Defense Mechanism Of HO-1mediated TRAP1 On Myocardial Cell Damage Induced By Intermittent Hypoxia

Background Obstructive sleep apnea syndrome(OSAS)is a common disease characterized by repeated partial or complete collapse of the upper respiratory tract during sleep,which is characterized by chronic intermittent hypoxia(CIH).Similar to ischemiareperfusion injury,CIH is the most important pathophysiological mechanism of OSAS related cardiovascular disease,which can cause myocardial injury and cardiac insufficiency.The cytological basis of the occurrence and development of heart failure is cardiomyocyte apoptosis,and mitochondrial apoptosis pathway is the main pathway of cell apoptosis.In addition,mitochondrial dysfunction will also produce a large number of reactive oxygen species(ROS),eventually leading to apoptosis.Heme oxygenase-1(HO-1)is a rate limiting enzyme for heme degradation.Its catalytic products,such as biliverdin,Fe2 + and carbon monoxide(CO),have the effects of anti-inflammatory response,antioxidant stress and inhibition of apoptosis.At the same time,a large number of literatures have confirmed that HO-1 has a protective effect on mitochondrial function.Tumor necrosis factor receptor associated protein 1(TRAP1),also known as heat shock protein 75,mainly exists in the inner membrane of mitochondria.It can maintain the integrity of mitochondrial respiratory chain and inhibit cardiomyocyte death caused by oxidative stress by reducing the production of ROS.Our previous research shows that: The damage of myocardial function caused by CIH is related to the imbalance of HO-1,and it is confirmed that the induction of high expression of HO-1 has a protective effect on the myocardial damage caused by CIH;It was also found that the expression trend of TRAP1 protein in cardiomyocytes induced by CIH was similar to that of HO-1.Therefore,we speculate that HO-1 can protect cardiomyocytes through TRAP1 and reduce the damage of cardiomyocytes caused by CIH.Objective To observe the damage of mitochondrial function and apoptosis of cardiomyocytes treated with different intermittent hypoxia(IH)time,and to explore whether HO-1 can inhibit IH induced cardiomyocyte apoptosis by mediating TRAP1,so as to reduce the damage of cardiomyocytes caused by CIH and play a protective role.Methods 1.Using Bio-Spherix Oxy Cycler,a system for controlling intermittent hypoxia,ac16 adult cardiomyocytes were cultured to establish IH cell model;CIH animal model was established by SD rats.2.To observe the changes of mitochondrial functional proteins,apoptosis and the expression of HO-1 and TRAP1 after IH treatment of cardiomyocytes at different times: cardiomyocyte culture dishes were randomly divided into 6 groups: normoxia circulation group(C group),intermittent hypoxia 16 circulation group(IH16 group),intermittent hypoxia 32 circulation group(IH32 group),intermittent hypoxia 64 circulation group(IH64 group)Intermittent hypoxia 96 cycle group(IH96 group)and intermittent hypoxia 128 cycle group(IH128 group).Western Blotting(WB)was used to observe the expression of mitochondrial fusion protein-2(Mfn2),dynamic related protein 1(Drp1),mitochondrial transcription factor A(Tfam),mitochondrial respiratory chain complex III(Complex III),HO-1,TRAP1 and apoptosis related proteins Caspase 3,Bcl-2 and Bax in cardiomyocytes after IH.3.Study myocardial histopathology and HO-1 and TRAP1 expression levels in SD rats with CIH(8 weeks): cut heart tissue from normoxia(Con)and CIH group,and left ventricular cross-section by hematoxylin-eosin(HE),and HO-1 and TRAP1 expression were observed by immunohistochemistry(IHC)staining.4.The expression of HO-1 protein in cardiomyocytes was induced by drug hemin(HO-1 inducer)or inhibited by Zn PP IX(HO-1 inhibitor).The effects of HO-1on cardiomyocyte TRAP1 and apoptotic protein Caspase3 were observed by WB technique.5.By treating cardiomyocytes with recombinant protein HO-1 or trap 1respectively,the effects of trap 1,HO-1 and apoptotic protein caspase 3 were observed.TDT mediated d UTP nick end labeling(TUNEL)was used to detect the protective effect of HO-1 and trap 1 recombinant protein on IH cardiomyocyte apoptosis.Results Part Ⅰ1.The SD rat model of chronic intermittent hypoxia was established.HE staining showed that the myocardial cells in the normoxia control group(Con group)had clear boundaries and central nuclei;The interstitial boundary of myocardial cells in chronic intermittent hypoxia group(CIH group)was unclear,and