Abstract Body: Background: Cardiovascular disease (CVD) is the number one cause of death globally, accumulating to over 17 million lives per year. Dilated cardiomyopathy (DCM) is a condition in which the chambers of the heart enlarge and lose their ability to contract, thereby impairing systolic function. The RNA-binding motif protein 20 (RBM20) gene, a nuclear protein that aids in the alternative splicing of genes, targets central cardiac genes like titin (TTN). Irregular splicing of TTN by pathogenic variants in RBM20 is thought to be linked to DCM.
Methods: To study this mutation, peripheral blood mononuclear cells were isolated from patients carrying the DCM mutations and reprogrammed using Sendai virus vectors to induced-pluripotent stem cell (iPSC) lines for generation. With these cells, we immunoassayed for the expression of pluripotency markers, SOX2, NANOG, and OCT3/4. Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), karyotyping, and sequencing were used to confirm the presence of pluripotency markers and RBM20-specific mutations.
Results: Mycoplasma free environment confirmed aseptic conditions in cells. The iPSCs transfected with Yamanaka factors displayed typical morphology as cardiomyocytes and expressed key markers after one week. Immunofluorescence (IF) analyses revealed the expression of the markers in iPSCs derived from both patients. IF analyses for OTX2, Brachyun, and SOX17 demonstrated appropriate formation of germ layers: ectoderm, mesoderm, endoderm, respectively. qPCR for SOX2 and NANOG confirmed their expression in both patients carrying RBM20 variants. Sanger sequencing analyses further confirmed the mutations (c.1901G>A and c.3595G>A). Karyotyping analysis in these cell lines with patient specific mutations revealed an absence of abnormalities at the chromosomal level. Thus, successful characterization of iPSC lines from DCM patients carrying different RBM20 mutations was achieved.
Conclusion: We established new cell lines with specific RBM20 mutation from DCM patients. These cells mimic disease criteria for DCM and provide a therapeutic rationale for clinical trials and drug development. Future directions will focus on using the iPSC cell lines to assess the pathophysiology of DCM.