Effect And Mechanism Of Therapeutic Hypothermia On Autophagic Flux Regulation In Hypoxic Cardiomyocytes

BackgroundWith the improvement of living standard, morbidity of coronary heart disease (CHD) have increased year by year and the age of onset to incline to the young. Acute myocardial infarction (AMI) is one of the most serious CHD, and epidemiological evidence indicates that approximately 3.5 million patients have died of AMI in China every year. Early and effective treatment and prevention is considered critical in treating AMI.Therapeutic hypothermia (TH) has been applied clinically to maintain the balance between catabolism and metabolism in order to help our body survive a crisis. Although TH has been considered a therapeutic adjunct for brain injuries such as stroke, applications of TH are quite limited especially in ischemic heart diseases due to unclear mechanisms. TH can significantly reduce ATP consumption, improve intracellular ion management and maintain intracellular pH balance, which alleviate hypoxia injury in myocardial cells. Autophagy is an evolutionarily conserved process to adapt to adverse conditions such as limited energy supplies. Essential autophagy is critical to cell survival and homeostasis maintenance, while excessive autophagy can be detrimental and induce cell death known as autophagic cell death. TH reportedly preserves organ functions and improves cell survival, however, the exact relationship between TH, autophagy and cell viability during hypoxia is seldom discussed. A simulated ischemia model of cardiac cells was established to explore the relationship between TH, autophagy and cell survival, In our experiment, H9c2 cells were exposed to hypoxia and cooled to 32℃. We firstly examined the effects of TH on autophagy regulation and cell survival. Then, by manipulation of autophagic activity with 3-MA or rapamycin, we explored possible underlying mechanisms how TH regulated autophagic flux and investigated whether autophagy affected by TH promoted cell viability during hypoxia.ObjectivesTherapeutic hypothermia (TH) has become an effective clinical intervention for decades, most commonly seen in open heart surgery and organ transplantion, although the underlying mechanisms remain unclear. Confined to insufficient clinical evidences and poor understanding of the mechanisms, TH has not been widely applied in clinical scenarios including ischemic cardiomyopathy. Thus, in the present study, we sought to assess effects of TH on hypoxic H9c2 cardiomyocytes.MethodsThe first part of the experiment:To explore the impacts of TH on cell viability of hypoxia H9c2 cells,3 groups were designed in the experiment:control group, hypoxia group, hypoxia+TH group, the cells were separately exposed to hypoxia for 1 h,2 h and 3 h with or without TH treatment. Optical microscope was utilized to value cell viability 。The second part of the experiment:To investigate the effects of TH and hypoxia on autophagy,3 groups were designed in the experiment:control group, hypoxia group, hypoxia+TH group. Protein indicators of autophagy such as LC-3B, p62 and Beclin-1 were examined by western blot. Transmission electron microscope was utilized to detect the formation of autophagosomes and autolysosomes. Autophagic flux was investigated by the tandem GFP-mRFP-LC3 adenovirus (Ad-tf-LC3) construct.The third part of the experiment:To elucidate the mechanisms of TH inhibitory effects on autophagy during hypoxia, we investigated the involvement of a mTOR-dependent pathway by rapamycin treatment.4 groups were designed in the experiment:Hypoxia, Hypoxia+TH, Hypoxia+rapamycin, Hypoxia+TH+ rapamycin. The cells were separately exposed to hypoxia for 3 h with or without TH treatment. Protein indicators of autophagy such as LC-3B, p62 and mTOR pathway relative protein phosphorylation of mTOR and S6 were examined by western blot.The fourth part of the experiment:Manipulation of autophagy by "3-MA or rapamycin was applied to elucidate the role of autophagy in cell viability regulation by TH during hypoxia.6 groups were designed in the experiment:Hypoxia, Hypoxia +TH, Hypoxia+3-MA, Hypoxia+TH+3-MA, Hypoxia+rapamycin, Hypoxia+ TH+rapamycin.3-MA (10 mM) or rapamycin (0.1 μM) pretreatment for 2 h decreased autophagy during hypoxia, protein indicators of autophagy such as LC-3B, p62 were examined by western blot and cell viability was examined by trypan blue exclusion method.Statistical analysisAll experiments were performed three times. All data were presented as the means ± standard deviation and were statistically analyzed using SPSS software, version 13.0. Comparisons among groups were analyzed using a one-way analysis of variance (ANOVA), followed by the Bonferroni test when appropriate to determine statistical significance. Probabilities of 0.05 or less were considered to be statistically significant.Results1. Therapeutic hypothermia significantly improved cell survival under hypoxiaTo explore the impacts of TH on cell viability of hypoxia H9c2 cells, the cells were separately exposed to hypoxia for 1 h,2 h and 3 h with or without TH treatment. Fig.1-1 showed changes in cell morphology under optical microscope for the indicated time. As hypoxia prolonged, more cells became shrinkage and turned round in the hypoxia group. Meanwhile, compared to hypoxia group, TH treatment during hypoxia improved cell viability showing more normal cellular shapes. Cell viability was examined by trypan blue exclusion method and TH treatment during hypoxia protected cells against hypoxia injury (Fig.1-1; Fig.1-2)2. Autophagic flux was inhibited via TH under hypoxia. And TH possibly improved cell viability during hypoxia in ways other than regulation on autophagyAs autophagy was often implicated in the survival regulation of cardiac cells, we investigated the effects of TH on autophagy during hypoxia. Protein indicators of autophagy such as LC-3B, p62 and Beclin-1 