µ-CALPAIN GENE KNOCKDOWN OF MUSCLE SATELLITE CELLS USING PSILENCER VECTOR Muthuraman Pandurangan, Hoa V. Ba, Dawoon Jeong, Beom-Young Park1, Soo-Hyun Cho1, Inho Hwang* Department of Animal Science and Institute of Rare Earth for Biological Aplication, Chonbuk National University, Jeonju, 561- 756, Korea 1Quality control and utilization of animal products division, National Institute of Animal Science, Suwon 441-706, South Korea Abstract – This study was designed to find the key called μ- and m-) which are the best-characterized role of μ-calpain and caspases using RNA calpains and calpain 3 (also called p94) which is interference mediated silencing of μ-calpain and highly expressed in this tissue. However, since caspase 9 during satellite cell proliferation. For this conventional inhibitors used for the studies of the experiment, four separate siRNA sequences were functions of these enzymes lack specificity, the screened for their ability to inhibit μ-calpain gene expression. Following optimizing the transfection individual physiological function and biochemical conditions for cell number, volume of transfection mechanism of these three isoforms, especially μ- agent used and the appropriate concentration of calpain, are not clear (3). In contrast, RNA each siRNA, quantitative real-time PCR were used interference (RNAi) has a great potential to to assess the level of inhibition of gene expression distinguish the functions of each member in a achieved by all siRNA sequences used. CAPN1, closely related gene family or to selectively target caspase 3, caspase 7, caspase 9, Hsp27, Hsp70 and a mutant gene, especially to study the functions of Hsp90 mRNA expression were quantified in a particular isoform. Recently numerous research CAPN1-siRNA treated cells by real-time PCR revealed that the potential role of calpains analysis. As a result, knock-down of μ-calpain lead involving apoptosis is indicated by a increasing to the reduction in caspase 3 and caspase 7 gene expression, suggesting that there were a cross-talk list of calpains substrates such as p53, PARP, Bax, between μ-calpain and the caspase enzyme systems. AIF and several cytoskeletal proteins (2). In addition, targeted suppression of caspase 9 gene Although calpains are known to contribute to expression reduced caspase 7 activation and we apoptosis, further studies are still needed to hypothesized that apoptosis take place through an precisely elucidate the role of calpains in apoptosis. intrinsic pathway during satellite cell proliferation. Recently a cross talk between the calpain and the caspase systems has been reported (4). Caspases is Key Words – µ -calpain, satellite cell, Hanwoo. another family of proteases which are involved in programmed cell death. The caspases that appear I. INTRODUCTION to play a role in these processes such as initiator caspases (caspase-2, -8, and-9) and effector Muscle tissue possesses the post-natal growth and caspases (caspase-3, -6, -7, and -14) (5). The role intrinsic regenerative capacity (1). Comprehensive of these caspases in muscle cell development or understanding of the satellite cells involvement in differentiation process in Hanwoo cattle is very postnatal myogenesis, skeletal muscle hypertrophy limited. Earlier studies from our reported that and myofiber regeneration are noteworthy issue elevation of caspase 9 expression during satellite for fundamental agricultural reasons. Calpains are cell proliferation and differen-tiation. In 2+ intracellular nonlysosomal Ca -regulated cysteine continuance to our earlier investigations, the proteases and it mediate regulatory cleavages of present work was designed to study the key role of specific substrates in various cellular processes μ-calpain and caspases using RNA interference such as signal transduction, cell proliferation and mediated silencing of μ-calpain and caspase 9 differentiation, apoptosis and necrosis in mammals during satellite cell proliferation. (2). Muscle tissue expresses mainly three distinct calpains: the ubiquitous calpains 1 and 2 (also II. MATERIALS AND METHODS 58th International Congress of Meat Science and Technology, 12-17th August 2012, Montreal, Canada increased in CAPN1-siRNA2, CAPN1-siRNA3 All chemicals and laboratory wares were and CAPN1-siRNA4 transfected satellite cells purchased from Sigma- Aldrich Chemical Co. (St. (Figure 7). Louis, MO, USA ) and Falcon Lab ware (Becton- In this study, primary satellite cell cultures are Dickinson, Franklin Lakes, Nj, USA), respectively. derived directly from adult Korean Hanwoo Satellite cells were isolated from 30-month-old longissimus dorsi muscle. The in vitro properties Korean Hanwoo cattle according to the method of exhibited by primary cultures of satellite cell more Dodson et al. (6). siRNA-mediated