241 Unfolded Protein Response in PLN R14del Cardiomyopathy 9 INTRODUCTION Phospholamban (PLN) encodes a critical regulatory protein of Ca2+ cycling and is a primary mediator of the β-adrenergic effects, resulting in enhanced cardiac output.1 The levels of PLN and its degree of phosphorylation profoundly influence the activation state of the sarcoplasmic reticulum calcium ATPase (SERCA2a). In the dephosphorylated state, PLN interacts with SERCA2a and shifts its Ca2+ activation toward lower apparent Ca2+ affinity. On protein kinase A–mediated phosphorylation, the inhibitory interaction between PLN and SERCA2a is abolished, and the apparent Ca2+ affinity is raised. Thus, PLN is the ratedetermining factor for Ca2+ reuptake by SERCA2a and a key regulator of contractility in the heart. Dilated cardiomyopathy (DCM) is the leading cause of heart failure, and familial DCM is responsible for up to a third of the reported cases.2 Various pathogenic genetic variants have been linked to DCM, including mutations in the coding region of the PLN gene. DCM caused by the deletion of the arginine 14 codon in the PLN gene (R14del) is associated with prevalent ventricular arrhythmias, heart failure, and sudden cardiac death.3,4 The limited mechanistic understanding of how the R14del contributes to the overall clinical presentation translates to the lack of disease-specific therapeutic strategies. In this study, we investigated the molecular underpinnings of PLN R14del-induced cardiomyopathy by leveraging the power of human induced pluripotent stem cells (hiPSCs), CRISPR/Cas9 genome editing, and single-cell RNA sequencing (scRNA-seq) technologies. PLN R14del hiPSC-derived cardiomyocytes (hiPSCCMs) faithfully recapitulated the contractile dysfunction observed in PLN R14del-induced cardiomyopathy. At the molecular level, we observed an elevated endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in the PLN R14del hiPSC-CMs compared with isogenic controls. Molecular and pharmacological modulation of the UPR pathway revealed a protective role of the UPR activation in PLN R14del hiPSC-CMs. Our findings suggest a mechanistic link between proteostasis and PLN R14del-induced pathophysiology that could be exploited to develop therapeutic strategies for PLN R14del cardiomyopathy. METHODS Human iPSC reprogramming and culture. Peripheral blood mononuclear cells (PBMCs) were reprogrammed to hiPSCs using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions with modifications. PBMCs were expanded in StemPro-34 SFM media (Life Technologies) supplemented with cytokines: SCF (100 ng/mL), FLT-3 (100 ng/mL), IL-3 (20 ng/mL), IL-6 (20 ng/mL) and EPO (2 U/mL). After six days, 2 x105 PBMCs were transduced with the three CytoTune® 2.0 reprogramming vectors in one well of a 24-well plate in 0.5 mL in complete StemPro-34 SFM. Twenty-four hours post-transduction the PBMCs were pelleted by centrifugation, resuspended in fresh complete StemPro-34 SFM and plated in one Matrigel-coated well of a 24-well plate. Three days later the media was replaced with StemPro-34 SFM without cytokines and cultured
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