Role Of Microtubule-associated Protein 4 In The Regulation Of Mitochondrial Permeability Transition Pore And Apoptosis In Hypoxic Cardiomyocytes

Cellular hypoxia is one of the most common pathologic phenomenon in severe burns. Cardiac damage exists in the early stage of severe burns. Mitochondria dysfunction in hypoxia plays an important role in the cardiac damage. Mitochondrial permeability transition pore (MPTP) openning is the key process of apoptosis, but its mechanism is still unclear. Voltage-dependent anion channel(VDAC) is a component protein of MPTP in mitochondrial outer membrane, which can modulate MPTP. Studies showed that MAP2 and Tau, which are the member of microtubule associated protein,can interact with VDAC.We presumed that MAP4 has an important role in modulating hypoxia-induced MPTP opening and apoptosis .. In this study, neonatal rat cadiocytes cultured in the hypoxic gas were employed as the hypoxic model. We construced wild-type and phosphorylation sites mutation as well as MAP4 RNA interference recombination adaenovirus to investigate the effects and mechanisms of MAP4 in hypoxia-induced MPTP openning and cardiocyte apoptosis.ObjectivesTo investigate the mechanism of microtubule-associated protein 4 on the opening of mitochondrial permeability transition pore and apoptosis in hypoxic cardiomyocytes.Methods1. Neonatal rat cadiocytes were isolated, then, cultured in the mixed gas (94%N₂,5%CO₂,1% O₂) to employ the hypoxic model.2. Rat myocard tissues and cadiocytes mitochondrial proteins were obtained after hypoxia treatment, and the expression of MAP4 protein was assayed using western- blot, normal myocard tissues and cadiocytes were used as control.3. Interactions between MAP4 and VDAC were evaluated by co-immunoprecipitation and immunofluorence co-localization. 4. We construced recombination plasmids of MAP4(Glu) and MAP4 SiRNA, and packaged recombinant adenovirus.5. Cadiocytes cytosol protein were obtained and cytochrome C content was evaluated by western-blot. The mitochondrial membrane potential was tested by TMRE and the apoptosis of cadiocytes were detected by TUNEL.Results1. MAP4 protein expresses in the mitochondria of both the neonatal rat cadiocytes and the adult rat myocardial tissues, and it increased after hypoxia treatement.2. MAP4 can bind to VDAC and they have well co-localization in cardiocyte, the binding of MAP4 to VDAC enhanced after 30-min hypoxia.3. Mitochondrial inner membrane potential loss was observed after 30-min hypoxia, and showed a dramatic reduction in TMRE fluorescence intensity. As the hypoxia time prolong, the decrease became more and more significant. MAP4 overexpression was founded to be effectively protective for mitochondrial membrane potential loss induced by hypoxia. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced mitochondrial inner membrane potential loss.4. Normal cadiocytes cytosol cytochrome C content was very low. However, cytosol cytochrome C was increased significant after 30-min hypoxia. With the hypoxia time prolong,the increase became more and more significant. MAP4 Overexpressed cadiocytes were protected from hypoxia-induced cytochrome C release from mitochondria. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced cytochrome c release from mitochondria of cadiocytes.5. Very little cadiocytes aptosis were observed under normal condition. After 3 h hypoxia treatment, cell death was increased significantly. As the hypoxia time prolong, the increase became more and more significant. MAP4 overexpressed cadiocytes were protected from hypoxia-induced cell death. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced incease of cadiocyte apoptosis.Discussion and Conclusions1. We first found that MAP4 protein expresses in the mitochondria of cadiocytes,and the expression of MAP4 increased after hypoxia.2. MAP4 can bind to VDAC, furthermore 30-min hypoxia enhances the binding between them.3. MAP4 phosphorylation, mitochondrial membrane potential lost, cytochrome C in cytosol increased and cardiocyte apoptosis were abserved in the early stage of hypoxia.4. MAP4 overexpression cadiocytes were protected from hypoxia-induced MPTP opening and cadiocyte apoptosis. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced MPTP opening and cadiocyte apoptosis. These results demonstrate that MAP4 has an important role in modulating hypoxia-induced MPTP opening and apoptosis, and MAP4 phosphorylation is the key mechanism of this signal pathway.5. The data presented in this study are consistent with a model in which MAP4 interacts with VDAC proteins to induce MPTP openning and ultimately, cardiocyte apoptosis. Hypoxia induced MAP4 phosphorylation which enhances interact between MAP4 and VDAC. These results demonstrate that MAP4 has an important role in modulating hypoxia-induced cardiocyte apoptosis.