 weeks. In G&agr;q mice, myocardial catalase overexpression had no effect on left ventricular end-diastolic dimension or fractional shortening at 4 weeks but prevented the subsequent deterioration in both. In G&agr;q mice, myocyte hypertrophy; myocyte apoptosis; interstitial fibrosis; and the progression to overt heart failure, as reflected by lung congestion and exercise intolerance, were prevented by catalase overexpression. Conclusion—In G&agr;q mice, myocyte-specific overexpression of catalase had no effect on the initial phenotype of left ventricular dilation and contractile dysfunction but prevented the subsequent progressive remodeling phase leading to heart failure. Catalase prevented the cellular hallmarks of adverse remodeling (myocyte hypertrophy, myocyte apoptosis, and interstitial fibrosis) and the progression to overt heart failure. Thus, H2O2, associated oxidant pathways, or both play a critical role in adverse myocardial remodeling and the progression to failure.

Hydrogen Peroxide–Mediated SERCA Cysteine 674 Oxidation Contributes to Impaired Cardiac Myocyte Relaxation in Senescent Mouse Heart

Background A hallmark of aging of the cardiac myocyte is impaired sarcoplasmic reticulum (SR) calcium uptake and relaxation due to decreased SR calcium ATPase (SERCA) activity. We tested the hypothesis that H2O2‐mediated oxidation of SERCA contributes to impaired myocyte relaxation in aging. Methods and Results Young (5‐month‐old) and senescent (21‐month‐old) FVB wild‐type (WT) or transgenic mice with myocyte‐specific overexpression of catalase were studied. In senescent mice, myocyte‐specific overexpression of catalase (1) prevented oxidative modification of SERCA as evidenced by sulfonation at Cys674, (2) preserved SERCA activity, (3) corrected impaired calcium handling and relaxation in isolated cardiac myocytes, and (4) prevented impaired left ventricular relaxation and diastolic dysfunction. Nitroxyl, which activates SERCA via S‐glutathiolation at Cys674, failed to activate SERCA in freshly isolated ventricular myocytes from senescent mice. Finally, in adult rat ventricular myocytes in primary culture, adenoviral overexpression of SERCA in which Cys674 is mutated to serine partially preserved SERCA activity during exposure to H2O2. Conclusion Oxidative modification of SERCA at Cys674 contributes to decreased SERCA activity and impaired myocyte relaxation in the senescent heart. Strategies to decrease oxidant levels and/or protect target proteins such as SERCA may be of value to preserve diastolic function in the aging heart.

Short Communication: Oxidative Posttranslational Modifications Mediate Decreased SERCA Activity and Myocyte Dysfunction in Gαq-Overexpressing Mice

Background: Myocyte contractile dysfunction occurs in pathological remodeling in association with abnormalities in calcium regulation. Mice with cardiac myocyte–specific overexpression of G&agr;q develop progressive left ventricular failure associated with myocyte contractile dysfunction and calcium dysregulation. Objective: We tested the hypothesis that myocyte contractile dysfunction in the G&agr;q mouse heart is mediated by reactive oxygen species, and in particular, oxidative posttranslational modifications, which impair the function of sarcoplasmic reticulum Ca2+-ATPase (SERCA). Methods and Results: Freshly isolated ventricular myocytes from G&agr;q mice had marked abnormalities of myocyte contractile function and calcium transients. In G&agr;q myocardium, SERCA protein was not altered in quantity but displayed evidence of oxidative cysteine modifications reflected by decreased biotinylated iodoacetamide labeling and evidence of specific irreversible oxidative modifications consisting of sulfonylation at cysteine 674 and nitration at tyrosines 294/295. Maximal calcium-stimulated SERCA activity was decreased 47% in G&agr;q myocardium. Cross-breeding G&agr;q mice with transgenic mice that have cardiac myocyte-specific overexpression of catalase (a) decreased SERCA oxidative cysteine modifications, (b) decreased SERCA cysteine 674 sulfonylation and tyrosine 294/295 nitration, (c) restored SERCA activity, and (d) improved myocyte calcium transients and contractile function. Conclusions: In G&agr;q-induced cardiomyopathy, myocyte contractile dysfunction is mediated, at least in part, by 1 or more oxidative posttranslational modifications of SERCA. Protein oxidative posttranslational modifications contribute to the pathophysiology of myocardial dysfunction and thus may provide a target for therapeutic intervention.

Short Communication: Oxidative Posttranslational Modifications Mediate Decreased SERCA Activity and Myocyte Dysfunction in G q-Overexpressing Mice

Background: Myocyte contractile dysfunction occurs in pathological remodeling in association with abnormalities in calcium regulation. Mice with cardiac myocyte–specific overexpression of G&agr;q develop progressive left ventricular failure associated with myocyte contractile dysfunction and calcium dysregulation. Objective: We tested the hypothesis that myocyte contractile dysfunction in the G&agr;q mouse heart is mediated by reactive oxygen species, and in particular, oxidative posttranslational modifications, which impair the function of sarcoplasmic reticulum Ca2+-ATPase (SERCA). Methods and Results: Freshly isolated ventricular myocytes from G&agr;q mice had marked abnormalities of myocyte contractile function and calcium transients. In G&agr;q myocardium, SERCA protein was not altered in quantity but displayed evidence of oxidative cysteine modifications reflected by decreased biotinylated iodoacetamide labeling and evidence of specific irreversible oxidative modifications consisting of sulfonylation at cysteine 674 and nitration at tyrosines 294/295. Maximal calcium-stimulated SERCA activity was decreased 47% in G&agr;q myocardium. Cross-breeding G&agr;q mice with transgenic mice that have cardiac myocyte-specific overexpression of catalase (a) decreased SERCA oxidative cysteine modifications, (b) decreased SERCA cysteine 674 sulfonylation and tyrosine 294/295 nitration, (c) restored SERCA activity, and (d) improved myocyte calcium transients and contractile function. Conclusions: In G&agr;q-induced cardiomyopathy, myocyte contractile dysfunction is mediated, at least in part, by 1 or more oxidative posttranslational modifications of SERCA. Protein oxidative posttranslational modifications contribute to the pathophysiology of myocardial dysfunction and thus may provide a target for therapeutic intervention.