Oxidative posttranslational modifications mediate decreased SERCA activity and myocyte dysfunction in Galphaq-overexpressing mice

Abstract

Background: Myocyte contractile dysfunction occurs in pathological remodeling in association with abnormalities in calcium regulation. Mice with cardiac myocyte-specific overexpression of Galphaq develop progressive left ventricular failure associated with myocyte contractile dysfunction and calcium dysregulation.

Objective: We tested the hypothesis that myocyte contractile dysfunction in the Galphaq 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 Galphaq mice had marked abnormalities of myocyte contractile function and calcium transients. In Galphaq 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 Galphaq myocardium. Cross-breeding Galphaq 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 Galphaq-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.

Figure 1

Abnormal contractile function and intracellular calcium transients in cardiac myocytes from Gαq overexpressing mice are ameliorated by cross-breeding with mice that overexpress catalase in the myocardium. Ventricular myocytes were isolated from wild-type (WT), Gαq (Gq) or Gαq/catalase (GqCat) mice. Panel A. Cell shortening (% of baseline). Panel B. Velocity of contraction (−dL/dt). Panel C. Velocity of relaxation (+dL/dt). Panel D. Calcium transient amplitude (delta of the ratio (R) of fluorescence 360/380nm). Panel E. Rate of calcium transient rise (+dR/dt). Panel F. Rate of calcium transient decline (−dR/dt). *p<0.05 vs. WT; **p<0.01 vs. WT; †p<0.05 vs. Gαq; ††p<0.01 vs. Gαq; 5–10 cells per heart, 4–5 hearts per group.

Figure 2

OPTM of SERCA in myocardium from Gαq mice. Panel A. Representative immunoblot for total and BIAM-labeled SERCA in WT, Cat, Gαq and Gαq/Cat mice. Panel B. Ratio of BIAM-labeled to total SERCA. Shown are mean data from 4 hearts in each group (*p<0.001 vs. WT; †p<0.05 vs. Gαq). Panel C and D. Representative micrographs showing increased levels of SERCA sulfonylated at cysteine 674 (Panel C) and nitrated at tyrosine 294/295 (Panel D) distributed diffusely in myocytes from Gαq mice, and the prevention of both OPTM by concurrent expression of catalase in Gαq/catalase mice (bar = 25 μM).

Figure 3

Decreased SERCA activity in Gαq myocardium is corrected by concurrent expression of catalase in Gαq/catalase mice. Maximum SERCA activity was assessed using maximum calcium-stimulated, thapsigargin-inhibited calcium uptake . Shown are mean data for 7 – 8 hearts in each group (*p<0.05 vs. WT; †p<0.05 vs. Gαq).