| Background:mesenchymal stem cells (MSCs) had been widely applied in regenerative medicine and exhibited beneficial effects on post-infarct hearts. However, the therapeutic potential of MSCs is hindered by their low survival rate after transplantation in damaged myocardium. Statins has been proved to reduce MSCs apoptosis during both ischemia and hypoxia/serum deprivation (H/SD), still the mechanism is uncertain. Autophagy is a complex "self-eating" process and has been involved in modulating cellular survival and apoptosis by statins in some cell lines. We have previously showed that atorvastatin (ATV) could regulate AMP-activated protein kinase (AMPK), a positive modulator of autophagy, in MSCs. Thus we hypothesized that autophagy activation through AMPK and its downstream target mammalian target of rapamycin (mTOR) may be a novel mechanism of ATV to protect MSCs from apoptosis during H/SD.Methods:MSCs separated from Sprague-Dawley rats were exposed to hypoxia and serum-deprivation (H/SD). After treated with different concentration of atorvastatin (ATV,0.001μM-10μM), autophagy inhibitor3-methyladenine (3-MA,5mM), autophagy promoter rapamycin (lOnM) or the inhibitor of AMPK Compound C (10μM), the autophagy and apoptosis rate, the protein pathways and the formation of autophagosomes were analyzed by flow cytometer, Western bolt and transmission electron microscopy, respectively.Results:ATV enhanced autophagy of MSCs under H/SD condition, as identified by increasing acidic vesicular organelles (AVO)-positive cells, type Ⅱ of light chain3(LC3-Ⅱ) expression and autophagosomes formation. Treatment with autophagy inhibitor3-MA attenuated ATV-induced autophagy and abrogated the protective effects of ATV on MSCs apoptosis, while rapamycin failed to cause additional effects on either autophagy or apoptosis compared with ATV alone. The phosphorylation of AMPK was upregulated whereas the phosphorylation of mTOR was downregulated in ATV-treated MSCs, which were both attenuated by AMPK inhibitor Compound C. Furthermore, treatment with Compound C reduced the autophagy observed in ATV-treated MSCs under H/SD. Conclusion:ATV could activate protective autophagy via AMPK/mTOR pathway to enhance MSCs survival during H/SD. Aims:To investigate whether RhoA/Rho associated coiled-coil forming protein kinase/extracellular signal-regulated kinase (RhoA/ROCK/ERK) pathway involved in the effects of atorvastatin (ATV) on improving the survival and therapeutic benefits of transplanted mesenchymal stem cells (MSCs) after acute myocardial infarction (AMI).Methods:In vitro, H9C2cells were exposed to hypoxia and serum-deprivation (H/SD) for24hours. After treated with different concentration of atorvastatin (ATV,0.001μM-10μM) and geranyl geranyl pyrophosphate (GGPP), the inflammation and fibrosis levels, and the related signal pathway were analyzed by Western Blot (WB) and Enzyme-Linked Immunosorbent Assay (ELISA). In vivo, female Sprague-Dawley rats were randomized into seven groups:sham group, AMI group (control), ATV group, Fasudil group, MSCs group, MSCs and ATV group, or Fasudil and MSCs group. AMI was created by the left anterior descending coronary artery ligation; MSCs from male rats were injected into the myocardium30minites after AMI. Cardiac function, histology, expression of Y-chromosomal genes and RhoA/ROCK/ERK signaling proteins were examined at four weeks after MSCs transplantation.Results:Under H/SD condition, ATV could significantly decrease the expression and secretion of interleukin6(IL-6), tumor necrosis factor a (TNF-a) and connective tissue growth factor (CTGF) in H9C2, which was abrogated by GGPP treatment. The phosphorylation of myosin phosphatase target subunit-1(MYPT) and extracellular signal-regulated kinases (ERK) was down-regulated in ATV-treated H9C2, which were both attenuated by GGPP. In vivo, as evaluated by echocardiography and left ventricular catheterization, compared with AMI group, the left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular end-diastolic pressure (LVEDP) and-dp/dt was decreased, whereas left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS) and dp/dt was increased significantly in ATV+MSCs group and Fasudil+MSCs group (P<0.001); However, MSCs alone has no effects on any of these cardiac function index (P>0.05). Histology analysis found that compared with AMI control group, the inflammation was attenuated and more small vessels were observed in all other groups, but the left ventricular fibrotic area was reduced markedly only in ATV+MSCs group and Fasudil+MSCs group (P<0.001). As detected by real-time Polymerase chain reaction and fluorescence in situ hybridization, the survival rate of implanted MSCs was significantly increased in ATV+MSCs group and Fasudil+MSCs group compared with AMI control group (P<0.001). WB showed that ATV and Fasudil treatment could decrease the expression of IL-6, TNF-a and CTGF in post-infarct hearts (P<0.001). Meanwhile, the phosphorylation of MYPT and ERK was significantly decreased by ATV and Fasudil treatment (P<0.001).Conclusion:ATV enhances the efficiency of MSCs transplantation in improving cardiac function after AMI. This cardioprotective role of ATV may be due to its anti-inflammation and anti-fibrosis effects through inhibiting RhoA/ROCK/ERK pathway.
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