Heterokaryons of Cardiac Myocytes and Fibroblasts Reveal the Lack of Dominance of the Cardiac Muscle Phenotype

The molecular characterization of a cardiac determination gene has been an elusive goal for the past several years. Prior to cloning of the skeletal muscle determination factor MyoD, the presence of a dominantly acting skeletal muscle determination factor had been inferred from the observation that the skeletal muscle phenotype was dominant in skeletal muscle-fibroblast heterokaryons (H. M. Blau, G. K. Pavlath, E. C. Hardeman, C.-P. Chiu, L. Siberstein, S. G. Webster, S. C. Miller, and D. Webster, Science 230:758-766, 1985). In these experiments, we have examined cardiac-fibroblast heterokaryons to investigate the existence of a dominantly acting cardiac determination factor. We have employed a novel experimental approach using primary embryonic fibroblasts from transgenic mice as a means of assaying for the activation of a cardiac promoter-luciferase reporter transgene within fibroblast nuclei. This approach provides a potential means of genetic selection for a dominantly acting positive factor and can be generalized to other systems. We have examined the expression of three markers of the cardiac lineage: a myofibrillar protein promoter (MLC2), a secreted protein (ANF), and a transcription factor (MEF2). MEF2 is specific to both cardiac and skeletal muscle cells. Our results indicate that in a majority of heterokaryons with an equal ratio of cardiac to fibroblast nuclei, none of these cardiac markers are expressed, indicating that the cardiac phenotype is not dominant over the embryonic fibroblast phenotype. The distinction from previous results with skeletal muscle is emphasized by our results with MEF2, which is dominantly expressed in skeletal muscle-fibroblast but not cardiac-fibroblast heterokaryons, supporting its divergent regulation in the two cell types.