Gene expression profiling of overexpression of myocardin

ABSTRACT Aims Forced differentiation of non-muscle cells into heart muscle cells using cardiac transcription factors (cTFs) may constitute a novel strategy to accomplish myocardial regeneration. Methods We investigated the potential of the cTF myocardin to induce cardiomyocyte differentiation and compared the myocardin-induced gene expression program to that of fully differentiated cardiomyocytes using oligonucleotide microarray hybridizations, quantitative RT-PCR analyses and immunofluorescence microscopy. The experiments were performed in the recently described human cardiomyocytes progenitor cells (hCMPCs), which differentiate into fully functional cardiomyocytes upon stimulation with 5-azacytidine and transforming growth factor β1. Results and Conclusions Forced myocardin expression stimulated transcription of a surprisingly large repertoire of heart muscle-specific genes in hCMPCs but did not cause their differentiation into functional cardiomyocytes. Specifically, myocardin gene transfer did not stimulate the synthesis of several sarcomeric (regulatory) proteins and ion channel constituents. The heart and smooth muscle-enriched isoforms of myocardin stimulate equally well the transcription of many of their cardiomyocyte-specific and virtually all of their smooth muscle target genes. However, the heart muscle-enriched myocardin species is a much more potent transactivator of a subset of genes encoding mainly cardiomyocyte-specific myofibrillar components. This may explain the underestimation of myocardin's cardiomyogenic potential in previous studies utilizing the smooth muscle-enriched isoform of this cTF. Experiment performed on primary cells in culture, the cardiomyocyte progenitor cells (CMPCs), ScaI+ cells isolated from human fetal heart. Cells tranduced with third gen lentivirus vectors, to allow stable overexpression of: 1.) CM-MYOCD cardiomyocyte specific isoform of human myocardin. n=4 2.) SM-MYOCD smooth muscle specific isoform of human myocardin (major open reading frame only). n=3 3.) SM-MYOCD smooth muscle specific isoform of human myocardin (both open reading frames). n=3 4.) LacZ transduction/vector/overexpression control. n=3 5.) untransduced. n=3 13 days post transduction, total RNA harvested for array.