Loss of GATA4 C-Terminus by p.S335X Mutation Modulates Coronary Artery Vascular Smooth Muscle Cell Phenotype.

Ting-Yan Yu,Hong-Lang Huang,Qing-Wen Liu,Fang-Fang Ma,Lei Zhou,Wei Zhang,Xin-Xin Chen,Azizah Ugusman

doi:10.1155/2021/3698386

Abstract

Coronary artery disease (CAD) has been the leading cause of morbidity and mortality worldwide, and its pathogenesis is closely related with the proliferation and migration of vascular smooth muscle cell (VSMC). We previously reported a truncated GATA4 protein lacking C-terminus induced by p.S335X mutation in cardiomyocyte from ventricular septal defect (VSD) patients. However, it is still unclear whether GATA4 p.S335X mutation could influence the development of CAD. GATA4 wild-type (WT) and p.S335X mutant (MU) overexpression plasmids were constructed and transfected transiently into rat coronary artery smooth muscle cell (RCSMC) to observe the proliferative and migratory abilities by MTS and wound healing assay, respectively. PCR array was used to preliminarily detect the expression of phenotypic modulation-related genes, and QRT-PCR was then carried out to verify the screened differentially expressed genes (DEGs). The results showed that, when stimulated by fetal bovine serum (10%) for 24 h or tumor necrosis factor-α (10 or 30 ng/ml) for 10 or 24 h, deletion of GATA4 C-terminus by p.S335X mutation in GATA4 enhanced the proliferation of RCSMC, without alteration of the migration capability. Twelve DEGs, including Fas, Hbegf, Itga5, Aimp1, Cxcl1, Il15, Il2rg, Il7, Tnfsf10, Il1r1, Irak1, and Tlr3, were screened and identified as phenotypic modulation-related genes. Our data might be beneficial for further exploration regarding the mechanisms of GATA4 p.S335X mutation on the phenotypic modulation of coronary VSMC.

Highlights

Coronary artery disease (CAD) is an atherosclerotic disease affecting the global human health and has been found to be the leading cause of death in both developed and developing countries [1]
QRT-PCR results showed that the expression of GATA4 mRNA in WT and MU groups was significantly higher than that in GATA4 empty vector (EV) group (P < 0:05, Figure 1(a)), indicating the successful transfection of GATA4 WT and MU plasmids
Western blot results demonstrated that deletion of GATA4 C-terminus induced by p.S335X mutation decreased the molecular weight of target protein in MU group (40 KDa) compared to that in WT (55 KDa), i.e., the GATA4 protein was truncated (Figures 1(b) and 1(c))

Summary

Introduction

Coronary artery disease (CAD) is an atherosclerotic disease affecting the global human health and has been found to be the leading cause of death in both developed and developing countries [1]. Vascular smooth muscle cell (VSMC) is a major cell type presenting at all stages of atherosclerosis [2]. Unlike skeletal muscle cells or cardiomyocytes, VSMCs are not terminally differentiated and maintain phenotypic plasticity [3]. VSMCs present as the contractile phenotype located in media and exhibit extremely low proliferative rate, as well as synthetic activity [4]. In the presence of environmental stimuli, such as inflammatory mediators, growth factors, and mitogens, VSMCs dedifferentiate into synthetic phenotype and lose the ability to contract, but migrate, proliferate, and accumulate into the intima [5]. VSMC dedifferentiation produces extracellular matrix and participates in fibrous cap formation [6] and subsequently accelerates the process of atherosclerosis [7]

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Discussion

Conclusion