Author + information
- Fanji Meng1
C1q/tumor necrosis factor-related protein-9 (CTRP9), a newly identified adiponectin (APN) paralog, was reported to reduce myocardial ischemia injury. However, the role of CTRP9 in hypoxia-induced cardiomyocytes autophagy remains completely unknown. This study determined whether CTRP9 protects cardiomyocytes from hypoxia-induced damage by inducing autophagy and elucidated the underlying mechanisms.
Cardiomyocytes were exposed to chronic hypoxia (1% O2, 5% CO2, 37°C) for 12h to establish a model of myocardial hypoxic-ischemic injury. The cells were pretreated with CTRP9 (5μg/ml) for 30 min followed by hypoxia 12h with or without autophagy inhibitor 3-MA (3- methyladenine), or AMPK inhibitor compound C. The viability and cellular damage of cardiomyocytes were assessed by CCK-8 staining and lactate dehydrogenase (LDH) assays, respectively. The protein expression level of p62, Beclin1, AMPK, p-AMPK, mTOR, p-mTOR,ULK, LC3-I and LC3-II were assessed by western blot assay.
After 12h hypoxia, CTRP9 markedly improved cell viability induced by hypoxia compared to that in hypoxia group (84.37 ± 3.11 vs. 55.93 ± 6.80%, p < 0.01), whereas compound C or 3-MA abolished the protective effect of CTRP9 on cell viability (61.34 ± 4.95%) (63.05 ± 5.65%) (p < 0.01) respectively. LDH (35.41±3.58 U/L) levels in hypoxia group were significantly increased compared to those in control group (5.81±0.53 U/L) (P<0.01). CTRP9 significantly inhibited the increase in LDH (15.76±2.35 U/L) levels (P<0.01). However, the protection of CTRP9 was partially blocked by pretreating cells with compound C (30.55±2.87 U/L) (p<0.01) or 3-MA (28.30±2.07 U/L) (p<0.01).The expression of p62,Beclin1,ULK,LC3-II/LC3-I ratio, which is correlated with the extent of autophagosome formation, in hypoxia group was significantly increased compared to the control group (P<0.05), whereas the expression of p62, Beclin1, ULK, LC3-II protein in the CTRP9 group was significantly induced compared to the control group (p<0.01) and hypoxia group (p<0.01). This effect was partially abolished by concurrent compound C or 3-MA treatment (p<0.01). In addition, we found that CTRP9 increased the expression of p-AMPK and decreased the expression of p-mTOR compared to the control group (p<0.01) and hypoxia group (p<0.01) respectively. Subsequently, upon inhibition of AMPK phosphorylation by AMPK inhibitors, CTRP9 mediated autophagy was abolished which further led to the increase in cell injury.
We demonstrate that adipokine CTRP9 attenuates hypoxia-induced cardiomyocytes injury by inducing autophagy via the AMPK signaling pathway.