Induced Pluripotent Stem Cells Technology For Investigating Familial Dilated Cardiomyopathy Due To Lamin A/C Mutations

Crasto, Silvia (2019). Induced Pluripotent Stem Cells Technology For Investigating Familial Dilated Cardiomyopathy Due To Lamin A/C Mutations. PhD thesis The Open University.



Mutations of LMNA gene, encoding the proteins Lamin A and C, are common causes of familial dilated cardiomyopathy (DCM), typically manifesting in association with cardiac conduction defects. LaminA/C regulate various nuclear functions, including gene transcription and chromatin organization. Most of human studies on LaminA/C so far were conducted on fibroblasts, while those focusing on cardiomyocytes (CMs) are scarce. Thus, we generated a human cardiac model of LMNA-dependent cardiomyopathy (LMNA-CMP) by differentiating CMs from induced pluripotent stem cells (iPSCs) carrying the K219T and R190W LMNA mutations (LMNA-CMs). In vitro, these cells recapitulate morphological features of DCM. RNA-sequencing experiments revealed profound differences between control and LMNA-CMs transcriptomes. Remarkably, the main cardiac sodium channel gene (SCN5A), was one of the most downregulated gene. Accordingly, electrophysiological studies in LMNA-CMs showed a significant reduction of the maximal upstroke velocity and the related properties of the action potential, accompanied by a reduction of the peak sodium currents and diminished conduction velocity. Functional experiments also revealed diminished contractile force in LMNA mutated cells. Biochemical studies confirmed the reduction of SCN5A gene, and its encoded protein Nav1.5, and showed an increased binding of LaminA/C to its promoter. A higher deposition of H3K27me3 marker, together with an increased binding of its catalysing complex PRC2, was also found at that genomic locus. Furthermore, 3D-FISH studies indicated a preferential localization of SCN5A gene at the nuclear periphery. Similar findings were obtained in relation to genes of the contractile pathways. Altogether, these data support a mechanism by which LaminA/C influence cardiac function by directly acting on transcriptional regulation of genes that control CMs functionality. Studies conducted on CMs from CRISPR/Cas9 gene-edited iPSCs proved the causality between the LMNA mutation and the observed phenotypes.

Finally, experiments for evaluating the efficacy of Remodelin molecule also demonstrated that the generated models are suitable for drug testing.

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