The Role of Heart Failure-Causing Phospholamban R9C Mutation in SERCA Dysfunction and Cardiac Ca2+ Mishandling

Abstract

The naturally occurring, heterozygous Arg9-to-Cys (R9C) missense mutation of phospholamban (PLB) triggers dilated cardiomyopathy, heart failure and premature death in humans. However, the fundamental molecular mechanism underlying the cardiotoxic role of PLBR9C in regulation of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and cardiomyocyte Ca2+ handling is not clear. The dynamic equilibrium between PLB monomer and pentamer plays a critical role in SERCA regulation. We compared the effect of R9C and pentamer-destabilizing transmembrane triple cysteine mutation (SSS) on PLB pentameric assembly. We co-expressed Cer- or YFP-tagged PLBWT, PLBR9C, PLBSSS, or PLBR9C+SSS in AAV-293 cells and measured intrapentameric Fluorescence Resonance Energy Transfer (FRET). PLBR9C and PLBR9C+SSS exhibited a significant increase in oligomerization as compared to PLBWT and PLBSSS, which was further enhanced by application of 100μM H2O2. In addition, we investigated the stability of PLB pentamer in WT, R9C, SSS, or R9C+SSS background by molecular dynamic simulation studies. Importantly, co-expression of CFP-PLBR9C and YFP-PLBR9C in adult rabbit ventricular myocytes exhibited a large and rapid increase in oligomerization over time after exposure to 100μM H2O2 as compared to PLBWT. To investigate the effect of PLBR9C on SR Ca2+ kinetics, we recorded Ca2+ transients in electrically paced adult cardiomyocytes overexpressing CFP-PLBR9C or CFP-PLBWT. PLBR9C overexpressing cardiomyocytes exhibited faster SR Ca2+ uptake as compared to PLBWT, and a blunted Ca2+ uptake/pacing frequency response. We propose that R9C mutation in PLB increases the stability of the PLB pentameric assembly by crosslinking of adjacent cysteine thiols via disulfide bonds, and these effects are enhanced during oxidative stress. The resultant decrease in availability of PLB monomeric species leads to decrease in inhibition of SERCA, inability to respond to stress, and eventual heart failure.