Yunle Wang, Zhijian Yang Exogenous HGF prevents cardiomyocytes from apoptosis after hypoxia via up-regulating cells autophagy Yunle Wang, Zhijian Yang Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, People's Republic of China Hi everyone, I am Yunle Wang. from cardiology department, the first affiliated hospital of Nanjing medical university. The topic I have here today is "exogenous HGF prevents cardiomyocytes from apoptosis after ischemia via up-regulating cells autophagy ".

Introduction The hepatocyte growth factor(HGF) has activities in various tissues for morphogenesis and anti-apoptosis. The functions of HGF is mediated by a specific tyrosine kinase receptor c-met phosphorylation Trusolino L, Bertotti A, Comoglio PM. Nature reviews Molecular cell biology 2010;11:834-848.

Introduction and by activation of downstream signaling pathway which include the MAPK cascades, the Pi3K–Akt axis. Both of them are important to energy metabolism and cellular homeostasis. Several studies have shown HGF/c-met protects heart from myocardial infarction, and protective effect of HGF includes various aspects such as angiogenesis, anti-fibrosis, anti-inflammation, myocardium protection and cell regeneration. However few study has been made to explore the relationship between this protection with cellular energy metabolism process, such as autophagy. Trusolino L, Bertotti A, Comoglio PM. Nature reviews Molecular cell biology 2010;11:834-848.

Introduction As we all known, autophagy is a highly reserved process of bulk degradation of cell organelles and long-lived proteins among eukaryotes, during which double-membrane autophagosomes engulf cell components and fuse with lysosomes for breakdown by hydrolases. It occurs in almost every tissues at base level and is induced by unfavorable condition such as nutrient deprivation. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Nature 2008;451:1069-1075

Introduction The nucleation and assembly of the initial phagophore membrane requires PI3K-Akt and MAPK. Interruption of autophagy process prevents cell survival in many organisms, whereas excessive autophagy also promotes programmed cell death known as autophagic apoptosis. In the heart, autophagy is stimulated by myocardial ischemia. At the early stage of ischemia, adaptable autophagy prevents cells from apoptosis and inhibition of autophagy increased cell death and heart dysfunction. In the present study, we chose Atg5, LC3 and the autophagosomes as the detected marker for autophagy. Cell Signaling Technology

Procedure Firstly we measured cell autophagy and apoptosis level after different time length of hypoxia and chose time points of autophagy and apoptosis for further study. Meanwhile we detected the expression of HGF and p-Met, and the release of HGF after hypoxia. Then we divided cells into control, HGF and HGF neutralizing antibody groups to detect autophagy and apoptosis process. To further explore the relationship between autophagy, apoptosis and cell mitochondria function, we divided cells into control, HGF, 3-MA and HGF-3-MA combination groups.

Results First of all, to explore the effect of hypoxia on NRVMs autophagy, we used mRFP-GFP-LC3 adenovirus infection, transmission electron microscope (TEM) and western blotting (WB) to detect cell autophagy at different time points after hypoxia. The results of TEM determined the formation of autophagosome in NRVMs (Figure 1B) at 2h of hypoxia. Results of TEM determined  the formation of autophagosome in cardiomyocytes at 2h of hypoxia.  

Results (scale bar: 5μm) This fluorescence microscope figures were about mRFP-GFP-LC3 adenovirus infection. Only when autophagy occurs, mRFP and GFP tagged LC3 gathered together and formed fluorescence points which could be counted under fluorescence microscope. The red points represented autolysosome while both green and red points were autophagosome. As expected, hypoxia stimulated NRVMs' autophagy process, and both autophagosome and autolysosome were increased after hypoxia. Results of mRFP-GFP-LC3 adenovirus infection indicated the formation of both autophagosome and autolysosome

Results The increased expression of both Atg5 and the ratio of LC3 II/LC3 I represented higher level of autophagy. After we repeated and detected autophagy process at different time points, the level of autophagy at 2h was selected to represent the early stage of hypoxia. Results of WB in detecting the change of autophagy process in different time points of hypoxia

Results Secondly, we employed flow cytometry, Hochest staining and WB to detecte cell apoptosis at different time points after hypoxia. Flow cytometry revealed that the rate of apoptotic cells increased significantly after 4h of hypoxia compared with 2h and kept increasing within 8h. Results of Flow cytometry in detecting cell apoptosis at different time points after hypoxia

Results (scale bar: 100μm) Hochest staining as well as WB were also consistent with the results. Based on these, we chose the time point of 4h for apoptosis detection in the following experiments. Results of Hochest in detecting cell apoptosis at different time points after hypoxia

Results Based on these, we chose the time point of 4h for apoptosis detection in the following experiments. Results of WB in detecting cell apoptosis at different time points after hypoxia

