pharmacologic agents or growth promotants
The use of pharmacologic agents or growth promotants is widespread in farm animals. In general, these agents are, or mimic, regulators normally present in an animal. Ractopamine, is an example of a beta- adrenergic agonist. In general, beta-adrenergic agonists partition the nutrient supply to increase muscle growth and decrease fat deposition. Use of exogenous agents such as beta-agonists allows measurement of changes occurring during manipulated growth as a model for physiological controls that may alter nutrient partitioning during normal growth. A better understanding of controls important in increasing lean tissue growth will increase our ability to alter the controls to stimulate lean growth.
The mechanisms of importance in regulation of nutrient partitioning during normal or manipulated growth may include a change in tissue sensitivity or responsiveness to a hormone or metabolite. Insulin is a key metabolic hormone in ruminant and nonruminant animals. Several studies suggest involvement of insulin in the mechanism of action of -adrenergic agonists.
Characterization of the Porcine Skeletal Muscle Calpain Gene and its Role in Lean Muscle Growth
From the 1995 Research Report
Christopher A. Bidwell, Ph.D. and Deana L. Hancock, Ph.D.
Purdue University
Abstract
Growth of muscle involves both synthesis of new proteins and degradation of old proteins. Calpains are a family of enzymes that degrade protein and are involved in protein turnover in muscle and therefore, directly affect nitrogen retention and feed efficiency. Skeletal muscle calpain is a member of the calpin family that is only found in skeletal muscle. We have isolated the pig gene for skeletal muscle calpain and measured expression of the gene in order to determine the role of the enzyme in muscle growth.
We have previously shown that skeletal muscle calpain expression was reduced in pigs treated with ractopamine which had leaner carcasses. We looked for naturally occuring differences in skeletal muscle calpain expression in a high lean gain line compared to a low lean gain line. There was a trend of lower skeletal muscle calpain expression in the high lean gain line but the differences were not large enough to be statistically significant. We have isolated the pig skeletal muscle gene and are determining the complete DNA sequence. We are developing a genetic test for use in gene mapping and marker assisted selection.
Introduction
Our research group is interested in understanding basic interactions between genetics and nutrition that lead to accumulation of protein in muscle. In muscle, both synthesis of new proteins and degradation of old proteins occur through out the life of the animal. Protein accumulation and net growth occur when protein synthesis is greater than protein degradation. There as been a great deal of research done on protein synthesis in muscle but very little research on the degradation component of muscle growth. With support from NPPC we are studying a family of enzymes, referred to as calpains, that break down muscle proteins and therefore influence net protein accumulation. There are three types of calpains, type I and type II-calpain which are found in most tissues such as the liver, lung, muscle, bones etc., and one which is found only in skeletal muscle and is referred to as skeletal muscle calpain.
The type I calpain and type II calpains have been studied and are known to be involved in degradation of muscle fibers (Goll et al., 1989) but the role of skeletal muscle-calpain in net protein accumulation has not been determined. However, since it is skeletal muscle specific and is expressed at greater levels than type I and type II calpain (Sorimachi et al., 1989; our unpublished data), skeletal muscle calpain may be more important in protein turnover than the other calpain types. We have developed a DNA based assay to measure the expression of skeletal muscle-calpain and have shown that the repartitioning agent ractopamine, can down regulate the expression of skeletal muscle-calpain in swine (Ji et al., 1992). Therefore, there is evidence that this gene is involved in the metabolic response that results in enhanced nitrogen retention and leaner swine carcasses. The isolation of the skeletal muscle-calpain gene and its regulation by ractopamine provides a novel experimental system to study the role of skeletal muscle-calpain in lean muscle growth. A thorough knowledge of the functional genetic elements of this gene can be utilized in nutrition research, gene mapping, marker assisted selection as well as biotechnological approaches to influencing muscle growth through transgenic swine.
Objectives
1. To determine the complete DNA sequence of the porcine skeletal muscle-calpain gene.
2. To measure mRNA abundance for skeletal muscle-calpain in skeletal muscle mRNA from lines of pigs with different genetic potentials (low and high) for lean growth.
