Abstract 3818: Notch1 Receptor Blockade Results in Dilated Cardiomyopathy in the Neonatal Mouse Heart
The objective of this study was to determine whether Notch1 receptor plays a crucial role in the commitment of c-kit-positive cardiac progenitor cells (CPCs) to the myocyte lineage. This possibility was tested in the neonatal mouse heart because of the dramatic increase in the myocyte compartment early postnatally. CPCs are located within the primitive heart and their number increases progressively with gestational age and after birth. c-kit colocalizes with transcription factors and contractile proteins specific of myocytes, pointing to a lineage relationship between CPCs and myocytes in the prenatal and postnatal heart. Notch1 intracellular domain (N1ICD) was consistently found together with Nkx2.5 suggesting that Notch1 receptor represents an early determinant of myocyte differentiation. The critical function of Notch1 in the generation of myocytes was established by interfering with this pathway through the administration of a γ-secretase inhibitor to newborn mice for 3–7 days; γ-secretase inhibition opposes the cleavage of the active fragment of Notch and initiation of transcription. Treated mice showed a dilated myopathy characterized by decreased muscle mass, wall thinning and impaired fractional shortening and ejection fraction. There was also a marked reduction in the fraction of CPCs and myocytes expressing the Notch1 active fragment N1ICD. Myocyte number decreased 28% while myocyte volume remained constant, suggesting that Notch1 blockade affects myocyte formation and conditions the development of a dilated myopathy. This hypothesis is supported further by a 57% decrease in replicating myocytes positive for Ki67. These anatomical and functional changes were accompanied by downregulation of Nkx2.5 transcripts in the pathologic heart. A perfect consensus site for the target gene of Notch, RBP-Jk, was recognized in the promoter of Nkx2.5. The formation of a functional complex between Nkx2.5 promoter and RBP-Jk protein was demonstrated by electrophoretic mobility assay, chromatin immunoprecipitation and luciferase reporter assay. In conclusion, Notch1 promotes the commitment of CPCs to the myocyte lineage and inhibition of Notch1 interferes with cardiomyogenesis leading to a dilated cardiomyopathy in the postnatal heart.