Essential Role Of Smad4 In Maintaining Cardiomyocyte Proliferation During Murine Embryonic Heart Development

Congenital heart disease is prevalent in newborn defects and affects about 1% of live birth. Although many factors were discovered related to heart malformation, the genetic basis of congenital heart disease remains unclear so far. Transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling pathway is essential for embryonic and postnatal heart development and remodeling. The intracellular factor Smad4 plays a pivotal role in mediating TGF-β/BMP signal transduction in the nucleus. However, the specific function of Smad4 in the development of heart has not been elucidated yet. In this study, we found that Smad4 played important roles during embryonic heart development by regulating BMP signaling and a series of pivotal factors involved in heart development.To examine the function of Smad4 in embryonic cardiac development during mid-gestation, we specifically deleted the Smad4 gene in embryonic cardiomyocytes using the Cre-LoxP system. Deletion of Smad4 as early as E9.5 led to embryonic lethality between E12.5 and E15.5. Mutant embryos exhibited severe morphological defects in the heart, including a thin compact layer, disorganized trabeculae, and ventricular septum defects (VSD). The few embryos survived to later stage also displayed VSD and severe outflow tract malformation.Smad4 deletion led to a dramatic decrease in cardiomyocyte proliferation but no alteration in apoptosis. Expression levels of contractile protein-encoding genes such as a-myosin heavy chain,β-myosin heavy chain, ventricular myosin light chain 2, and a-cardiac actin were down-regulated as measured by QPCR. In addition, deletion of Smad4 resulted in perturbation of TGF-β/BMP signaling, including decreased Smad2 and increased Smad1/5/8 activity in cardiomyocyte, and a higher expression level of BMP10, which is a cardiomyocyte-specifically expressed BMP ligand. Expression levels of several cardiac transcription factor genes such as Nkx2.5, GATA4, and MEF2c were also impaired in Smad4 deficient myocardium.These results provide direct genetic evidences that Smad4 is essential for regulating cardiocmyocyte proliferation and differentiation during murine cardiogenesis, and provide new insights into potential causes of congenital heart disease.