Abstract 3259: Reversal of Ischemic Cardiomyopathy Using Reprogrammed Skeletal Myoblasts Overexpressing Multiple Therapeutic Growth Factor Genes
Background: We hypothesize that a combinatorial approach involving skeletal myoblasts (SkMs) as carriers of multiple therapeutic genes will be more effective in preservation of heart function after ischemic injury through myoangiogenesis and paracrine effects.
Methods and Results: SkMs purified from GFP transgenic male rats were >90% for desmin expression. Plasmids encoding human IGF1, VEGF, SDF1α and HGF were prepared and transfected into SkMs (TransSkMs). Western blotting showed significantly higher expression of the growth factors in TransSkMs. Multiple angiogenic and prosurvival factors such as SFRP1, SFRP2, SFRP4, SFRP5, MMP3, MMP9, Cx43, Netrin1, Nos2, and Wnt3 were observed in TransSkMs. Western blotting showed activation of Akt, MAPK42/44, Stat3, NFκB, HIF1α, and PKC in TransSkMs. LDH and TUNEL assays showed higher survival of TransSkMs under anoxia (p<0.01 vs non-TransSkMs). Conditioned medium from TransSkMs was also protective for H2C9 and HUVEC under anoxia and induced higher tube formation on Matrigel (47.8±1.9, p<0.01) and migration of HUVEC in Transwell system (223.3±1.8; p<0.01). For in vivo study, 70μl DMEM without cells (group-1) or containing 1.5×106 non-TransSkMs (group2) or 1.5×106 TransSkMs (group3) were injected intramyocardially in a female rat model of acute LAD ligation. Real-time PCR for sry-gene (n=5/group) showed better survival of SkMs in group3 on day7 (p<0.05 vs group2). Immunohistology for GFP expression (n=4/group) combined with Ki67 and TUNEL showed extensive proliferation (p<0.01) and reduced apoptosis (p<0.01) in group3 as compared with group2. Massive mobilization of stem and progenitor cells (cKit+, Sca-1+, CD31+, CXCR4+, CD45+, CD44+) was observed in the infarcted heart. Confocal imaging of heart tissue sections immunostained for myosin heavy chain combined with Cx43 at 8 weeks post engraftment showed extensive myogenesis. Infarct size was attenuated in group3 (22.2±1.7%, p<0.01 vs group2). Blood vessel density (23.7±2.2/400× field, p<0.01) and heart function indices (fractional shortening 19.5±0.4%) were improved in group3 (p<0.01 vs group2).
Conclusions: SkMs engraftment combined with multiple gene delivery will be a novel approach to regenerate cardiovascular tissue in infarcted heart.