Experimental Studies On The Effects Of MAP4 On Microtubule Stabilization Of Hypoxic Cardiomyocytes

Objectives:To determine the effects of microtubule-associated protein 4 (MAP4) on microtubule stabilization of hypoxic cardiomyocytes.Methods:Neonatal murine cardiomyocytes cultured in the mixed gas (1﹪O2, 5﹪CO2, 94﹪N2) were employed as the hypoxic model. The expression ofα-tubulin and MAP4 proteins were determined by Western Blot. The replication defective recombinant adenovirus vector containing MAP4 cDNA was constructed and transfected (used as the treated group). After 48h trasfection, the energy metabolism (with HPLC), the cell viability (with MTT), and the apoptosis (with TUNEL) were determined. The expression of MAP4 protein and its effects on microtubule were observed through laser scanning confocal microscopy.Results:1. In the normal culture condition, microtubules were well-distributed in myocardial cell and the cell kept in complete shape. MAP4 was fixed on the polymerizing microtubules beam formed byαandβ-Tubulin proteins. MAP4 expression decreased markedly at 6 hours. At 12 hours, microtubules were destructed markedly, and a great deal of microtubule fragment were seen around the cell nucleus. The immunofluorescence staining intensity of MAP4 in microtubules decreased significantly. This phenomenon showed that no apparent polymerized microtubules existed in myocardial cell, and the cystoskeleton was destroyed severely. The contents ofα-tubulin protein and MAP4 decreased gradually.2. The cDNA part of MAP4 containing 3174bp was constructed successfully, then it was inserted in adeno-virus gene group to form the replication defective Ad-MAP4 vector.3. The results showed that up-regulation of MAP4 expression had a certain effect on microtubule stabilization.4. The ATP content in cardiomyocytes decreased markedly after hypoxia of 0.5~3.0 hours, and remained at a relative lower level during the following 6~12 hours of hypoxia.