The Protective Effect And Mechanism Of Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1) On Hypoxic Damage In Cardiomyocytes

Myocardial ischemic/hypoxic damage and cardiac functional impairment precede the significant decrease in blood volume that occurs after a severe burn. In the early stages following a burn (before the decrease in blood volume), myocardial damage not only causes cardiac insufficiency but also induces or aggravates burn shock, which can cause or aggravate ischemic/hypoxic injury to other organs. Studies have demonstrated that hypoxia is one of the main causes of myocardial damage after a burn. Hence, it is important to protect cardiomyocytes from hypoxic damage. Mitochondria are the primary target of hypoxic damage in cardiomyocytes. Mitochondrial dysfunction in hypoxic cardiomyocytes can directly lead to cell death. Following hypoxia, the opening of mitochondrial permeability transition pore (MPTP) leads to the release of apoptogenic proteins (i.e., cytochrome c) into the cytoplasm, and the activation of caspase-dependent apoptotic pathways; these steps result in cell necrosis and apoptosis. The irreversible mitochondrial injury caused by MPTP opening is critical to induce the hypoxic damage of cardiomyocytes. MPTP is a nonspecific pore consisting of several proteins. Some models have proposed the presence of other molecular components of the pore, but there is still no consensus as to the exact components. However, cyclophilin D (CypD) is generally accepted as a critical regulatory component of MPTP and plays an important role in regulating MPTP opening.Tumor necrosis factor receptor-associated protein 1 (TRAP1) localises to the mitochondria and its targeting sequence has been found in the N-terminus of the protein. An analysis of the cDNA sequences reveals that TRAP1 is identical to heart shock protein 75 (HSP75), which is a member of the HSP90 family. The ATPase activity of TRAP1 is inhibited by Geldanamycin, which is a specific inhibitor of HSP90. Despite its ATP-binding activity, TRAP1 seems have specific functions that are different from those of other well-characterised HSP90 homologues. After hypoxia treatment, HSP90 expression increases, and it plays a protective role against damage. However, the changes induced in TRAP1 expression in the cardiomyocytes under hypoxic conditions are still unclear. TRAP1 is a mitochondrial chaperone that is critical for maintaining mitochondrial homeostasis and regulating apoptosis and necrosis of tumor cells. Furthermore,there is an interaction between TRAP1 and CypD in mitochondria of tumor cells. Since MPTP is recognized as a key player in cell death, so we hypothesise that TRAP1 protects cardiomyocytes from hypoxic damage by regulating MPTP opening, and CypD may be the target protein through which TRAP1 modulates MPTP opening. The present study is designed to test this hypothesis.Objectives:The purpose of this study was to observe the changes in TRAP1 expression after hypoxia treatment and investigate the protective effect and mechanism of TRAP1 on hypoxic damage in primary cultured cardiomyocytes.Methods:1. Primary cultured cardiomyocytes were prepared from neonatal Sprague-Dawley rats (days 1 to 3) and hypoxic conditions were established by using an anaerobic jar and a vacuum glove box filled with a mixed gas containing 94% N₂, 5% CO₂, and 1% O₂.2. TRAP1 expression was detected by western blot and immunofluorescence in cardiomyocytes under normoxic conditions and hypoxic conditions (for times of 1, 3, 6, and 12 hours respectively). Then, a recombinant adenovirus vector for TRAP1 overexpression and a recombinant adenovirus vector for silencing of TRAP1 expression were constructed. To evaluate the role of TRAP1 changes in cardiomyocytes under hypoxic conditions, we investigated the effects of TRAP1 overexpression or TRAP1 depletion on cell viability and cell death after 6 hours of hypoxia in cardiomyocytes.3. The role of TRAP1 on MPTP opening in hypoxic cardiomyocytes was observed by investigating the effects of TRAP1 overexpression or TRAP1 depletion on mitochondrial membrane potential (Δψ) and cytochrome c content in cytosol fraction. Meanwhile, the changes of caspase 3 activity in cardiomyocytes were examined. We next investigated the effects of TPAP1 depletion on cell viability, caspase 3 activity and cell death by inhibiting MPTP opening using cyclosporine A (CsA) in hypoxic cardiomyocytes.4. Mass spectrography analysis and co-immunoprecipitation were used to determine whether there was an interaction between TRAP1 and CypD in cardiomyocytes. Then, we investigated the effects of TRAP1 overexpression or TRAP1 depletion on CypD expression both in protein levels and mRNA levels under normoxic conditions. Meanwhile, we also observed the roles of hypoxia and CsA on CypD protein expression and mRNA expression.Results:1. Western blot and immunofluorescence showed that TRAP1 content increased after 1 hour of hypoxia and continued to increase until for up to 12 hours compared with the normoxic group. Meanwhile, longer hypoxic treatments yielded higher TRPA1 expression2. After 6 hours of hypoxia, there were an increase in cell death and a decrease in cell viability. Overexpressing TRAP1 prevented hypoxia-induced damage to cardiomyocytes, while silencing TRAP1 expression aggravated an increase in cell death and a decrease in cell viability in hypoxic cardiomyocyte.3. Hypoxia caused a decrease inΔψand increased cytochrome c content in cytosol fraction and caspase 3 activation. Overexpressing TRAP1 abolished hypoxia-induced effects to cardiomyocytes and silencing TRAP1 expression aggravated hypoxia-induced damage. Furthermore, the cell damage induced by the silencing of TRAP1 was prevented by MPTP inhibitor CsA in hypoxic cardiomyocytes.4.There wasn't an interaction between TRAP1 and CypD both in normoxic conditions and hypoxic conditions. Overexpression TRAP1 increased mRNA levels of CypD and silencing TRAP1 decreased its mRNA levels in normoxic cardiomyocytes. However, there was no effect of TRAP1 on CypD protein levels. Furthermore, hypoxia decreased CypD mRNA levels and CsA increased its mRNA levels, while they also had no effect on CypD protein levels.Conclusions1. Hypoxia treatment (for times of 1, 3, 6, and 12 hours respectively) induced a time-dependent increase in the levels of TRAP1 protein. 2. Hypoxia-induced TRAP1 increase was a protective role against hypoxic damage to cardiomyocytes.3. TRAP1 regulated the activation of mitochondrial-dependent apoptotic pathway by modulating MPTP opening in hypoxic cardiomyocytes, and then played a protective role.4. The mechanism that TRAP1 regulated MPTP opening might be though influencing CypD expression.