Cardiac muscle fibers consist of highly aligned cardiomyocytes containing myofibrils oriented parallel to the fiber axis, and successive cardiomyocytes are interconnected at their ends through specialized junctional complexes (intercalated disks). Cell culture studies of cardiac myofibrils and intercalated disks are complicated by the fact that cardiomyocytes become extremely flattened and exhibit disorganized myofibrils and diffuse intercellular junctions with neighboring cells. In this study we sought to direct the organization of cultured cardiomyocytes to more closely resemble that found in vivo. Lanes of laminin 5-50 microm wide were microcontact-printed onto nonadhesive (BSA-coated) surfaces. Adherent cardiomyocytes responded to the spatial constraints by forming elongated, rod-shaped cells whose myofibrils aligned parallel to the laminin lanes. Patterned cardiomyocytes displayed a striking, bipolar localization of the junction molecules N-cadherin and connexin43 that ultrastructurally resembled intercalated disks. When laminin lanes were widely spaced, each lane of cardiomyocytes beat independently, but with narrow-spacing cells bridged between lanes, yielding aligned fields of synchronously beating cardiomyocytes. Similar cardiomyocyte patterns were achieved on the biodegradable polymer PLGA, suggesting that patterned cardiomyocytes could be used in myocardial tissue engineering. Such highly patterned cultures could be used in cell biology and physiology studies, which require accurate reproduction of native myocardial architecture.

Skeletal myoblast grafts can form contractile tissue to replace scar and repair injured myocardium. Although potentially therapeutic, generating reproducible and sufficiently large grafts remains a challenge. To control myoblast proliferation in situ, we created a chimeric receptor composed of a modified FK506-binding protein (F36V) fused with the fibroblast growth factor receptor-1 cytoplasmic domain. Mouse MM14 myoblasts were transfected with this construct and treated with AP20187, a dimeric F36V ligand, to induce receptor dimerization. Transfected myoblasts proliferated in response to dimerizer (comparable with basic fibroblast growth factor (bFGF) treatment), whereas the dimerizer had no effect on non-transfected cells. Similar to bFGF treatment, dimerizer treatment blocked myotube formation and myosin heavy chain expression and stimulated mitogen-activated protein (MAP) kinase phosphorylation in transfected cells. Non-transfected cells differentiated normally and showed no MAP kinase phosphorylation with dimerizer treatment. Furthermore, myoblasts treated with dimerizer for 30 days in culture reduced MAP kinase phosphorylation, withdrew from the cell cycle, and differentiated normally upon drug withdrawal, demonstrating reversibility of the effect. Thus, forced dimerization of the fibroblast growth factor receptor-1 cytoplasmic domain reproduces critical aspects of bFGF signaling in myoblasts. We hypothesize that in vivo administration of AP20187 following myoblast grafting may allow control over graft size and ultimately improve cardiac function.Skeletal myoblast transplantation is a promising technique that may allow delivery of therapeutic genes (1) and can serve as a source of contractile muscle to repair damaged tissue, such as infarcted myocardium (2-5) and dystrophic skeletal muscle (6, 7). The success of such cell-based therapies is dependent on transplanting an appropriate number of myoblasts to elicit the desired therapeutic effect, which remains a significant challenge. One strategy for improving the outcome of myoblast grafting involves the implantation of a smaller number of cells initially, followed by in vivo expansion using mitogenic factors to increase graft size and improve integration with host tissue.

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