299 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 INTRODUCTION The pathogenic Phospholamban (PLN) p.Arg14del (PLN-R14del) founder mutation accounts for approximately 10% of all cases of dilated cardiomyopathy (DCM) and 15% of all cases of arrhythmogenic cardiomyopathy (ACM) in the Netherlands.1 PLN is a sarcoplasmic reticulum (SR) protein that reversibly binds to SERCA2a pumps, reducing its affinity for calcium (Ca2+), and thereby decreasing Ca2+ re-uptake into the SR. PLN is phosphorylated by protein kinase A (PKA) or Ca2+-calmodulin-dependent protein kinases (CaMKII) during -adrenergic signaling.2 Phosphorylation abolishes PLN's inhibitory effects, resulting in increased Ca2+ flux into the SR, which transports approximately 70% of all Ca2+ ions in the human heart.4 Dysregulation of SERCA and PLN and concomitant Ca2+ dysfunction have been correlated with dysfunctional contractility and heart diseases.3 The clinical spectrum of disease phenotypes caused by PLN-R14del ranges from early-stage ECG and ultrasound strain abnormalities to a moderate disease stage consisting of decreased left ventricular function and, ultimately, progression into congestive biventricular failure.5–7 Moreover, a more severe biventricular phenotype is characterized by dilation of the heart and/or myocardial fibrofatty tissue replacement, which provides the risk for ventricular arrhythmias.8 The previously described disturbed Ca2+ regulation9, identification of protein aggregates10, and increase in fibrosis11 in PLN-R14del patient hearts have been observed in young homozygous PLN-R14del mice, which also demonstrated inducibility of ventricular arrhythmias (VAs), suggestive of aberrant Ca2+ handling.12 Recent in vitro studies by us and others have shown reduced contractility in human induced pluripotent stem cells (hiPSC)-derived cardiomyocytes (hiPSC-CM) from individuals carrying the R14del heterozygous mutation, which was mediated by an elevated Unfolded Protein Response (UPR)/Endoplasmic Reticulum (ER) stress response13,14 and impairment of cardiac metabolism.14,15 Moreover, frequent episodes of irregular Ca2+ waves have been observed in hiPSC-CMs harboring the PLN-R14del mutation.16 The mechanistic understanding of how the PLN-R14del mutation contributes to disease progression and whether this can be mitigated via disease-specific therapeutic strategies remains unknown. Three-dimensional (3D) cardiac cell culture and tissue engineering techniques reflect better human cardiac physiology compared to regular 2D culturing. Human cardiac spheroids (hCSs) are small and have been proven to recapitulate developmental stages, tissue organization, and function, as seen in whole organs.17–19 Moreover, the development of an hCS screening platform could be essential not only to better recapitulate pathophysiological phenotypes and study molecular mechanisms but also to allow testing therapeutic strategies in an unbiased, high-throughput manner. In this study, we established a human cardiac spheroid model of PLN-R14del patient-derived hiPSC and observed the presence of various pathophysiological phenotypes of the PLNR14del cardiomyopathy, including fibrosis, metabolic dysfunction, UPR/ER stress, and Ca2+ handling/contractile dysfunction. We explored the therapeutic potential of a constitutively active inhibitor-1 (I-1c) delivered by AAV2/8 to improve PLN phosphorylation and restore cardiomyocyte function.
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