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

  • Molecules. 2019 Apr 8;24(7):1376. doi: 10.3390/molecules24071376.
Meng-Luen Lee  1  2 ,  Erna Sulistyowati  3  4 ,  Jong-Hau Hsu  5  6  7 ,  Bo-Yau Huang  8 ,  Zen-Kong Dai  9  10  11 ,  Bin-Nan Wu  12  13 ,  Yu-Ying Chao  14 ,  Jwu-Lai Yeh  15  16  17  18
Affiliations
  • 1. Division of Pediatric Cardiology, Department of Pediatrics, Changhua Christian Children's Hospital, Changhua 50050, Taiwan. [email protected].
  • 2. School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan. [email protected].
  • 3. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. [email protected].
  • 4. Faculty of Medicine, University of Islam Malang, Malang city, East Java Province 65145, Indonesia. [email protected].
  • 5. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. [email protected].
  • 6. Department of Pediatrics, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. [email protected].
  • 7. Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan. [email protected].
  • 8. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. [email protected].
  • 9. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. [email protected].
  • 10. Department of Pediatrics, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. [email protected].
  • 11. Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan. [email protected].
  • 12. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. [email protected].
  • 13. Department of Pharmacology, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. [email protected].
  • 14. Department of Public Health, College of Health Sciences, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. [email protected].
  • 15. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. [email protected].
  • 16. Department of Pharmacology, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. [email protected].
  • 17. Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan. [email protected].
  • 18. Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan. [email protected].
PMID: 30965668 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.

Keywords
KMUP-1; cardiomyocyte apoptosis; hypoxia; nitric oxide.
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