Sik1 Controls Cardiomyogenesis Figure 6. Expression profile of CDKIs during both cardiomyocyte and neuron differentiation. (A) Expression profile of genes encoding for CDK inhibitors belonging to the KIP/CIP family (Cdkn1a, Cdkn1b and Cdkn1c) measured by quantitative real-time PCR at different time points during cardiomyocyte differentiation of wild-type and sik1flpflp ES cells, n = 3, *, P,0.05, **, P,0.001 vs. control cells. P values were calculated using a two-tailed, unpaired t test. (B) Western blots analysis of CDKIs protein level during in vitro cardiomyocyte differentiation of wild-type and sik1flpflp ES cells. (C) p21, p27 and p57 protein levels were expressed as the ratio between their arbitrary densitometric units (ADU) and b-actin. Densitometry analyses were performed using the ImageQuant 5.2 software (GE Healthcare). (D) Expression profile of Cdkn1c gene measured by quantitative real- time PCR at different time points during neuronal differentiation of wild-type and sik1flpflp ES cells. doi:10.1371/journal.pone.0009029.g006 Discussion Sik1 (also known as SNF1LK, sucrose nonfermented 1-like kinase) encodes a serine/threonine kinase that belongs to a family of AMP-activated protein kinases (AMPK) involved in the regulation of metabolism during energy stress [25]. The expression pattern of sik1 during embryogenesis suggests its involvement in cardiomyocyte differentiation and/or heart development. However, despite the significant research efforts toward the comprehension of the function of such a protein, the definition of its biological significance during cardiac development is still unknown. We decided to investigate the role of sik1 gene during cardiac differentiation using a gene trap ES cell line and revealed a novel role of sik1 in controlling the cell cycle progression of cardiomyoblasts. Using the ES/EB model, we found that the absence of sik1 produced a consistent delay of terminal cardiac differentiation and most remarkably, that this delay can be explained with the failure of the transcriptional regulation of the cyclin-dependent kinase inhibitor p57Kip2. It is worth noting here that the levels of p57Kip2 are very low in proliferating cells, and that differentiative signals result in its accumulation, which is necessary to withdraw cells from the cell cycle and to move them toward differentiation [8]. These data are in agreement with our observation that, during cardiac differentiation, the expression of p57Kip2 increases with the appearance of cardiomyocytes. Given that sik1 deficiency completely suppressed the expression of p57Kip2, we speculated that defective signalling in the cell-cycle exit of these cells causes the delayed phenotype. The findings presented in this study indicate that sik1 signaling contributes to the cardiogenic program through the coordinated action of p57Kip2. Moreover, p57Kip2 over-expression experiment indicates that p57Kip2 is a downstream mediator of sik1, acting specifically in the cardiac differentiation process. It is worth noting that the absence of cdkn1c transcriptional activation during differentiation of sik1flp/flp ES cells does not prevent the formation of mature cardiomyocytes. This might be PLoS ONE | www.plosone.org 8 February 2010 | Volume 5 | Issue 2 | e9029