Abstract 12646: Oxidation-Dependent Phosphomimetic Effect of a Human Heart Failure Mutation of Phospholamban
Rationale: A naturally-occurring, missense Arg9-to-Cys (R9C) mutation of phospholamban (PLB) triggers cardiomyopathy and premature death in humans. However, the fundamental molecular mechanism underlying the cardiotoxic role of R9C-PLB in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) regulation and cardiomyocyte Ca2+ handling is not clear.
Objective: The goal of this study was to investigate the acute physiological consequences of R9C-PLB mutation on cardiomyocyte Ca2+ kinetics and contractility and identify the molecular mechanism underlying R9C pathology.
Methods and Results: We measured the physiological consequences of R9C-PLB mutation on Ca2+ transients and sarcomere shortening in adult cardiomyocytes at increasing pacing frequencies. 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 fused fluorescent protein tags to PLB and SERCA, and compared the effect of R9C and pentamer-destabilizing mutation (SSS) on PLB oligomerization and PLB-SERCA interaction. Fluorescence resonance energy transfer (FRET) measurements in live cells revealed that R9C exhibited an increased affinity of PLB oligomerization, and a decreased binding affinity to SERCA due to an oxidative modification which mimics phosphorylation. Real-time FRET analysis in cardiomyocytes revealed that R9C-PLB exhibits enhanced sensitivity to oxidative stress, which is a prevailing condition in heart failure.
Conclusions: We conclude that the primary mechanism of R9C pathology is a phosphomimetic effect of PLB cysteine oxidation, manifested as increased oligomerization and a change in the structure of the PLB-SERCA regulatory complex.
Author Disclosures: N. Abrol: None. P.P. de Tombe: None. S.L. Robia: None.
- © 2014 by American Heart Association, Inc.