Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy

Abstract

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.

Characterization of the PLN p.Arg14del Mutation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

“…Nonetheless, lack of CaT amplitude changes has been consistently reported under conditions of perturbed SERCA2a modulation by PLN, even if the latter was associated with significant mechanical derangement [18,19]. In particular, engineered heart tissue (EHT), also generated by our group from the same hiPSC-CMs used in the present study, was characterized by a marked decrease in force development in the face of normal CaT amplitude [20,21]. While the resilience of CaT amplitude to perturbations may be accounted for by a robust homeostatic control [22], deranged contraction in spite of unchanged Ca 2+ signal would require additional explanations, such as a decrease in myofilament response to Ca 2+ , a hindrance to cell shortening or, as suggested by findings in EHTs [20], energetic incompetence.…”

“…Altogether, an alternative hypothesis for the pathogenesis of the clinical phenotype should be considered. An intriguing one is that the effect of PLN p.Arg14del on myocardial function may be independent of PLN role in modulating EC coupling, as recently suggested by the observation that PLN R14del phenotype may be reverted by enhancement of the "unfolded protein response" [21].…”

Antisense Therapy Attenuates Phospholamban p.(Arg14del) Cardiomyopathy in Mice and Reverses Protein Aggregation

“…Although it is yet unclear if protein aggregates are a cause or consequence of this disease, PLN aggregation clearly is a pathological biomarker as it preceded other cardiac abnormalities [ 15 ], and was resolved along with attenuation of HF. Along the same line of reasoning, Feyen et al recently demonstrated that knockdown of endoplasmic reticulum (ER) stress sensors exaggerated contractile dysfunction of PLN-R14del-induced pluripotent stem cell-derived cardiomyocytes, while stimulation of the unfolded protein response improved contractility and force development to amplitudes that were similar to cardiomyocytes derived from healthy donors [ 32 ].…”

Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy

“…This approach is similar to previous studies, whereby isogenic gene corrected iPSC lines were generated to study disease biology using patient-derived iPSC-CMs. [87][88][89][90] Further, the use of patient-derived iPSCs presumes that the genetic background is permissive for the disease without major limitations from disease-modifying effectors. 91 Finally, we employed snRNA-seq because conventional microfluidics-based single-cell RNA-sequencing technologies are not suitable for adult cardiomyocytes.…”