Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation

Abstract

The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca 2+ transient decay time, Ca 2+ -load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow-up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non-failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca 2+ -binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca 2+ -scavenging, suggesting impaired local Ca 2+ cycling as an important disease culprit.

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.…”

“…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. Since we did not measure contraction, this issue is not directly relevant to the present data; however, it should be considered when interpreting the pathophysiology of mutations leading to major contractile failure.…”

Recent Advances in Modeling Mitochondrial Cardiomyopathy Using Human Induced Pluripotent Stem Cells

“…A recent study by Cuello and co-workers described a tight correlation between phospholamban gene mutations and mitochondrial dysfunction. Particularly, patients’ iPSC-CMs harboring the PLN R14del variant depicted a detrimental mitochondrial function by displaying a low oxygen consumption rate as well as elevated mitochondrial ROS production ( Cuello et al, 2021 ). Notably, whereas sarcoplasmic reticulum function remained unaltered, impairments of the endoplasmic reticulum to mitochondria crosstalk were detected resulting in lipid accumulation, mitochondrial dysfunction and degeneration, suggesting a cause-consequence correlation provided by a deleterious cytoplasmic Ca 2+ signaling ( Cuello et al, 2021 ).…”

Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction

“…Furthermore, PLA has been widely utilized to study ER-mitochondria interactions in disease. Decreased MERCS were observed in cardiomyopathy caused by phopsholamban p. Arg14del mutation ( Cuello et al, 2021 ), in Charcot-Marie-Tooth type 2A (CMT2A, a dominant axonal form of peripheral neuropathy due to mutation in MFN2 ( Bernard-Marissal et al, 2019 )) and also in conditions such as amyotrophic lateral sclerosis and frontal dementia (ALS/FTD) due to defects in fused in sarcoma (FUS) ( Stoica et al, 2016 ). In addition, overexpression of α-Synuclein, a protein that accumulates in patients with Parkinson’s disease, disrupts binding between tethering proteins VAPB and PTPIP5 at MERCS ( Paillusson et al, 2017 ).…”