Gunturiz María Luz, Gómez Luis Alberto
Grupo de Fisiología Molecular, Subdirección de Investigación Científica y Tecnológica, Dirección de Investigación en Salud Pública, Instituto Nacional de Salud.
Biomedica. 2014 Jul-Sep;34(3):387-402. doi: 10.1590/S0120-41572014000300009.
The microphthalmia -associated transcription factor ( MITF ) regulates the expression of specific genes and its cardiac expression and function is not known.
To identify the expression of MITF in hearts and isolated cardiomyocytes from Guinea pigs, to describe morphological changes associated with mRNA interference of MITF and to evaluate their relative changes in expression in isolated cardiomyocytes under ischemic preconditioning.
The cardiac specific isoform, MITF-H, and relative expression level analysis, was determined by semi-quantitative real time PCR, sequencing and Western blotting. Reduction of mRNA-MITF-H was induced by transduction of specific-MITF-shRNAi interference. The cardiac morphological changes, diameter and number of cardiac fibers were evaluated by direct observation and light microscopy.
A cDNA fragment of 281 bp was amplified from heart and isolated ventricular cardiac myocytes. Sequence analysis confirmed the identity of the isoform MITF-H, exon 1. The MITF silencing was associated with an increase in cardiac index (heart weight/body weight vs . 5.46 x 10 -3 vs 4.6 x 10 -3 ) and higher diameter of cardiac fibers (50.2±16 µ m vs 38,7±14,7 µ m p<0.05, n=150). In isolated cardiac myocytes under ischemic preconditioning we observed a higher relative expression compared with that measured in myocytes exposed to normoxia and simulated ischemia (eighty and one hundred times, p <0.05, n = 5). Conclusion. The results suggest that MITF-H isoform may be involved in Guinea pig cardiac hypertrophy, response to stress by ischemia and cardiomyocytes survival.
小眼畸形相关转录因子（MITF）调节特定基因的表达，其在心脏中的表达及功能尚不清楚。
鉴定MITF在豚鼠心脏及分离的心肌细胞中的表达，描述与MITF mRNA干扰相关的形态学变化，并评估缺血预处理下分离的心肌细胞中其表达的相对变化。
通过半定量实时PCR、测序和蛋白质印迹法测定心脏特异性异构体MITF-H及其相对表达水平。通过转导特异性MITF-shRNAi干扰诱导mRNA-MITF-H的减少。通过直接观察和光学显微镜评估心脏形态变化、心肌纤维直径和数量。
从心脏和分离的心室心肌细胞中扩增出一个281 bp的cDNA片段。序列分析证实了异构体MITF-H外显子1的身份。MITF沉默与心脏指数增加（心脏重量/体重，分别为5.46×10-3和4.6×10-3）和心肌纤维直径增加（50.2±16 µm对38.7±14.7 µm，p<0.05，n = 150）有关。在缺血预处理下的分离心肌细胞中，我们观察到与暴露于常氧和模拟缺血的心肌细胞相比，相对表达更高（分别为80倍和100倍，p<0.05，n = 5）。结论。结果表明，MITF-H异构体可能参与豚鼠心脏肥大、对缺血应激反应及心肌细胞存活。
