Myocardin Expression Is Regulated by Nkx2.5, and Its Function Is Required for Cardiomyogenesis

Abstract

Nkx2.5 (also known as Csx) is an evolutionarily conserved cardiac transcription factor of the homeobox gene family. Nkx2.5 is required for early heart development, since Nkx2.5-null mice die before completion of cardiac looping. To identify genes regulated by Nkx2.5 in the developing heart, we performed subtractive hybridization by using RNA isolated from wild-type and Nkx2.5-null hearts at embryonic day 8.5. We isolated a mouse cDNA encoding myocardin A, which is an alternative spliced isoform of myocardin and the most abundant isoform in the heart from embryo to adult. The expression of myocardin A and myocardin was markedly downregulated in Nkx2.5-null mouse hearts. Transient-cotransfection analysis showed that Nkx2.5 transactivates the myocardin promoter. Inhibition of myocardin function in the teratocarcinoma cell line P19CL6 prevented differentiation into cardiac myocytes after dimethyl sulfoxide treatment. Myocardin A transactivated the promoter of the atrial natriuretic factor gene through the serum response element, which was augmented by bone morphogenetic protein 2 and transforming growth factor β-activated kinase 1. These results suggest that myocardin expression is regulated by Nkx2.5 and that its function is required for cardiomyogenesis.

ACKNOWLEDGMENTS

We thank W. Pu, J. R. McMullen, and E. Hücht for critical reading of the manuscript. We also thank I. Chen for preparing the figures.

This study was supported by Japan Heart Foundation and Bayer Yakuhin Research Grant Abroad, a postdoctoral fellowship from the American Heart Association (AHA; New England affiliate) to T.U., an AHA (Massachusetts Affiliate) Beginning-Grant-in-Aid to H.K., and SCOR in congenital Heart Disease grant from the NIH (P50-HL61036) to S.I.