Combination Of D942with Curcumin Protects Cardiomyocytes From Ischemic Damage Through Promoting Autophagy

Myocardial ischemia is one of the main causes of sudden cardiac death. Autophagy has been demonstrated to protect cardiomyocytes from ischemia/reperfusion-induced damage. A small molecule compound D942has been previously shown to specifically activate AMP-activated protein kinase (AMPK) in cancer cells. Another reagent, curcumin, has been shown to inhibit mammalian target of rapamycin (mTOR) signal pathway in tumor cells. Since AMPK signalling induces autophagy, while mTOR signaling inhibits autophagy, here we tested the potential protective efficacy of D942with curcumin for cardiomyocytes under oxygen-glucose deprivation and reoxygenation (OGD/R) in vitra and in vivo.Part one:the protective effect of D942and curcumin on cardiocytes under oxygen-glucose deprivation and reoxygenation in vitro.Cardiomyocytes subjected to hypoxic substrate-free solution for24h and reinstated with normal culture conditions for3h to mimic Ischemia-Reperfusion injury. After be treated with D942or curcumin and other inhibitors such as compound C or Pervanadate or chloroquine, the ratio of autophagy/apoptosis cells were measured with flow cytometry; Cardiomyocytes vitality were measured with MTT; LDH was used to determine the cell injury; AMPK signaling, mTOR signaling and LC3B conversion were determined by Immunobloting. Results show that:Part one:OGD/R robustly increased both early and late apoptosis, while D942and curcumin significantly inhibited cell death as well as promoted cell survival. Notably, combination treatment with D942and curcumin was the most effective in inhibiting OGD/R-induced apoptosis and maintaining cell survival. The increase of early apoptosis in the treated groups is probably not due to enhanced induction of early apoptosis, but instead reflects blocked entry into late apoptosis and necrosis, since late apoptosis and necrosis was decreased and living cells were increased. Cell viability assay also confirmed the protective effects of D942and curcumin. Cardiomyocytes were treated with D942and/or curcumin under normal culture condition for24hours. OGD/R was performed as a positive control since it has been well documented as an autophagy inducer. After treatment, autophagic markers were detected by Western blot. D942or curcumin significantly induced conversion of LC3B-I to LC3B-Ⅱ under normal culture condition, which is a marker for autophagosome formation. p62was decreased by D942or curcumin. OGD/R exposure also induced similar but weaker changes of above markers in comparison with D942or curcumin treatment, suggesting either agent is more potent in evoking autophagy. It is noteworthy that combined treatment with D942and curcumin after OGD/R induced the most significant changes of autophagic markers among all treated groups, suggesting combination of D942and curcumin is the most potent autophagy activator under our conditions. Chloroquine has been shown to effectively inhibit autophagy by imparing lysosomal acidification and subsequent protein degradation. Chloroquine itself increased the levels of LC3B-Ⅱ and p62in comparison with vehicle control, due to inhibited degradation of these autophagic proteins. Activating phosphorylation level of AMPK was significantly higher in the presence of D942than those in non-treated group and curcumin alone group. both D942and curcumin decreased phosphorylation of p70S6kinase and Eukaryotic translation initiation factor4E-binding protein1(4EBP1) at their activating sites, suggesting mTOR signaling is substantially inhibited after treatment. It is noteworthy that combination of D942with curcumin induced a more robust decrease of activating phosphorylation of p70S6K and4EBP1, suggesting the most potent inhibitory effect of combination treatment.Part two:the effect of D942and curcumin on myocardial injure from ischemia/reperfusion in vivo.IR model was establised by ligating the left anterior descending artery for30min, followed by loosening the ligature. D942and curcumin were injected into8-12week old male C57BL/6mice via tail vein once a day for three consecutive days. AMPK signaling, mTOR signaling and LC3B conversion were determined by Immunobloting; RT-PCR were used in the detection of beclinl mRNA to evaluate the induction of autophagy after ischemia/reperfusion; Infarct size of Heart tissue were detected by TTC. Results show that:IR was induced by ligating the left anterior descending artery for30min, followed by loosening the ligature for120min. D942and curcumin were injected via tail vein and induced a higher level of autophagy in comparison with vehicle control, demonstrated by higher level of LC3B-Ⅱ and lower level of p62. Taken together, our data suggest that combination treatment with D942and curcumin could be therapeutical for myocardial I/R. The entire ventricular tissue was cut into six horizontal slices and was incubated in1%TTC solution. It was found that infarct size of vehicle control group (29.3±4.7%) was higher than D942and curcumin group (18.2±3.1%)(P<0.05). These suggested that the myocardium was injuried by IR, and was protected by D942and curcumin.Taken together our data suggested that both D942and curcumin enhanced cell survival after OGD/R. D942and curcumin induced autophagy in cardiomyocytes through activating AMPK pathway, or inhibiting mTOR signaling, respectively. Induction of autophagy by D942and curcumin was the cause of cardiaoprotection, since inhibition of autophagy abolished the protective efficacy. Furthermore, combination treatment with D942and curcumin profoundly up-regulated autophagy after OGD/R and significantly promoted cell survival. Treatment with D942and curcumin significantly up-regulated autophagy in a murine myocardial ischemia/reperfusion model. Our research suggests that D942and curcumin could be promising therapeutic agents for myocardial ischemia/reperfusion.