Effects of cigarette smoke components on myocardial differentiation of mouse embryonic stem cells

Abstract

The heart is the first organ formed in the developing fetus, and abnormal development of the heart is a major cause of fetal death. The adverse effects of cigarette smoke on the heart have been well established, but it is not well understood how cigarette smoke components regulate signaling molecules and cardiac specific functions during the early differentiation stage of the embryonic heart. In this study, we identified changes in the size of mouse embryoid bodies (mEBs) in response to treatment with cigarette smoke extract (CSE) via regulation of HDAC2, p53, p21, and cyclin D1 protein expression, which are cardiac differentiation and cell-cycle markers, respectively.In addition, exposure of mouse embryonic stem cells (mESCs) to cigarette smoke components inhibited myocardial differentiation and development through the expression of HDAC1, HDAC2, GATA4, NKX2-5, TBX5, HAND1, and Troponin I. Long-term exposure studies showed that CSE and nicotine may delay the development of mouse cardiomyocytes from mESCs and inhibit the contractibility, which is a fundamental function of the heart. Taken together, these findings suggest that cigarette smoke components, including nicotine, may affect abnormal myocardial differentiation and development. K E Y W O R D S cardiomyocyte, cell cycle, cigarette smoke extract, HDACs, mouse embryonic stem cells, myocardial differentiation, nicotine 1 | INTRODUCTION Embryonic stem cells isolated from the inner cell mass in the blastocyst stage can differentiate into three germ layers, the endoderm, mesoderm, and ectoderm. 1 The mesoderm layer forms connective tissues and muscles including cardiac muscle throughout the body. The heart is the first organ formed and differentiated in the developing embryo and fetus. 2,3 In early embryonic development, cardiac cells begin to develop through specific protein signaling. During embryogenesis, NK2 homeobox 5 (NKX2-5) is expressed in early cardiac mesoderm cells throughout the left ventricle and atrial chambers. 4,5 NKX2-5 activates a number of downstream transcription factors (such as myocyte enhancer factor-2 [MEF2] and GATA), which activate the expression of cardiac muscle specific proteins. Mutations in NKX2-5 result in heart development defects and congenital heart malformations. 6-8 The heart tube undergoes right-ward looping to change from anterior/posterior polarity to left/right polarity. 9 Looping alsodepends on heart specific proteins activated by NKX2-5 such as heartand neural crest derivatives-expressed protein (HAND) 1 and HAND2. 10,11 HAND1 is localized to the left ventricle while HAND2 is localized to the right ventricle. 12 When the heart is developed through this specific protein signaling, it starts to beat and pump blood at about 22-23 days, with blood flow beginning in week 4. 2,3 It is well known that cigarette smoke adversely affects the development of early embryos. [13][14][15] Cigarette smoke contains over 4000

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