were examined. Fig.2B showed that compared to control, expression of LC-3B elevated as hypoxia prolonged in the hypoxia group. Furthermore, p62 expression also decreased time-dependently (Fig. 2-1C), indicating that hypoxia activated autophagy via a time-dependent manner. While hypoxia increased autophagy in our experiment, TH treatment during hypoxia reduced autophagic activity. Difference of p62 as well as LC-3B expression between hypoxia+TH group and hypoxia group peaked at 3 h (Fig.2-IB, C). However, protein expression of Beclin-1 was not affected by either hypoxia or treatment with TH (Fig.2-1D).In addition, it was confirmed by transmission electron microscope. Autophagosomes are characterized by double-and multiple-membrane structures containing cytoplasm or undigested organelles including mitochondria. Conversely, autolysosomes are identified as single-membrane structures with remnants of cytoplasmic components.Compared with the basal level of autophagic vacuoles in control, the number of autophagic vacuoles increased following hypoxia, which was attenuated by TH (Fig.2-2). To provide a more accurate analysis of how autophagy was regulated by TH during hypoxia, autophagic flux was investigated by the tandem GFP-mRFP-LC3 adenovirus (Ad-tf-LC3) construct. As depicted in Fig.2-3, compared to control, hypoxia increased autophagic flux as indicated by red and yellow dots. Furthermore, TH treatment during hypoxia partly reduced autophagic flux. The above suggested that compared to control group, hypoxia could efficiently increased autophagic flux, while presence of TH partly reduced autophagosome formation and thereby inhibited autophagic flux induced by hypoxia.Manipulation of autophagy by 3-MA or rapamycin was applied to elucidate the role of autophagy in cell viability regulation by TH during hypoxia. As a PI3K inhibitor,3-MA is always recognized as an effective way to explore autophagy for its ability to inhibit sequestration of autophagosomes at an early stage resulting in reduced LC-3B expression and decreased number of autophagy structures. As shown by immunoblot assays in our research,3-MA (10 mM) pretreatment for 2 h decreased autophagy during hypoxia, and 3-MA treatment further reduced the autophagy in presence of TH during hypoxia by detecting the expression LC-3B and p62(Fig. 2-4A.B.C). Cell viability was examined by trypan blue exclusion method and TH treatment during hypoxia protected cells against hypoxia injury. However, TH treatment further impaired cell viability in the presence of 3-MA during hypoxia (Fig. 2-4D).Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), serves as an effective agent to activate autophagy both in vitro and in vivo. Immunoblot analysis in our research showed that rapamycin (0.1 μM)[1] elevated autophagy during hypoxia and also restored the TH-inhibited autophagy (Fig.2-4E.F.G). Interestingly, rapamycin possibly abolished the inhibitory effect of TH on autophagy during hypoxia. Meanwhile, rapamycin improved cell viability during hypoxia and quite contrary to the case of 3-MA, TH even significantly increased cell viability in the presence of rapamycin during hypoxia(Fig.2-4H). All the above indicated that TH could improve cell viability in ways other than autophagy regulation.3. TH Inhibited autophagy via a mTOR-dependent manner during hypoxiaTo elucidate the mechanisms of TH inhibitory effects on autophagy during hypoxia, we investigated the involvement of a mTOR-dependent pathway. mTOR has been identified as a negative regulator of autophagy in mammalian cells. The ribosomal protein S6 (S6) is a downstream effector of mTOR and phosphorylation of S6 is commonly considered as a surrogate marker for mTOR activation. Thereby, phosphorylation of mTOR and S6 was evaluated. Our results indicated that 3 h hypoxia significantly inhibited both mTOR and S6 activation while TH treatment during hypoxia restored (Fig.3-1D, E). As a potent inducer of autophagy by suppressing mTOR and its downstream effectors such as S6, rapamycin was utilized to confirm the mTOR-dependent inhibition of autophagy by TH during hypoxia. First, effect of rapamycin on autophagy induction was confirmed by western blot. Treatment with rapamycin increased autophagy as determined by the protein level of LC-3B and p62. Specifically, autophagy activity was restored as utilization of rapamycin attenuated this decrease in LC-3B and p62 afforded by TH during hypoxia (Fig.3-1B, C). Furthermore, phosphorylation of mTOR and S6 was significantly reduced with the presence of rapamycin compared to TH+hypoxia group (Fig.3-ID, E).Conclusions1. This study simulated myocardial ischemia injury induced by hypoxia myocardial cells of H9c2, after 3 hours hypoxiam, can obviously induced the injury of H9c2, suggested myocardial hypoxia injury model was constructed successfully, which made a foundation of investigating myocardial ischemia injury and autophagic mechanism of TH under hypoxia.2. This study investigated hypoxia myocardial cells of H9c2 by TH treatment, detection of autophagy and autophagic flux, shows that autophagy was inhibited by TH under hypoxia as well as autophagic flux with reduced autophagosome formation. This study investigated hypoxia myocardial cells of H9c2 by TH treatment and manipulated autophagy by 3-MA or rapamycin was applied to elucidate the role of autophagy in cell viability regulation by TH during hypoxia. In the present research, we found that down-regulation of autophagy by TH during hypoxia may not the only mechanism for myocardial cell protection.3. This study investigated hypoxia myocardial cells of H9c2 treated by TH and added the inhibition of mTOR, rapamycin to detect autophagy, thereby to elucidate the mechanisms of TH inhibitory effects on autophagy during hypoxia. In the present research, we found that inhibition of autophagic activity by TH was probably performed via a mTOR-dependent pathway.