Sik1 Controls Cardiomyogenesis Figure 7. Over-expression of p57 in sik1flp/fl cells. (A) Western blot analysis with the anti-p57 antibodies to detect the expression of transgene in three different clones in sik1flpflp ES cells. (B) Measurement of myosin positive cells in wild-type, sik1flp/flp and sik1flp/flp overexpressing p57 EBs by FACS, analyzed at days 0, 5, 6 and 7 of differentiation with the antibody MF20. Data represent results of one out of two independent experiments (C) Q-PCR analysis of terminal cardiac markers (a-MyHC, Mlc2V, aCach and cTnI). doi:10.1371/journal.pone.0009029.g007 explained by the expression of other members of the KIP/CIP family, as p21 and p27, that is not abolished during differentiation and which can likely compensate for the lack of cdkn1c. In addition, our results show that sik1 specifically regulates the appearance of cardiac cells, since the timing of appearance of neurons is not affected by sik1 deficiency. Our findings clearly shown that sik1 is one of the regulators in the cascade of events leading to cardiomyogenesis through the modulation of p57Kip2 expression. Notably, the balance between proliferation and terminal differentiation is a critical point in organ development [26], especially for the heart that is the first organ to form in the embryo [27]. Indeed, cardiac growth and chamber maturation are regulated by temporally and spatially controlled processes, resulting in a precise regulation of cell cycle exit and cell differentiation of cardiac cells. The ability of these cells to undergo cell division decreases progressively during heart development, such that adult myocytes cannot divide [7]. In this respect, cell cycle regulatory proteins are essential in orchestrating this process. Although the identities of signals involved in cardiac specifica- tion are far to be fully elucidated, p57Kip2 is one of the intracellular candidates initially expressed in the myocardium at E10.5 [8]. Embryos lacking p57Kip2 exhibit hyperplasia in several organs and delayed differentiation, probably due to failure to exit from the cell cycle in a timely fashion. Surprisingly, cardiac defects have not been reported in p57Kip2 deficient mice [28], suggesting that other regulators are likely to be involved in a cooperative way with p57Kip2 in the cardiac cell cycle withdrawal and terminal differentiation. The gene profiling study performed on sik1 deficient ES cells revealed that some of the transcriptional affected genes are expressed in the heart or involved in cardiac defects. This data suggests that sik1 provide a critical growth signal for cardiomyoblasts, acting not only on cdkn1c gene, to promote cell cycle exit, but also on other genes involved in cardiomyocyte differentiation and/or heart development. The observation that sik1 plays an important role in muscle function as HDAC5 kinase raises interesting questions regarding the possible link between HDAC5 and cell cycle in ES cells. HDAC5 belongs to class II HDAC enzymes and is highly enriched in muscles, heart and brain, as HDAC9 [29]. Although transgenic mice lacking either HDAC5 or HDAC9 are viable, mice lacking both HDAC5 and 9 show a propensity for lethal ventricular septal defects and thin-walled myocardium, which tipically arise from abnormalities in growth and maturation of cardiomyocytes [30]. The development of cardiac defects in these double KO mice probably results from a precocious differentiation and cell cycle withdrawal of cardiomyocytes, causing hypocellularity of the myocardium. Is sik1 involved in this intrigued mechanism? Our results shed light on the role of sik1 in cardiomyogenesis and, although further investigation is required to solve this issue, our data provide a new piece of puzzle in the understanding of the molecular mechanisms regulating cardiomyocyte cell cycle, which is of vital importance for future clinical approach. To our knowledge, our manuscript provides the first evi- dence that sik1 via p57Kip2 plays a central role in the time clock mechanism that regulates cardiac cell proliferation and differentiation. PLoS ONE | www.plosone.org 9 February 2010 | Volume 5 | Issue 2 | e9029