Expression and localization of intermediate filament synemin isoforms in human pluripotent stem cells and their cardiac derivates

Abstract

Synemin is an intermediate filament family involved in the cytoarchitecture of skeletal, cardiac and smooth muscle cells. In mammals, the SYNM gene encodes 3 isoforms: H (α, 170 kDa), M (β, 140 kDa) and L (Low, 41 kDa) through alternative splicing. In mouse, H and M isoform expression begins in the embryo while L isoform is only found in adult. Mouse embryonic pluripotent stem cells express M isoform as well as Oct-3/4 and SSEA-1. Two missense mutations have been recorded in patients with dilated cardiomyopathy and, α and β synemin levels increased in failing human hearts. Little is known about synemin expression in human induced pluripotent stem cells (hiPSC) and their cardiac derivatives. The aims are: (1) To characterize expression of synemin isoforms in hiPSC differentiation towards the cardiac lineage; (2) To analyze the impact of synemin in a human in vitro model of cardiac hypertrophy using hiPSC-derived cardiomyocytes (hiPSC-CMs). Expression of synemin isoforms was investigated in 2 lines of healthy donor-derived hiPSC and hiPSC-CMs by RT-qPCR, Western blot and immunofluorescent analysis. Synemin and α-actinin immunostaining was carried out on hiPSC-CMs treated 7 days with 200 nM AngII or 100 nM Iso following by an estimate of cell size and contraction measurements (ImageJ). Two different lines of healthy donor-derived hiPSC express the 3 isoforms of synemin at mRNA and protein levels. Synemin protein is detected in the cytoplasm and potentially in the nucleus of hiPSC expressing Nanog and Oct3/4. In hiPSC-CMs at 30 days of differentiation, expression of α, β and L isoforms increased with a fold change of 35, 80 and 114 respectively compared with hiPSC. Immunostaining reveals a cytoplasmic localization of synemin with striated pattern. An increase of synemin protein expression and nuclear localization was shown in hiPSC-CMs treated by hypertrophic agents. We showed for the first time that hiPSC and hiPSC-CMs express all known synemin isoforms. Moreover, subcellular localization of this protein change under hypertrophic conditions. Based on the literature, these data could suggest a potential role of synemin in cell motility, proliferation and also in the modulation of cardiac hypertrophy. To better understand these functions we have generated two synemin KO hiPSC lines using Dual-Guide CRISPR-Cas9 system of which characterization is ongoing.