Cobellis1,4* Abstract Lack of Sik1 in Mouse Embryonic Stem Cells Impairs Cardiomyogenesis by Down-Regulating the Cyclin- kip2 Dependent Kinase Inhibitor p57 Antonio Romito1, Enza Lonardo 2 , Guglielmo Roma 3 , Gabriella Minchiotti2, Andrea Ballabio1, Gilda 1 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy, 2 Stem Cell Fate Laboratory, Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche (CNR), Naples, Italy, 3 Centre for Genomic Regulation (CRG), Pompeu Fabra University (UPF), Barcelona, Spain, 4 Dipartimento di Patologia Generale, Seconda Universita` di Napoli, Naples, Italy Sik1 (salt inducible kinase 1) is a serine/threonine kinase that belongs to the stress- and energy-sensing AMP-activated protein kinase family. During murine embryogenesis, sik1 marks the monolayer of future myocardial cells that will populate first the primitive ventricle, and later the primitive atrium suggesting its involvement in cardiac cell differentiation and/or heart development. Despite that observation, the involvement of sik1 in cardiac differentiation is still unknown. We examined the sik1 function during cardiomyocyte differentiation using the ES-derived embryoid bodies. We produced a embryonic stem cell using a gene-trap cell line carrying an insertion in the sik1 locus. In absence of the sik1 protein, the temporal appearance of cardiomyocytes is delayed. Expression profile analysis revealed sik1 as part of a genetic network that controls the cell cycle, where the cyclin-dependent kinase inhibitor p57Kip2 is directly involved. Collectively, we provided evidence that sik1-mediated effects are specific for cardiomyogenesis regulating cardiomyoblast cell cycle exit toward terminal differentiation. Citation: Romito A, Lonardo E, Roma G, Minchiotti G, Ballabio A, et al. (2010) Lack of Sik1 in Mouse Embryonic Stem Cells Impairs Cardiomyogenesis by Down- Regulating the Cyclin-Dependent Kinase Inhibitor p57kip2. PLoS ONE 5(2): e9029. doi:10.1371/journal.pone.0009029 Editor: Rory Edward Morty, University of Giessen Lung Center, Germany Received April 20, 2009; Accepted January 11, 2010; Published February 3, 2010 Copyright: 2010 Romito et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Project was supported by the grant from 6th Framework of EU Commission, Contract no.: 037627 to A.B., A.R. and G.C. and the Italian Association for Cancer Research (AIRC) and Telethon Foundation grant to G.M. and E.L. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cobellis@tigem.it Introduction The formation of the heart involves a precisely orchestrated series of molecular and morphogenetic events, and even a subtle perturbation of this process can have catastrophic consequences for cardiac function. The specification of the appropriate numbers and types of cardiac cells is an early event during embryogenesis. These cells then migrate to form a simple, yet functional, heart tube. Further morphogenesis transforms this heart tube into morphologically and functionally discrete cardiac chambers [1]. Cardiomyogenesis depends on the regulated activities of numerous specific transcription factor genes, which encode members of the zinc finger [2], homeodomain [3], T-box [4], bHLH [5] and MADS domain families [6]. These factors act in a combinatorial way to create a positive feed-forward regulatory circuitry that controls the development of cardiac myocytes. A concomitant regulation in the expression and activities of cell- cycle regulatory molecules (cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors [CDKIs]) is essential for the control of cell proliferation that is concurrent with differentiation [7]. Among multiple cell-cycle regulators, CDKI p57Kip2 is the first to be detectable in the developing heart, at E10.5, and is involved in cardiac cell-cycle exit during chamber maturation [8]. The sik1 protein was identified in a screen for kinases specifically expressed in the heart of the mouse embryo [9]. During mouse embryogenesis, sik1 expression is detected at 8.0 d.p.c. in the monolayer of the future myocardial cells; it is rapidly down- regulated at 8.5 d.p.c. upon formation of the primitive ventricle, although it is still present in the myocardial cells that will populate the primitive atrium and bulbus cordis. At 9.5 d.p.c. sik1 expression is down-regulated in the primitive atrium but still detected in the sinus venosus and truncus arteriosus. The expression pattern of sik1 gene suggests a role during the earliest stages of myocardial cell differentiation and/or primitive chamber formation [10]. Recent studies have demonstrated that sik1 protein phosphorylates class II HDACs in vivo, triggering the cytoplasmic export of the HDACs and activation of MEF2-dependent transcription [11,12]. The specific expression pattern of sik1 during mouse develop- ment prompted us to investigate the role of sik1 in the regulation of cardiac lineage commitment in a stem-cell model system. Embryonic stem (ES) cells can differentiate into derivatives of all three of the primary germ-cell layers, including cardiomyocytes, and previous studies have suggested that early steps in embryonic cardiomyogenesis take place during embryoid body (EB) differentiation of ES cells [13]. Using an ES cell line carrying a gene-trap insertion in the sik1 gene, we produced a sik1-deficient cell line. The cardiac developmental program in the ES cell/EB model is affected in the absence of sik1. The PLoS ONE | www.plosone.org 1 February 2010 | Volume 5 | Issue 2 | e9029