μ-calpain gene closely reflect their in vivo properties than those silencing was carried out according to method of exhibited by transformed cell lines (8). Furthmore, Honda et al (7). psilencer 2.1 hygro vector is used for Primary cell culutres, transformed cell lines this study and primer sequences used is listed in cutures and isolated myofiber cultures have all table 1. been well used to undertake general aspects of Statistical analysis satellite cell physilogy and satellite cell regulation. All the values are expressed as means ± SEM. Even though, there are remains hotly debated on Statistical analysis was performed using SPSS the use of these in vitro systems, both critics and version 16.0 (Statistical Package). Student’s t-test proponents have tended to agree that the use of in was performed to determine the differences vitro systems for determining the developmental between control and treatments. p ≤0.05 was biology of satellite cells has resulted in considered to be significant. considerable, and useful, data (9). Numerous papers have been published involving the animal III. RESULTS AND DISCUSSION as a major experimental unit that were to develop species-specific satellite cell. However, all of these In present study, there are four separate siRNA satellite cell researchers worry about few sequences were screened for their ability to inhibit contaminating cells such as fibroblasts, which co- μ-calpain gene expression. Following optimizing isolated with satellite cells during cell isolation the transfection conditions for cell number, regimens because these contaminating cells volume of transfection agent used and the protentially could overrun cultures and provide appropriate concentration of each siRNA, biased measures of cell activity in vitro (9). In this quantitative real-time PCR were used to assess the study, to decrease the presence of non-myogenic level of inhibition of gene expression achieved by cells in primary cultures, the cell suspensions were all siRNA sequences used. CAPN1, caspase 3, loaded onto a magnetic cell sorting system, caspase 7, caspase 9, Hsp27, Hsp70 and Hsp90 AutoMACS (Milteny Biotec, Germany) to isolate mRNA expression were quantified in CAPN1- the satellite cells which strengthened insure that siRNA treated cells by real-time PCR analysis. culture systems were controlled and interpretable. CAPN1-siRNA3 and CAPN1-siRNA4 mRNA Herein, caspase 9 acts as an initiator caspase and expressions significantly reduced compared to caspase 7 acts as an effector caspase which are control (Figure 1). Caspase 3 expression not possibly involved in satellite cell proliferation. significantly changed in CAPN1-siRNA However, in present study knock-down of caspase transfected satellite cells (Figure 2). Caspase 7 9 expression in satellite cells led to an increase in expression significantly reduced in CAPN1- caspase 3 expressions, probably satellite cells were siRNA2, CAPN1-siRNA3 and CAPN1-siRNA4 stressed from exposure to high concentration (30 transfected satellite cells (Figure 3). Caspase 9 nM) transfection agent/siRNA complex and expression significantly increased in CAPN1- different pathways of apoptosis happened. There is siRNA2, CAPN1-siRNA3 and CAPN1-siRNA4 still a possibility that other initiator/effector transfected satellite cells (Figure 4). Hsp27 caspases and proteinases carried out the role of expression significantly increased in CAPN1- caspase-3 in proliferation muscle satellite cell. siRNA2, CAPN1-siRNA3 and CAPN1-siRNA4 Taken together, herein we hypothesized that transfected satellite cells (Figure 5). Hsp70 apoptosis take place via a mitochondrial pathways expression significantly increased in CAPN1- during cattle muscle satellite cell proliferation. siRNA3 and CAPN1-siRNA4 transfected satellite Apoptosis is a genetically programmed form of cells (Figure 6). Hsp90 expression significantly cell death that can be triggered through death 58th International Congress of Meat Science and Technology, 12-17th August 2012, Montreal, Canada receptors such as the TNF-receptor or via like growth factor II with skeletal muscle mitochondrial pathways (10)). The intrinsic death satellite cells during aging. Mechanisms of signaling pathway is induced by the release of Ageing and Development. 39:121-128. cytochrome c from mitochondria. Cytochrome c, 7. Honda M, Masui F, Kanzawa N, Tsuchiya T apoptosis-activating factor-1 (APAF-1) and and Toyo-oka T (2008). Specific knockdown of m-calpain blocks myogenesis with cDNA caspase 9 then form a complex called the deduced from the corresponding RNAi. Am J apoptosome which results in the activation of Physiol Cell Physiol . 294( 4): C957-C965 caspases 7 as the down-stream effector caspases. 8. Allen RE (1987) Muscle cell culture as a tool Our