RESUMEN
Cardiomyocytes are post-mitotic, long-lived cells until disruptions to pro-survival factors occur after myocardial ischemia. To gain an understanding of the factors involved with ischemic injury, we examined expression changes in pro-survival and opposing pro-apoptotic signals at early and chronic periods of ischemia using an in vivo murine model. Alterations of pro-survival proteins such as the inhibitor of apoptosis protein on chromosome X (xIAP) and the apoptotic repressor protein (ARC) have not been evaluated in a murine model of cardiac ischemia. Early ischemia (1 day) resulted in a 50% reduction in ARC protein levels relative to sham-operated left ventricles, without significant changes in the expression of xIAP or other pro-survival factors. In contrast, a deficiency of xIAP expression was found in cardiac infarcts starting after 1 week, concomitant with significant evidence of apoptotic cell death and an up-regulation of pro-apoptotic signals including Bax, tumor necrosis factor-a, and caspase-8 activation. Chronic ischemia (after 2 weeks) was associated with elevated levels of other pro-survival factors such as Bcl-xL and the phosphorylated form of Akt, as part of the adaptive remodeling of the myocardium. Altogether, these findings suggest that strategies to increase IAP expression may promote myocyte survival after chronic ischemia.
Asunto(s)
Regulación de la Expresión Génica , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Miocardio/metabolismo , Enfermedad Aguda , Animales , Proteínas Reguladoras de la Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Western Blotting , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Supervivencia Celular , Enfermedad Crónica , Modelos Animales de Enfermedad , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos ICR , Proteínas Musculares/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
Two-component regulatory systems are involved in processes important for bacterial pathogenesis. The proposed misR/misS (or phoP/phoQ) system is one of four two-component systems of the obligate human pathogen Neisseria meningitidis. Inactivation of this system results in loss of phosphorylation of the lipooligosaccharide inner core and causes attenuation in a mouse model of meningococcal infection. MisR and the cytoplasmic domain of MisS were purified as His6 and maltose binding protein fusion proteins, respectively. The MisS fusion was shown to be autophosphorylated in the presence of ATP, and the phosphoryl group was subsequently transferred to MisR. The phosphotransfer reaction was halted with a MisR/D52A mutation, while a MisS/H246A mutation prevented autophosphorylation. Specific interaction of phosphorylated MisR (MisR approximately P) and MisR with the misR promoter was demonstrated by gel mobility shift assays, where MisR approximately P exhibited higher affinity than did the nonphosphorylated protein. The transcriptional start site of the misRS operon was mapped, and DNase I protection assays revealed that MisR interacted with a 15-bp region upstream of the transcriptional start site that shared no similarity to binding motifs of other two-component systems. Transcriptional reporter studies suggested that MisR phosphorylation is critical for the autoinduction of the misRS operon. Limited Mg2+ concentration failed to induce expression of the misRS operon, which is the only operon now proven to be under the direct control of the MisRS two-component system. Thus, these results indicate that the meningococcal MisRS system constitutes a functional signal transduction circuit and that both components are critical in the autoregulation of their expression.