Changes of cytoskeleton and polarity in cardiomyocyte-like cells derived from left ventricular noncompaction patients

Abstract

Objective To investigate the changes and mechanism of the cytoskeleton and polarity in cardiomyocyte-like cells derived from patients with left ventricular noncompaction (LVNC). Methods After induced pluripotent stem cells (iPSCs) were isolated from LVNC patients and normal individuals, respectively, the obtained cells were induced to differentiate into cardiomyocyte-like cells. Then transcriptome sequencing was performed. Transmission electron microscopy (TEM) was employed to observe the changes of the myocardial cytoskeleton. Immunofluorescence assay was used to detect the location and expression of cytoskeleton-associated proteins, fibrous actin (F-actin), tubulin dimer (ɑ/β-tublin), and polar protein Par6 and vinculin. Western blotting was used to detect the expression of Rho/Rock pathway key protein, Ras homolog gene family member A (RhoA). Results Both the obtained iPSCs derived from LVNC patients and normal persons positively expressed the surface molecular markers of embryonic stem cells, Oct4, Sox2, SSEA4, and TRA1-81, and could be induced into cardiomyocyte-like clusters with autonomous pulsating rhythm. Immunofluorescence assay indicated that the cardiomyocyte-like cells expressed the surface markers of cardiomyocytes, cTnT and ɑ-actinin. The results of transcriptome sequencing showed that there were multiple differential genes related closely to microtubules between the 2 kinds of cardiomyocyte-like cells. Compared with the cells derived from the normal individuals, there was a disordered arrangement of the cytoskeleton with TEM, significantly decreased expression levels of ɑ/β-tublin (n=62, 226.4±65.1, P < 0.01, Z=-3.451), F-actin (n=62, (236.4±33.2), P < 0.001), and vinculin (n=57, 137.2±35.9), P < 0.001, Z=-3.852). and it was unevenly distributed and obviously concentrated in the cell membrane, and obviously up-regulation of Par6 (n=64, -132.7±43.5, P < 0.01, Z=-3.148) with the aid of immunofluorescence assay. Western blotting demonstrated that the expression of RhoA was significantly increased in the cells derived from the patient group (P < 0.01). Conclusion The cytoskeleton and polarity of cardiomyocyte-like cells derived from LVNC patients are indeed abnormal.

Keywords

• left ventricular noncompaction
• induced pluripotent stem cells
• cytoskeleton
• polarity
• ras homolog gene family member a)