Abstract Background Phospholamban (PLN) is a key regulator of cardiac function connecting adrenergic signaling and calcium homeostasis. The R9C mutation of PLN is known to cause early onset dilated cardiomyopathy (DCM) and premature death, yet the detailed mechanisms underlie the pathologic remodeling process are not well defined in human cardiomyocytes. The aim of this study is to unravel the role of PLN R9C in DCM and identify potential therapeutic targets. Methods PLN R9C knock-in (KI) and patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated and comprehensively examined for their expression profile, contractile function, and cellular signaling under both baseline conditions and following functional challenges. Results PLN R9C KI iPSC-CMs exhibited near-normal morphology and calcium handling, slightly increased contractility, and an attenuated response to β-adrenergic activation compared to wild-type (WT) cells. However, treatment with a maturation medium (MM) has induced fundamentally different remodeling in the two groups: while it improved the structural integrity and functional performance of WT cells, the same treatment result in sarcomere disarrangement, calcium handling deficiency, and further disrupted adrenergic signaling in PLN R9C KI cells. To understand the mechanism, transcriptomic analysis showed the enrichment of protein homeostasis signaling pathways specifically in PLN R9C KI cells in response to the MM treatment and increased contractile demands. Further studies also indicated elevated ROS levels, interrupted autophagic flux, and increased pentamer PLN aggregation in functionally challenged KI cells. These results were further confirmed in patient-specific iPSC-CM models, suggesting that functional stresses exacerbate the deficiencies in PLN R9C cells through disrupting protein homeostasis. Indeed, treating stressed patient cells with autophagy-accelerating reagents, such as metformin and rapamycin, has restored autophagic flux, mitigated sarcomere disarrangement, and partially rescued β-adrenergic signaling and cardiac function. Conclusions PLN R9C leads to a mild increase of calcium recycling and contractility. Functional challenges further enhanced contractile and proteostasis stress, leading to autophagic overload, structural remodeling, and functional deficiencies in PLN R9C cardiomyocytes. Activation of autophagy signaling partially rescues these effects, revealing a potential therapeutic target for DCM patients with the PLN R9C mutation. Graphic abstracts A graphic abstract is available for this article.

Mitigation of Stress-induced Structural Remodeling and Functional Deficiency in iPSC-CMs with PLN R9C Mutation by Promoting Autophagy