RESUMO
The ability to control proliferation of grafted cells in the heart and consequent graft size could dramatically improve the efficacy of cell therapies for cardiac repair. To achieve targeted graft cell proliferation, we created a chimeric receptor (F36Vfgfr-1) composed of a modified FK506-binding protein (F36V) fused with the cytoplasmic domain of the fibroblast growth factor receptor-1 (FGFR-1). We retrovirally transduced mouse C2C12 and MM14 skeletal myoblasts with this construct and treated them with AP20187, a dimeric F36V ligand ("dimerizer"), in vitro and in vivo to induce receptor dimerization. Dimerizer treatment in vitro activated the mitogen-activated protein kinase pathway and induced proliferation in myoblasts expressing F36Vfgfr-1 comparable with the effects of basic FGF. Wild-type myoblasts did not respond to dimerizer. Subcutaneous grafts composed of myoblasts expressing F36Vfgfr-1 showed a dose-dependent increase in DNA synthesis with dimerizer treatment. When myoblasts expressing F36Vfgfr-1 were injected into infarcted hearts of nude mice, dimerizer treatment resulted in a dose-dependent increase in graft size, from 20 +/- 3 to 42.9 +/- 4.3% of the left ventricle. Blinded echocardiographic analysis demonstrated that larger graft size was associated with a dose-dependent reduction in ventricular dilation after myocardial infarction, although animals with the largest grafts showed an increased incidence of ventricular tachycardia. Thus, selective proliferation of genetically modified graft cells can be induced with a systemically administered synthetic molecule in vitro or in vivo. Control of intramyocardial graft size by this approach may allow optimization of cell-based therapy to obtain desired cardiac function postinfarction.
Assuntos
Infarto do Miocárdio/terapia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Animais , Linhagem Celular , Proliferação de Células , Dimerização , Terapia Genética/métodos , Técnicas In Vitro , Camundongos , Camundongos Nus , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Infarto do Miocárdio/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/uso terapêutico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução GenéticaRESUMO
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.