Results It is known that the concentration of HGF in patients’ serum increases significantly after myocardial infarction occurs. We achieved similar results that HGF level in medium increased rapidly after hypoxia. The concentration of HGF in medium increased to almost 2 times shortly after hypoxia compared with non-hypoxia while the expression of HGF in NRVMs decreased within 1h. Results revealed inconsistency of HGF concentration in medium and in NRVMs within 1h of hypoxia, indicating that the release of HGF was before its synthesis and both of them were activated by hypoxia. C-Met receptor significantly phosphorylated at 2h compared with other time points (p < 0.05) and inactivated soon. Up to now, we found that extracellular HGF and phosphorylation of C-Met (p-Met) peaked at 1h and 2h after hypoxia respectively, which was consistent with the change of autophagy level Results of Elisa and WB in detecting and the release of HGF and the expression of HGF and p-Met

Results (scale bar: 5μm) To identify whether extracellular HGF and p-Met was related with autophagy of NRVMs, we added HGF or HGF neutralizing antibody (HA) into medium before hypoxia. Exogenous HGF increased cell autophagy at early stage as the fluorescence points were significantly increased compared with control group. HGF neutralizing antibody on the contrary decreased NRVMs autophagy compared with control group and HGF group. Results of mRFP-GFP-LC3 adenovirus infection in detecting autophagy between group con, HGF, HA

Results The expression of Atg5 and the ratio of LC3 II/LC3 I were both higher in HGF group than the other two groups. On the other side, the expression of apoptosis-related proteins showed that exogenous HGF decreased NRVMs' apoptosis compared with control group. HGF neutralizing antibody markedly increased cell apoptosis compared with the control group and HGF group. Results of WB in detecting autophagy and apoptosis between groups con, HGF and HA

Results (scale bar: 5μm) To elucidate the relationship between autophagy and apoptosis, we respectively added 3-methyladenine (3-MA, an autophagy inhibitor), HGF, or a combination of 3-MA and HGF into medium and then induced hypoxia. Results revealed that 3-MA inhibited autophagy process, evidenced by significantly decreased fluorescence points number compared with control and HGF groups. However, the number of fluorescence points in the combination group was more than 3-MA group but still fewer than HGF group, which indicated that exogenous HGF could reverse the effects of 3-MA to a certain degree. Results of mRFP-GFP-LC3 adenovirus infection in detecting autophagy between group con, HGF, 3-MA and combination.

Results WB analysis revealed consistent results. Meanwhile, we found that inhibition of autophagy using 3-MA increased cells' apoptosis and additional HGF could also attenuate this effect to some extent. Now we could come to the conclusion that HGF protects cardiomyocytes from apoptosis via up-regulating auphagy after hypoxia. Results of WB in detecting autophagy and apoptosis between groups con, HGF, 3-MA and combination.

Results A C D After looking through the former researches about cardiomyocyte autophagy, we came to a hypothesis that in cardiomyocytes, mitochondrial may be a main target of autophagy, and reduce autophagy in myocardium lead to accumulation of dysfunctional mitochondrial. To further investigate the potential biological mechanism involved in the process, we explored mitochondrial membrane potential and detected injured mitochondria rate. The results showed that mitochondrial membrane potential decreased after hypoxia and HGF abated this change and decreased the rate of injured mitochondria. 3-MA increased the mitochondrial injury compared with HGF group and control group, while adding HGF reversed this change. The expression of Parkin increased in HGF group compared with control group (p < 0.05) and also increased in HGF+3-MA group compared with 3-MA group. Compared with the control group, adding 3-MA slightly decreased Parkin expression. Results of JC1 and WB in detecting mitochondrial function and Parkin expression between groups con, HGF, 3-MA and combination.

Summary Parkin HGF autophagy P-Met 3-MA apoptosis HGF-ab extracellular HGF could increase NRVMs autophagy via activation of c-Met while neutralizing antibody inhibited the process of autophagy exogenous HGF protected NRVMs from apoptosis and neutralizing antibody inhibited this protection cell apoptosis markedly increased after inhibition of NRVMS autophagy during hypoxia HGF prevented NRVMs from death through improving autophagy at early stage of hypoxia HGF increased NRVMs autophagy partially via increasing Parkin and the ratio of damaged mitochondria clearance HGF-ab

Limitations The changes of HGF expression and p-Met were contrary between group control, HGF and HGF-ab. The transfer of Parkin from cytoplasm to mitochondrion was more important than the expression of Parkin. The changes of HGF expression and p-Met were contrary between group control, HGF and HGF-ab. HGF expression was higher in HGF-AB group than other two groups and the activation of c-met was lower than them. In HGF group the results were opposite. This may be related with p-met negative feedback regulation and may provide a small amount of evidence that the extracellular HGF played a more important role in HGF/C-Met axis than the intracellular HGF. The transfer of Parkin from cytoplasm to mitochondrion was more important than the expression of Parkin. There is much work left to ensure that HGF induced clearance of damaged mitochondria was via increasing Parkin expression and transfer.

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