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

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.