Sarcosin (Krp1) in skeletal muscle differentiation: gene expression profiling and knockdown experiments LEONIE DU PUY 1,# , ABDELAZIZ BEQQALI 2,## , HELENA TA VAN TOL 1 , JANTINE MONSHOUWER-KLOOTS 2 , ROBERT PASSIER 2 , HENK P. HAAGSMAN 3 and BERNARD A.J. ROELEN *,1,4 1 Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, 2 Department of Anatomy & Embryology, Leiden University Medical Center, Leiden, 3 Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht and 4 Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands. ABSTRACT SARCOSIN, also named Krp1, has been identified as a protein exclusively expressed in striated muscle tissue. Here we report on the role of SARCOSIN in skeletal muscle development and differentiation. We demonstrate, by means of whole-mount in situ hybridization, that Sarcosin mRNA is expressed in the myotome part of the mature somites in mouse embryos from embryonic day 9.5 onwards. Sarcosin is not expressed in the developing heart at these embryonic stages, and in adult tissues the mRNA expression levels are five times lower in the heart than in skeletal muscle. SARCOSIN protein partially co-localizes with the M-band protein myomesin and between and below laterally fusing myofibrils in adult skeletal muscle tissue. RNA interference mediated knock-down of SARCOSIN in the C2C12 myoblast cell line appeared to be stimulatory in the early phase of differentiation, but inhibitory at a later phase of differentiation. KEY WORDS: mouse, skeletal muscle, sarcosin, differentiation, RNAi Introduction SARCOSIN, also named kelch related protein 1 (Krp1) was originally identiied and described to be exclusively expressed in sarcomeric muscle (Taylor et al., 1998). Northern hybridization experiments revealed high expression in adult human skeletal and heart muscle, with lower levels of expression in prostate muscle. The levels in skeletal muscle were found to be about 15 fold greater than the levels in cardiac muscle. No hybridization was detected in muscle samples from uterus, colon, intestine, bladder and stomach (Taylor et al., 1998). In skeletal muscle differentiation, important processes take place such as withdrawal from the cell cycle, fusion, de novo myoibril- logenesis and myotube formation that do not occur in primary cardiomyocyte cultures but the function of SARCOSIN in these processes is relatively unknown. Skeletal muscle originates from the mesoderm. During embry- onic development, paraxial mesoderm is irst present on either side of the neural tube and notochord after which it will undergo segmentation to form somites. The somites are further speciied into Int. J. Dev. Biol. 56: 301-309 doi: 10.1387/ijdb.113327lp www.intjdevbiol.com *Address correspondence to: Bernard A. J. Roelen. Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University. Yalelaan 104, 3584 CM Utrecht, The Netherlands. Tel: +31-30-253-3352. Fax: +31-30-253-4811. e-mail: b.a.j.roelen@uu.nl Present addresses: # Kinesis-Pharma, Breda, the Netherlands; ## Heart Failure Research Center, Academic Medical Center, Amsterdam, the Netherlands Accepted: 3 August 2011. Final, author-corrected PDF published online: 23 April 2012. ISSN: Online 1696-3547, Print 0214-6282 © 2012 UBC Press Printed in Spain Abbreviations used in this paper: ISH, in situ hybridisation; N-RAP, nebulin-related anchoring protein; siRNA, small interfering RNA. ventral sclerotome, which gives rise to the axial skeleton, and dorsal dermomytome responsible for the formation of dermal precursors and trunk, limb and several head muscles (Buckingham, 2001). Because of relatively high expression levels in skeletal muscle, SARCOSIN is thought to be important in muscle physiology but little is known about its expression during embryonic development. Several indications for SARCOSIN’s functions are presented by its structure. The amino acid sequence encodes ive Kelch repeats at its carboxyl terminus and a BTB/POZ domain at the amino terminus (Spence et al., 2000; Taylor et al., 1998). Kelch repeats form a b-propeller which is important for interactions with proteins of the cytoskeleton (Gray et al., 2009) and proteins containing Kelch repeats indeed have diverse functions in cell morphology and organization (Adams et al., 2000). Several proteins have been reported as binding partners for SARCOSIN, all alluding to a role in cell structure and in particular in myoibril function. One of such binding partners of SARCOSIN is