KMUP-1 Ameliorates Ischemia-Induced Cardiomyocyte Apoptosis through the NO⁻cGMP⁻MAPK Signaling Pathways.

Meng-Luen Lee,Yu-Ying Chao,Zen-Kong Dai,Jwu-Lai Yeh,Bo-Yau Huang,Bin-Nan Wu,Erna Sulistyowati,Jong-Hau Hsu

doi:10.3390/molecules24071376

Abstract

To test whether KMUP-1 (7-[2-[4-(2-chlorophenyl) piperazinyl]ethyl]-1,3-dimethylxanthine) prevents myocardial ischemia-induced apoptosis, we examined KMUP-1-treated H9c2 cells culture. Recent attention has focused on the activation of nitric oxide (NO)-guanosine 3’, 5’cyclic monophosphate (cGMP)-protein kinase G (PKG) signaling pathway triggered by mitogen-activated protein kinase (MAPK) family, including extracellular-signal regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 in the mechanism of cardiac protection during ischemia-induced cell-death. We propose that KMUP-1 inhibits ischemia-induced apoptosis in H9c2 cells culture through these pathways. Cell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and apoptotic evaluation was conducted using DNA ladder assay and Hoechst 33342 staining. The level of intracellular calcium was detected using-Fura2-acetoxymethyl (Fura2-AM) staining, and mitochondrial calcium with Rhod 2-acetoxymethyl (Rhod 2-AM) staining under fluorescence microscopic observation. The expression of endothelium NO synthase (eNOS), inducible NO synthase (iNOS), soluble guanylate cyclase α1 (sGCα1), PKG, Bcl-2/Bax ratio, ERK1/2, p38, and JNK proteins were measured by Western blotting assay. KMUP-1 pretreatment improved cell viability and inhibited ischemia-induced apoptosis of H9c2 cells. Calcium overload both in the intracellular and mitochondrial sites was attenuated by KMUP-1 pretreatment. Moreover, KMUP-1 reduced intracellular reactive oxygen species (ROS), increased plasma NOx (nitrite and nitrate) level, and the expression of eNOS. Otherwise, the iNOS expression was downregulated. KMUP-1 pretreatment upregulated the expression of sGCα1 and PKG protein. The ratio of Bcl-2/Bax expression was increased by the elevated level of Bcl2 and decreased level of Bax. In comparison with the ischemia group, KMUP-1 pretreatment groups reduced the expression of phosphorylated extracellular signal-regulated kinases ERK1/2, p-p38, and p-JNK as well. Therefore, KMUP-1 inhibits myocardial ischemia-induced apoptosis by restoration of cellular calcium influx through the mechanism of NO-cGMP-MAPK pathways.

Highlights

Ischemic heart disease is the leading cause of global cardiovascular-related mortality with some 15.2 million deaths in 2016 [1,2]
We made a different exposure; normoxia and hypoxia, we no differences are denoted on cell viability rate among control, solvent, and KMUP-1 (1 and 10 μM)
These results suggest that KMUP-1 pretreatment inhibits the inhibits the elevation of [Ca2+ ]i cardiomyocytes under hypoxic stimulation

Summary

Introduction

Ischemic heart disease is the leading cause of global cardiovascular-related mortality with some 15.2 million deaths in 2016 [1,2]. The prediction made by the World Health Organization shows that ischemic heart disease will be the most common cause of death in the world until 2030 while the cause of disability will rise from sixth to third position [1]. A multitude of recent studies suggests that ischemia injury leads to undergoing cardiomyocyte apoptosis and results in myocardial cell loss. It develops cardiac dysfunction, ventricular remodeling, and contributes to deterioration with heart failure [3]. The signaling cascades of the mitogen-activated protein kinase (MAPK)

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Conclusion