Characterisation of hypertrophic response and the effect of a GATA4-targeting compound on the phenotype of hypertrophic cardiomyopathy patient-derived hiPSC-cardiomyocytes

Abstract

Abstract Introduction Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. Patient-derived human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an informative platform to study disease pathomechanisms. Transcription factor GATA4 serves a significant role in cardiomyocyte hypertrophy, and GATA4-targeted compounds inhibit hypertrophy [1]. Purpose This study aims at identifying phenotypic differences between patient-derived hiPSC-CMs and control hiPSC-CMs and at investigating the effect of a novel GATA4-targeting compound C-2021 on the patient-derived hiPSC-CMs phenotype. Methods hiPSC-CMs were differentiated from a healthy, control line (iPS(IMR90)-4; here, control hiPSC-CMs) and from HCM patient-derived hiPSCs (HCM hiPSC-CMs) that carry a mutation in the MYBPC3 gene. Two types of hypertrophic stimuli were applied to hiPSC-CMs: mechanical stretch and endothelin-1 (ET-1) treatment. For pharmacological assays, compound C-2021 (at 30 µM) or vehicle (0.1 % DMSO) were introduced to hiPSC-CMs one hour prior to the stimuli. hiPSC-CMs were exposed to cyclic equibiaxial mechanical stretching for 24 h or 72 h using Flexcell FX-5000 Tension System (Flexcell International Corporation) [2]. The expression of hypertrophic markers (such as NPPB, NPPA) and structural proteins (including α-myosin and β-myosin heavy chains; MYH6 and MYH7, respectively) was investigated with qPCR. The relative gene expression was analysed using the 2-ΔΔCt method using 18S and ACTB as reference genes. Furthermore, hiPSC-CMs were subjected to ET-1 at 100 nM for 24 h and proBNP expression was quantified using high-content analysis (HCA). Results HCM hiPSC-CMs displayed increased basal expression of NPPB (Fig. 1A-B), lower expression of MYH6 (Fig. 1C) and higher expression of MYH7 (Fig. 1D) compared to control hiPSC-CMs, which is in line with the hypertrophic disease phenotype. Control and HCM hiPSC-CMs responded to 24 h and 72 h mechanical stretching by increased NPPB expression (Fig. 1A-B). At 72 h, the increase in NPPB expression was more pronounced in the HCM hiPSC-CMs (Fig. 1B). The compound C-2021 inhibited stretch-induced NPPB expression after 72 h stretching. HCA results indicate that ET-1 induced a 17-fold increase in proBNP-positive control hiPSC-CMs, and this effect was attenuated by C-2021 treatment. Interestingly, ET-1 did not enhance proBNP expression in HCM hiPSC-CMs (Fig. 2). Conclusions Our results show that the HCM hiPSC-CMs differ from the control hiPSC-CMs in response to hypertrophic stimuli and in myosin heavy chain isoform expression. Additionally, C-2021 may have anti-hypertrophic effects in both in control and HCM hiPSC-CMs based on the downregulation of NPPB expression, and the decrease in proBNP-positive hiPSC-CMs. Further studies are needed to elucidate the exact mechanisms underlying HCM and the action of C-2021 on the HCM hiPSC-CM phenotype.