LUMA p.S358L mutation and cardiac diseases: a molecular and functional analysis in induced Pluripotent Stem Cell (iPSC)-based cardiac models

BACKGROUND: LUMA (also called Transmembrane protein 43) is a well-conserved gene that encodes a protein of 400 amino acids. Mutations in LUMA have been associated with Arrhythmogenic Right Ventricular Dysplasia and Emery-Dreifuss muscular dystrophy, which cause cardiomyopathy, arrhythmia, and muscular dystrophy in patients who carry the variants. Despite this evidence, very few studies were conducted to investigate the pathogenic role of the LUMA gene and to better characterize the biological function of its encoded protein. METHODS: We developed a human model of LUMA p.S358L-dependent cardiomyopathy through induced pluripotent stem cells (iPSCs) technology. iPSCs were generated from skin fibroblasts of the patient (carrying the mutation) and from a family-matched control (WT), using a lentiviral-based strategy. iPSCs were then differentiated into cardiomyocytes (iPSCs-CMs). Molecular studies addressed localization and expression levels of LUMA, Lamin A/C, and Emerin, as well as the effect of the mutation on nuclear morphology and chromatin organization, by immunofluorescence, Western blot and real-time PCR. Functional properties of LUMA p.S358L CMs were determined by conventional whole-cell patch-clamp techniques. Expression levels of genes encoding the main cardiac ionic currents were analyzed through real-time PCR. RESULTS: We generated a human model of LUMA p.S358L-dependent cardiomyopathy, demonstrating that this specific mutation does not prevent the ability of mutant iPSCs to differentiate into beating iPSC-CMs. We found that LUMA is mostly a cytoplasmic protein abundantly located on the cell membrane, in particular at the cell-to-cell junction, when both WT and mutant iPSC-CMs are organized in clusters. Interestingly, the mutation significantly alters the nuclear morphology of the cells that show misshapen nuclei and an irregular lamina with invaginations, lobulations, and herniations. In addition, a loss of heterochromatin has been observed in mutant cells. At the functional level, LUMA p.S358L mutation associates with a more depolarized phenotype in iPSC-CMs and altered properties of cardiac excitability in CMs electrically forced to -85 mV. Results from gene expression studies indicates a profound downregulation of the ATP1A2 gene (encoding for the catalytic subunit of the Na+/K+-ATPase) in mutant CMs, while expression levels of genes encoding for the main cardiac ionic currents, LUMA itself and the nuclear proteins Emerin and Lamin A/C, are unchanged. CONCLUSIONS: Through the reprogramming of fibroblasts from two siblings, a proband and the related healthy control, we generated a cardiac human model of LUMA p.S358L-dependent cardiomyopathy, in which we characterize LUMA localization and started to address its role in the human CM by investigating molecular and functional effects of the p.S358L mutation. Our results support a link between the LUMA p.S358L mutation, nuclear defects and heterochromatin loss in patient’s cells, and suggest that the downregulation of the ATP1A2 gene in mutant CMs could underlie the electrophysiological defects recorded in human CMs differentiated from patient-specific iPSCs.