Abstract Body: Background: Dilated cardiomyopathy (DCM) is characterized by impaired myocardial function, leading to high morbidity and mortality, and the only curative treatment is heart transplantation. Genetic mutations predispose approximately 40% of DCM cases; however, this knowledge has not yet translated into mechanism-based therapeutics. This project follows up on promising therapeutic targets that were identified in a proteome-wide screen using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).

Objective: To elucidate the role of SR/ER stress in the pathogenesis of inherited DCM in a patient-specific hiPSC-CM model.

Methods: A cardiac-specific protein-protein-interaction (PPI) network analysis was conducted to identify physiological processes capable of restoring contractility of DCM cardiomyocytes. Metabolically matured iPSC-CMs were generated from patients with inherited DCM harboring pathogenic variants in the TNNT2, PLN, RBM20, and LMNA genes. Isogenic control (IC) lines were created for each disease variant to establish baseline contractility and ER stress assessment for comparative analyses. Synthetic siRNAs were employed to selectively knock down individual target genes. High-throughput kinetic imaging-based contractility measurements of iPSC-CMs were performed utilizing Kinetic Imaging Cytometer (KIC). ER stress was evaluated through the implementation of both an XBP1 splicing reporter system and a fluorescent genetically encoded SR/ER calcium indicator, R-CEPIA.

Results: Cardiac-PPI network analysis identified a set of target genes related to SR/ER stress homeostasis. Individual knockdown of such target genes with synthetic siRNAs restored key contractile parameters, including peak contraction amplitude, contractile rate, and relaxation rate.

Conclusions: This study builds upon a large-scale, functional genomics screen, which identified a set of genes that ameliorate contractile dysfunction in hiPSC-CMs carrying pathogenic DCM variants. Informatics analysis revealed that a significant subset of target genes are involved in the regulation of SR/ER stress homeostasis. This was further validated by in vitro data using siRNA targeting individual genes in various patient-specific iPSC-CM lines. These results linked genes that regulate ER/SR stress to physiological processes relevant to heart failure, thereby defining novel therapeutic target space to treat diverse forms of inherited DCM.