the myocardial fibers were significantly widened.Statistical analysis showed that the relative cardiomyocyte area was significantly higher than that in con group(P < 0.001).2.In CIH rat model,the apoptotic protein of rat cardiomyocytes was detected by Western Blotting.We found that compared with con group,the ratio of Bcl-2 / Bax in CIH group decreased(P < 0.01),and the expression of caspase 3 protein increased significantly(P < 0.01);TUNEL fluorescence test showed that the apoptosis rate of cardiomyocytes in CIH group increased significantly(P < 0.01).3.IH cardiomyocyte model was constructed.Through Western Blotting Technology,we found that with the gradual increase of IH cycle times,the expression levels of apoptotic proteins Bcl-2 and caspase 3 increased gradually(P < 0.05);Anti apoptotic protein Bcl-2 decreased significantly in the later stage of IH(P < 0.05);The ratio of Bcl-2 / Bax was significantly lower than that in group C(P < 0.05).4.In IH cardiomyocyte model,the mitochondrial dynamics related proteins drp1 and Mfn2 were measured by Western Blotting.We found that Mfn2 protein increased in stress in the early stage of IH(P < 0.05),but decreased significantly in the later stage of IH(P < 0.01);Drp1 protein increased in late IH(P < 0.05).In the late stage of IH,the proportion of Mfn2 / drp1 protein was unbalanced,mitochondria were abnormal,and mitosis increased.5.Further observe the effect of IH on the mitochondrial function of cardiomyocytes.Through Western Blotting Technology,we found that in the later stage of IH,the expression level of TFAM protein in cardiomyocytes was significantly lower than that in group C(P < 0.01),indicating the dysfunction of mitochondrial gene transcription in cardiomyocytes;The expression level of complex III protein was significantly lower than that in group C(P < 0.05),indicating that the function of oxidative phosphorylation was impaired.Part II1.In the CIH rat model,in order to observe the effect of CIH on the expression of HO-1 and TRAP1 protein in SD rats,Western Blotting showed that the expression of HO-1 and TRAP1 in CIH group decreased compared with con group(all P < 0.01);Immunohistochemical staining showed that the relative positive area ratios of HO-1and TRAP1 in ventricular myocardium in CIH group were significantly lower than those in con group(P < 0.001).2.In IH cardiomyocyte model,the protein expression of cardiomyocytes measured by Western Blotting showed that the expression levels of HO-1 and TRAP1 were consistent,the compensatory expression level increased in the early stage of IH(all P < 0.05),and the decompensated expression level decreased in the later stage of IH(all P < 0.01).3.In order to verify the relationship between HO-1 and TRAP1,hemin was used to induce the high expression of HO-1 in cardiomyocytes(P < 0.01).Western Blotting was used to observe the changes of TRAP1 protein: the up regulation of HO-1expression in late IH(IH96 group)increased the expression of TRAP1(P < 0.01).Zn PP IX was used to inhibit the expression of HO-1.The results showed that the expression of TRAP1 decreased with the decrease of HO-1 level in the early stage of IH(IH64 group)(P < 0.001).4.In order to observe the effects of the above two drugs on cardiomyocyte apoptosis,we found that hemin could inhibit the increase of caspase 3 protein induced by IH(P < 0.01);Zn PP IX could increase caspase 3 protein in group C and IH(P <0.05).5.In order to further verify the relationship between HO-1 and TRAP1,HO-1recombinant protein was used to treat cardiomyocytes.Western Blotting showed that HO-1 recombinant protein could increase the expression of TRAP1 in IH group(P <0.001);However,the expression level of HO-1 in IH group did not change significantly after using TRAP1 recombinant protein(P > 0.05).6.To observe the protective effect of HO-1 or trap 1 on cardiomyocyte apoptosis.The results of Western Blotting showed that the apoptotic protein caspase 3 in IH group decreased significantly under the treatment of HO-1 and trap 1 recombinant protein(P < 0.01).Using TUNEL fluorescence technique,the detection results were consistent with WB results.HO-1 and trap 1 could significantly reduce the apoptosis rate of cardiomyocytes(all P < 0.01).Conclusion 1.The mechanism of cardiac insufficiency caused by IH is related to mitochondrial dysfunction of cardiomyocytes and apoptosis of cardiomyocytes2.The HO-1/TRAP1 signaling pathway is an important protection mechanism for IH to cause cardiac insufficiency...