RESUMEN
OBJECTIVE: To investigate the role of MyD88-dependent nuclear factor-kappaB (NF-(B) activation signaling pathway in the development of cardiac hypertrophy in vivo. METHODS: Dominant negative myeloid differentiation protein (dn-MyD) 88 fragment was inserted into pShuttle plasmid and then fused into adenovirus so as to construct Ad5-dn-MyD88. Some Sprague-Dawley (SD) rats underwent banding of aorta (aorta binding group) and some SD rats underwent sham operation (sham operation group). Part of the rats in the aorta banding group were transfected with Ad5-dn-MyD88 into the myocardium tissue (Ad5-dn-MyD88 transfection group) or adenovirus expressing dnMyD88 (Ad5-GFP) (control group) so as to determine the effect of blocking down stream of MyD88 signaling on the development of cardiac hypertrophy. Three days after the hearts of some rats from the 4 groups were collected and Western blotting was used to detect the expression of dn-MyD88 protein and fluorescent microscopy was used to detect the expression of GFP. Three weeks after the beginning of experiment the hearts were collected to calculate the heart weight/body weight (HW/BW) ratio and extract the plasma protein and nuclear protein. Electrophoretic mobility shift assay (EMSA) was used to determine the NFkappaB binding activity. Western blotting was used to examine the phosphorylation of IkappaBalpha and IKKalpha/beta with appropriate specific anti-phospho antibodies. RESULTS: Flag and dn-MyD88 were effectively expressed 3 days after the transfection of Ad5-dn-MyD88 into the myocardium. Three weeks after the HW/BW ratio was 0.47 +/- 0.01 in the aorta banding group, significantly higher, by 37.8%, than that of the sham operation group (0.34 +/- 0.01, P < 0.01), and was 0.41 +/- 0.02 in the Ad5-dn-MyD88 transfection group, significantly lower, by 11.58%, than that of the aorta banding group (P < 0.01); the myocardial ANP protein expression level of the aorta binding group was significantly higher, by 43.5%, than that of the sham operation group (P < 0.01) and 36.2% higher than that of the Ad5-dnMyD88 transfection group (P < 0.01); the ANP/GAPDH in the aorta binding group was significantly higher than that of the sham operation group (P < 0.01) and that of the Ad5-dn-MyD88 transfection group (P < 0.01); the NF-kappaB binding activity in the myocardium of the aorta banding group was 9.94 +/- 1.58, significantly higher, by 144.8%, than that of the sham operation group (4.06 +/- 0.52, P < 0.01) and significantly lower, by 41.8%, than that of the Ad5-dn-MyD88 transfection group (5.79 +/- 0.52, P < 0.05); the phospho-(p-) IkappaBalpha level and p-IkappaBalpha/IkappaBalpha of the aorta binding group were significantly higher than those of the sham operation group (P < 0.01) and significantly higher, by 26.7%, than that of the Ad5-dn-MyD88 transfection group (P < 0.05); the p-IKKalphabeta/IKKalphabeta in the myocardium of the aorta binding group was significantly higher, by 318.0%, than that of the sham operation group (P < 0.01), and significantly higher, by 77.4%, than that of the Ad5-dn-MyD88 transfection group (P < 0.01). CONCLUSION: MyD88-dependent NFkappaB signaling is a novel pathway for inducing the development of cardiac hypertrophy in vivo and blocking MyD88 mediated signaling pathway attenuates the development of cardiac hypertrophy.