Myoblasts for survive 16 weeks after intracardiac transfer and start differentiation

INTRODUCTION: Skeletal myoblasts have been shown to survive transfer into myocardial scar tissue. The purpose of this study was to investigate differentiation after intramyocardial transplantation. Additionally, we evaluated an intravital fluorescence dye.

MATERIALS AND METHODS: Skeletal myoblasts were harvested from 8 male inbred Lewis rats and expanded in culture. For each transplantation planned, 10(6) cells were trypsinized and incubated for three minutes with 2 ml of buffered PKH-26 solution (Sigma, St. Louis). This dye was integrated into the cell membrane. After washing three times, the cells were plated again for two more days. The cells were then transplanted into the hearts of 60 female Lewis-rats, mean weight 380 g (260 - 450 g). At implantation, 10(6) cells suspended in 100 microliters were injected into the apex region of the left ventricle. 12 animals served as the control group with only cell medium injected. Animals were sacrificed after 1, 2, 6, 8, 12, and 16 weeks (n = 10 each). The hearts were explanted and serial frozen sections of the hearts were prepared for detecting labeled cells. Sections with labeled cells were stained immunohistochemically for Myo D1 (myogenic origin), n-cam (early myotubes), desmin (muscular filament), myosin light chain (muscular contractile protein), and connexin 43 (tight junction).

RESULTS: Cell labeling was successful in all cases. After two days, the myoblasts had recovered from the staining procedure. The fluorescing dye, however, was only rarely transmitted by cell division. Marked cells were found in the intercellular spaces between the cardiac myofibers in at least 8 animals from each group. No fibrotic reaction or inflammation was seen surrounding the transplanted cells. Up to 6 weeks after implantation, the cells stained positive for n-cam and Myo D1, and particularly for desmin. More n-cam positive cells were found than labeled cells, indicating cell division after the cell transfer. Two animals suffered sudden death after a follow-up time of 8 and 10 weeks, which was possibly due to cardiac arrhythmia. After 8 weeks, the cells formed conglomerates and stained positive for desmin, myosin light chain, and connexin 43. The cells were not structurally integrated into the recipient myocardial tissue, however.

CONCLUSIONS: Myoblasts divided further after transplantation into rat myocardium. Positive staining for desmin demonstrated the development of myofibers. Starting at 8 weeks after transplantation, the cells started differentiation without reaching structural integration during follow-up. Labeling the cells with PKH-26 proved to be a reliable method to detect the cells.