255 Unfolded Protein Response in PLN R14del Cardiomyopathy 9 treated with BiX (Figure VI in the Data Supplement). In agreement, the expression levels of PLN and other key calcium handling proteins, including SERCA2a, NCX1 (sodium-calcium exchanger), RYR2 (ryanodine receptor 2), and CASQ2 (calsequestrin 2), were similar between BiX and dimethyl sulfoxide–treated PLN R14del hiPSC-CMs (Figure VII and Table VI in the Data Supplement). Collectively, these data suggest that stimulating the UPR pathway in PLN R14del hiPSC-CMs restored contractility to levels of isogenic controls without affecting calcium homeostasis. DISCUSSION Human iPSCs provide an opportunity for modeling DCM in vitro to understand the molecular consequences of pathogenic mutation such as PLN R14del. However, the inherited heterogeneity caused by a mixture of cell types in hiPSC-CM differentiation cultures and the genetic background of the donor present confounding factors for defining disease-specific phenotypes. To overcome these challenges, we generated multiple isogenic hiPSC lines carrying the R14del mutation and performed scRNA-seq on these genotype-defined hiPSC lines. This experimental approach can be widely used to define the specific contributions of pathogenic mutation in DCM and cardiomyopathies more broadly. Using this approach, we revealed that the PLN R14del mutation activates the UPR, an integrative intracellular signaling pathway that plays a critical role in the maintenance of proteostasis in the ER.25 Proteostasis, the balance among protein synthesis, folding, refolding, and degradation, is essential for the long-term preservation of cell and tissue function. With age, the ability of many cells and organs to maintain proteostasis is gradually compromised.26 Accumulating evidence suggests that UPR is activated in response to a loss of proteostasis in the ER and the corresponding accumulation of protein aggregates, which characterizes age-related diseases and protein folding disorders such as Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis.10 Reminiscent of these diseases, aggregation of PLN protein is a hallmark of PLN R14del DCM,12 suggesting that the disruption of proteostasis plays a role in disease presentation. It is tempting to speculate that the altered stability and folding kinetics of the PLN R14del mutant directly activate the UPR in mutant hiPSC-CMs and human heart tissues. It will be of interest to explore this hypothesis further in the future. Our isogenic models allowed us to perform high-throughput genetic and pharmacological assays both to understand the underlying pathological mechanisms and to identify therapeutic targets to prevent or treat such diseases. We found that activation of UPR in PLN R14del hiPSC-CMs is protective because molecular inhibition of each of the 3 UPR sensors, IRE1, PERK, and ATF6, exacerbated the contractile dysfunction. Conversely, pharmacological enhancement of UPR by the small molecule agonist BiX ameliorated the contractility defect in PLN R14del hiPSCCMs. BiX activates the chaperone BiP, thereby increasing the activity of the UPR pathway. These observations are consistent with previous studies showing that activation of UPR suppressed disease onset and progression in cellular and animal models of neurodegenerative
RkJQdWJsaXNoZXIy MTk4NDMw