Abstract
A naturally occurring R9C mutation of phospholamban (PLB) triggers cardiomyopathy and premature death by altering regulation of sarco/endoplasmic reticulum calcium-ATPase (SERCA). The goal of this study was to investigate the acute physiological consequences of the R9C-PLB mutation on cardiomyocyte calcium kinetics and contractility. We measured the physiological consequences of R9C-PLB mutation on calcium transients and sarcomere shortening in adult cardiomyocytes. In contrast to studies of chronic R9C-PLB expression in transgenic mice, we found that acute expression of R9C-PLB exerts a positively inotropic and lusitropic effect in cardiomyocytes. Importantly, R9C-PLB exhibited blunted sensitivity to frequency potentiation and β-adrenergic stimulation, two major physiological mechanisms for the regulation of cardiac performance. To identify the molecular mechanism of R9C pathology, we quantified the effect of R9C on PLB oligomerization and PLB-SERCA binding. FRET measurements in live cells revealed that R9C-PLB exhibited an increased propensity for oligomerization, and this was further increased by oxidative stress. The R9C also decreased PLB binding to SERCA and altered the structure of the PLB-SERCA regulatory complex. The structural change after oxidative modification of R9C-PLB was similar to that observed after PLB phosphorylation. We conclude that R9C mutation of PLB decreases SERCA inhibition by decreasing the amount of the regulatory complex and altering its conformation. This has an acute inotropic/lusitropic effect but yields negative consequences of impaired frequency potentiation and blunted β-adrenergic responsiveness. We envision a self-reinforcing mechanism beginning with phosphomimetic R9C-PLB oxidation and loss of SERCA inhibition, leading to impaired calcium regulation and heart failure.
Highlights
We observed that the localization of endogenous WT-PLB labeled with Alexa Fluor 532 secondary antibody was similar to CFP-R9C-PLB (Fig. 1C)
Effect of frequency potentiation on the sarcomere shortening parameters in R9C-PLB-expressing cardiomyocytes compared with WT-PLB-expressing cardiomyocytes
We observed a 52% increase in the amplitude of the peak of the Ca2ϩ transient in R9C-PLB-expressing cells compared with WT-PLB (Fig. 2E), suggesting an increase in myocyte sarcoplasmic reticulum Ca2ϩ load
Summary
Conclusion: Loss of phospholamban inhibitory function acutely increases SERCA function but impairs SERCA regulation, resulting in a blunted stress response, leading to cardiomyopathy. A naturally occurring R9C mutation of phospholamban (PLB) triggers cardiomyopathy and premature death by altering regulation of sarco/endoplasmic reticulum calcium-ATPase (SERCA). We conclude that R9C mutation of PLB decreases SERCA inhibition by decreasing the amount of the regulatory complex and altering its conformation. This has an acute inotropic/lusitropic effect but yields negative consequences of impaired frequency potentiation and blunted -adrenergic responsiveness. We envision a self-reinforcing mechanism beginning with phosphomimetic R9C-PLB oxidation and loss of SERCA inhibition, leading to impaired calcium regulation and heart failure
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