UCLA

The formation of the heart, cardiomyogenesis, is highly regulated to ensure proper formation and function. The objective of this study was to examine the role of bone morphogenetic protein (BMP) signaling in cardiomyogenesis. In order to accomplish this, we used an adipocyte-derived multipotent cell model termed dedifferentiated (DFAT) fat cells which readily differentiate into cardiomyocytes. Specifically, we investigated the effects of treating DFAT cells with BMP10 and crossveinless-2 (CV2), a BMP inhibitor. BMP10 is expressed exclusively in the heart and activates the activin receptor-like kinase 1 (ALK1) which is a BMP receptor that induces expression of cardiogenic factors such as Nkx2.5 through the SMAD signaling pathway. CV2 was investigated because it has been shown to modulate BMP signaling. We chose to examine BMP10's role in cardiomyogenesis because it is structurally similar to BMP9, which regulates vascular cell differentiation, and is expressed early in the developing heart. We hypothesized that BMP10 induced proliferation of cardiac progenitor cells and CV2 induced their differentiation.

We demonstrated that CV2 binds directly to BMP10 and inhibits BMP10 from initiating SMAD signaling using co-immunoprecipitation and a luciferase reporter gene assay. We showed that BMP10 treatment induced proliferation of DFAT cells using a 3H-thymidine proliferation assay, whereas CV2 proliferation levels were similar to those produced by control treatments. However, CV2 induced differentiation of DFAT cells into cardiomyocytes, as demonstrated by the induction of expression of cardiomyocyte markers by real-time PCR. Expression levels of Nkx2.5 and troponin I, which are early and late cardiomyocyte markers, respectively, were studied. Treatment with BMP10 showed an increase in Nkx2.5 expression at Day 7 after treatment, whereas CV2 treatment showed an increase in troponin I expression at Day 14. These results were corroborated by immunohistochemistry studies using antibodies against troponin I, which indicates the presence of cardiomyocytes, and α-myosin heavy chain (α-MHC) which indicates the presence of muscle tissue. The results indicated that BMP10 increased the number of early cardiomyocyte progenitor cells, whereas CV2 induced differentiation of DFAT cells into cardiomyocyte-like cells that were electrically active and beating. Taken together, these results supported a model where cardiomyogenesis occurs via a two-step mechanism. First, BMP10 increases the pool of cardiac progenitor cells. Then, once the progenitor pool has been expanded sufficiently, CV2 inhibits BMP10 and triggers cardiomyocyte differentiation.

Our findings may improve our understanding of the mechanism involved in cardiomyogenesis and enhance our ability to generate cardiomyocytes from DFAT cells or other stem cells.