Induction of autophagy by KMUP-3 in cardiac fibroblasts and cardiomyocytes

• Main Authors: Bo-Wen Shiau, 蕭柏文
• Other Authors: Jwu-Lai Yeh
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/36097008057560594225

碩士 === 高雄醫學大學 === 藥理學研究所 === 101 === Autophagy is an evolutionary conserved process involved in the degradation of long-lived proteins and excess or dysfunctional organelles in eukaryotic cells. In the heart, autophagy functions predominantly as a pro-survival pathway during cellular stress by removing protein aggregates and damaged organelles, protecting the heart against famine, ischemia/reperfusion and heart failure. Cardiac remodeling involves increased rates of cardiomyocyte cell death and precedes heart failure. Meanwhile, cardiac fibroblasts play important role by regulating structure, biochemical, mechanical and electrical propertied of the heart. Therefore, we aimed to investigate signal transduction pathways involved in the induction of myocardial autophagy by KMUP-3. KMUP-3 is a chemically synthetic xanthine-based derivative. It has been demonstrated to have phosphodiesterase inhibition, endothelial nitric oxide synthase (eNOS) enhancement and KATP channel opening activities. When cardiac fibroblasts and cardiomyocytes were treated with KMUP-3 for different time, we found that the expression of LC3-II protein and autophagy-related genes including Beclin-1 and Atg7 were markedly upregulated in time- and dose-dependent manner. In addition, cells treated by KMUP-3 developed autophagosome-like characteristics, including single and double membrane vacuoles containing intact and degraded cellular debris. To investigate the ability of KMUP-3 to stimulate autophagy as a potential mechanism involved in eNOS phosphorylation. We treated cells with NOS inhibitor L-NAME, and found that L-NAME significantly inhibited KMUP-3-induced LC3-II expression and eNOS phosphorylation. Because it was recently reported that phosphatidylinositol 3-kinase (PI3K) and its downstream effector Akt are implicated in the activation of eNOS. We treated cells with PI3K inhibitor wortmannin, and found that wortmannin abolished KMUP-3-induced eNOS phosphorylation and Akt phosphorylation, providing the first evidence that KMUP-3 stimulates autophagy probably through PI3K/Akt/eNOS signaling. Besides, enhancement of AMPK activity also induces eNOS activity. We treated cells with KMUP-3 for different time, and found that the phosporylation of AMPK was markedly upregulated in time-dependent manner. Taken together, our results show that KMUP-3 induces autophagy through AMPK/PI3K/Akt/eNOS signaling in cardiac fibroblasts and cardiomyocytes.