Reinecke Hans, Minami Elina, Virag Jitka I, Murry Charles E
Department of Pathology, University of Washington, Seattle, 98195, USA.
Hum Gene Ther. 2004 Jul;15(7):627-36. doi: 10.1089/1043034041361253.
Cellular cardiomyoplasty using skeletal myoblasts may be beneficial for infarct repair. One drawback to skeletal muscle cells is their lack of gap junction expression after differentiation, thus preventing electrical coupling to host cardiomyocytes. We sought to overexpress the gap junction protein connexin43 (Cx43) in differentiated skeletal myotubes, using retroviral, adenoviral, and plasmid-mediated gene transfer. All strategies resulted in overexpression of Cx43 in cultured myotubes, but expression of Cx43 from constitutive viral promoters caused significant death upon differentiation. Dye transfer studies showed that surviving myotubes contained functional gap junctions, however. Retrovirally transfected myoblasts did not express Cx43 after grafting into the heart, possibly due to promoter silencing. Adenovirally transfected myoblasts expressed abundant Cx43 after forming myotubes in cardiac grafts, but grafts showed signs of injury at 1 week and had died by 2 weeks. Interestingly, transfection of already differentiated myotubes with adenoviral Cx43 was nontoxic, implying a window of vulnerability during differentiation. To test this hypothesis, Cx43 was expressed from the muscle creatine kinase (MCK) promoter, which is active only after myocyte differentiation. The MCK promoter resulted in high levels of Cx43 expression in differentiated myotubes but did not cause cell death during differentiation. MCK-Cx43-transfected myoblasts formed viable cardiac grafts and, in some cases, Cx43-expressing myotubes were in close apposition to host cardiomyocytes, possibly allowing electrical coupling. Thus, high levels of Cx43 during skeletal muscle differentiation cause cell death. When, however, expression of Cx43 is delayed until after differentiation, using the MCK promoter, myotubes are viable and express gap junction proteins after grafting in the heart. This strategy may permit electrical coupling of skeletal and cardiac muscle for cardiac repair.
使用骨骼肌成肌细胞进行细胞心肌成形术可能对梗死修复有益。骨骼肌细胞的一个缺点是分化后缺乏缝隙连接表达，从而阻止了与宿主心肌细胞的电偶联。我们试图使用逆转录病毒、腺病毒和质粒介导的基因转移，在分化的骨骼肌肌管中过表达缝隙连接蛋白连接蛋白43（Cx43）。所有策略均导致培养的肌管中Cx43过表达，但组成型病毒启动子驱动的Cx43表达在分化时导致大量细胞死亡。然而，染料转移研究表明，存活的肌管含有功能性缝隙连接。逆转录病毒转染的成肌细胞移植到心脏后不表达Cx43，可能是由于启动子沉默。腺病毒转染的成肌细胞在心脏移植物中形成肌管后表达大量Cx43，但移植物在1周时出现损伤迹象，2周时死亡。有趣的是，用腺病毒Cx43转染已分化的肌管是无毒的，这意味着在分化过程中有一个易损期。为了验证这一假设，从肌肉肌酸激酶（MCK）启动子表达Cx43，该启动子仅在心肌细胞分化后才活跃。MCK启动子在分化的肌管中导致高水平的Cx43表达，但在分化过程中不引起细胞死亡。MCK-Cx43转染的成肌细胞形成了存活的心脏移植物，在某些情况下，表达Cx43的肌管与宿主心肌细胞紧密相邻，可能实现电偶联。因此，骨骼肌分化过程中高水平的Cx43会导致细胞死亡。然而，当使用MCK启动子将Cx43的表达延迟到分化后时，肌管是存活的，并且在移植到心脏后表达缝隙连接蛋白。这种策略可能允许骨骼肌和心肌之间实现电偶联以进行心脏修复。
