Effects Of The Microtubule Damage On The Opening Of Mitochondrial Permeability Transition Pore In Hypoxic Cardiomyocytes.

Objectives:To clarify if microtubule damage of cardiomyocyte induced by hypoxia plays an important role in the openning of the mitochondrial permeability transition and its pathogenesis.Methods:1. Neonatal rat cardiomyocytes cultured in the mixed gas (94%N2,5%CO2,1% O2) were employed as the hypoxic model.2.Cardiomyocytes in primary culture were randomized as normoxia group(A), hypoxic group (B), normoxia treated with microtubule destabilizing agent(colchicine ) group (C), and hypoxia treated with microtubule stabilizing agent(taxol) group (D). After 0.5h,1h,3h,6h and 12h treatment, polymeric tubulin was detected by immunocytochemistry and western blotting.3. Open of mitochondrial permeability transition pore were observed by coloading with calcein AM and cobalt chloride, the mitochondrial inner membrane potential loss by TMRE and the activity of cells by measuring the mitochondrial-dependent reduction of MTT to formazan.4. The changes of free calcium ions and ROS were determined using fluorescent probe Fluo-3/F-127 and DCFH-DA.Results:1. Early disassemble and decrease of fluorescence intensity of polymeric microtubule were observed after 30min of hypoxia. The dissemble and decrease became more and more significant with the duration of hypoxia from 30min to 12h, which was confirmed by the results of western blotting of polymeric microtubule.2. Fluorescence intensity alterations of polymeric microtubule were quantified using a...