Background: Phospholamban (PLN) is a critical regulator of calcium cycling and contractility in the heart. The loss of arginine at position 14 in PLN (R14del) is associated with dilated cardiomyopathy (DCM) with a high prevalence of ventricular arrhythmias. How the R14 deletion causes DCM is poorly understood and there are no disease-specific therapies. Methods: We used single-cell RNA sequencing to uncover PLN R14del disease-mechanisms in human induced pluripotent stem cells (hiPSC-CMs). We utilized both 2D and 3D functional contractility assays to evaluate the impact of modulating disease relevant pathways in PLN R14del hiPSC-CMs. Results: Modeling of the PLN R14del cardiomyopathy with isogenic pairs of hiPSC-CMs recapitulated the contractile deficit associated with the disease in vitro . Single-cell RNA sequencing revealed the induction of the unfolded protein response pathway (UPR) in PLN R14del compared to isogenic control hiPSC-CMs. The activation of UPR was also evident in the hearts from PLN R14del patients. Silencing of each of the three main UPR signaling branches (IRE1, ATF6, or PERK) by siRNA exacerbated the contractile dysfunction of PLN R14del hiPSC-CMs. We explored the therapeutic potential of activating the UPR with a small molecule activator, BiP protein Inducer X (BiX). PLN R14del hiPSC-CMs treated with BiX showed a dose-dependent amelioration of the contractility deficit of in both 2D cultures and 3D engineered heart tissues without affecting calcium homeostasis. Conclusions: Together, these findings suggest that the UPR exerts a protective effect in the setting of PLN R14del cardiomyopathy and that modulation of the UPR might be exploited therapeutically.

These findings implicate pulmonary endothelial cells as key regulators of pulmonary artery smooth muscle cell and monocyte behavior in chronic thromboembolic pulmonary hypertension and suggest a potential mechanism for the progression from unresolved thrombus to fibrotic vascular remodeling.