Abstract 530: Mechanisms Underlying Phospholamban L39 Stop (PLN L39X) Cardiomyopathy

Abstract

Background: Defective calcium (Ca++) handling is a hallmark of HF across species. Together with the Sarco/Endoplasmic Reticulum Ca-ATPase (SERCA2a), Phospholamban (PLN) has emerged as a critical regulator of Ca++homeostasis. Worldwide, PLN mutations are identified with increasing frequency in patients with dilated, hypertrophic and arrhythmogenic cardiomyopathy (CMPs) but the causative defects leading to the CMP remain incompletely understood. While preclinical studies have unequivocally shown that absence of PLN (PLNKO) is therapeutic in rodent HF models, the discovery of a human pathogenic mutation (L39X) presumed to be the human equivalent of the PLNKO lead to the conclusion that PLN ablation was lethal in human, mitigating any enthusiasm in targeting PLN inhibition in PLN-associated disease or HF treatment. The objective of this proposal is to levarge the use of “induced-pluripotent” stem cells (iPSCs) derived cardiomyocytes (CM) from homozygote L39X carriers to elucidate the role of PLN (L39) in human pathophysiology. Methodology and Results: We obtained mononuclear cells from Homozygotes (Hom) L39 carriers and generated 11 iPSC clones. To derive CMs, we used the direct differentiation method temporally modulating the Wnt/β-catenin signaling. Immunocytostaining revealed positive expression of cardiac troponin T as well as PLN. In Hom L39 derived CMs, PLN showed an abnormal cytoplasmic distribution, formed intracellular aggregates and there was loss of perinuclear localization when compared to matched WT iPSC-CMs. Using fura-2AM, we observed decreased calcium transient amplitude in iPSC-CMs from L39 compared to WT with prolongation of the time constant of relaxation and early after depolarization (EAD). Lastly, we saw a 70% and 50% reduction in the protein and mRNA expression of PLN and SERCA2a respectively. Conclusions: Our data suggest that the L39 PLN mutant is expressed but mis-located within the cardiomyocytes. The mis-location of PLN was associated to decreased SERCA2a expression impaired Ca++ handling and increased arrhythmogenicity. Further studies will be required to fully elucidate the impact of the mutation in HF pathophysiology

Phospholamban p.Leu39* Cardiomyopathy Compared with Other Sarcomeric Cardiomyopathies: Age-Matched Patient Cohorts and Literature Review

“…Different studies suggest that the p.Leu39* PLN mutant is expressed but mis-located within the cardiomyocytes [22,23]. The mis-location of PLN was associated to decreased SERCA2a expression and impaired Ca 2+ handling in human pathophysiology.…”