Exploring the pathophysiological role of the PLN-R14del mutation in a novel heterozygous mouse model of dilated cardiomyopathy.

Abstract

The sarco/endoplasmic Ca2+-ATPase (SERCA2a) and its natural inhibitor phospholamban (PLN) play a pivotal role in cardiac excitation-contraction coupling. A heterozygous deletion of arginine 14 of the PLN protein (R14del) is associated with a dilated cardiomyopathy (DCM). It has been previously shown that overexpression of PLN-R14del leads to a substantial decrease in SERCA2a affinity for Ca2+, thus proposing that a “superinhibitory” effect of mutant PLN on SERCA2a may lead to the DCM phenotype. This theory is not consistently supported by recent experimental evidences, thus suggesting alternative pathogenetic mechanisms. To shed light on the debated pathophysiological mechanisms of PLN-R14del DCM exploiting a novel heterozygous transgenic model (Mut) that recapitulates the clinical human phenotype. SERCA2a activity and intracellular Ca2+ dynamics have been measured in cardiomyocytes (CMs) isolated from 8-12 weeks old Mut mice and WT littermates. To compare mutation effect with pharmacological SERCA2a modulation, CMs were perfused with the pure SERCA2a activator PST-3093. To test the involvement of metabolic alterations, oxidative phosphorylation and glycolysis have been investigated. In myocardial homogenates, the KdCa of SERCA2a ATPase activity was lower in Mut. In Mut CMs, the Ca2+ τdecay was shorter and diastolic Ca2+ was lower compared to WT cells. When applied to WT CMs, PST-3093 decreased τdecay to approach the value found in Mut. When applied to Mut, PST-3093 had no effect on CaT parameters. In Mut cells O2 consumption and glycolytic acidification were reduced. These results indicate enhancement of SR Ca2+ uptake rate in Mut, which is compatible with diminished PLN inhibitory function. Future work will be directed to establish if the metabolic abnormalities may be related to altered Ca2+ dynamics, or be an independent mechanism for PLN-R14del DCM.