Procedures
A porcine gene library was constructed that contains greater than 99% of all the DNA in the pig genome (Briley and Bidwell, 1994). Three gene fragments that contain the skeletal muscle-calpain gene were isolated from the gene library. The boundaries of the protein coding regions were identified using sequencing primers derived from our pig cDNA as well as the rat and human cDNA's. Five restriction enzyme fragments that contain the gene are being sequenced using a series of progressive deletions from each end of the fragment. DNA sequencing is being carried out by the Biochemistry Department Center for DNA Sequencing by automated fluorescence DNA sequencing.
Thirty-two barrows from two lines with different lean gain potentials were tested with feed intake and live weight recorded weekly in a study conducted by Dr. Alan Grant and Dr. Schinkel of as part of the Purdue-Cooperative Research Swine Lean Growth Consortium. Four animals from each line were slaughtered at 8 different liveweights and skeletal muscle, fat and liver samples were collected. Total RNA was isolated and the level of skeletal muscle calpain mRNA was determined by slot blot hybridization with the skeletal muscle-calpain cDNA probe.
Results
Analysis of the isolated skeletal muscle calpain gene indicate that it is nearly 20,000 base pairs in length which is about twice as large as an average mammalian gene. We have sequenced 11,000 base pairs to date and sequencing continues with a second year of support from NPPC. At this time we have enough sequence data to develop a genetic test for the skeletal muscle calpain gene. We have identified DNA sequences unique to skeletal muscle calpain that can detect the gene in pig DNA and we are cooperating with other researchers involved in pig gene mapping in order to improve the assay to distinguish different skeletal muscle calpain alleles. A portion of the gene was sent to the laboratory of Dr. James Womack at Texas A&M University and Ms. Penny Riggs has determined that the skeletal muscle calpain gene is located on the q arm of pig chromosome 1. Therefore, we have made significant progress in characterizing this gene and developing a genetic test to aid in selection of the most favorable alleles for skeletal muscle calpain.
For the second objective we looked at the expression of the skeletal muscle calpain gene in two different lines of pigs with high and low potential for lean gain (Figure 1). At several time points the high lean gain line (line 7) had reduced levels of skeletal muscle-calpain mRNA that was of the same magnitude of reduced skeletal muscle-calpain expression as seen in ractopamine treated vs. control pigs (Ji et al., 1992). However, due to large variation in individuals as evidenced by the standard error, the differences were not statistically significant.
The results of this project provide further insight into the role of skeletal muscle-calpain on protein accretion and will result in a DNA marker sequence that can be used in marker assisted selection of pigs with enhanced propensity for lean tissue deposition. In a discussion of protein degradation on overall growth rate and efficiency, Goll et al., (1989) calculated that a slight reduction (10%) in protein degradation would more than double average daily gain in cattle and the same would be true for swine. Furthermore, 15-25% of energy intake is used to replace muscle protein, therefore, a reduction in protein degradation would decrease the energy required for protein replacement and would directly improve efficiency (Goll et al., 1989). Since Ractopamine decreases skeletal muscle-calpain expression and results in increase lean tissue deposition, selection for reduced skeletal muscle-calpain expression could result in increased lean growth potential. In our second year of funding from NPPC we will complete the DNA sequence for the skeletal muscle calpain gene and develop a rapid assay for use in gene mapping and marker assisted selection. This project benefits pork producers by laying the foundations for a better understanding of metabolic processes in muscle that either promote or antagonize protein deposition in muscle. Additionally this project has contributed to training of new scientists interested in the interactions of genetics and nutrition to increase the efficiency of pork production.
Bibliography
Briley, G.P. and C.A. Bidwell. 1994. Biotechniques 17, 278-280
Goll, D.E., W.C. Kleese, A. Szpacenko. 1989. In "Animal Growth Regulation". (D.R. Campion G.J. Houseman and R.J. Martin, eds.). Plenum Press NY
Ji S.Q., D.L. Hancock, C.A. Bidwell, and D.B. Anderson. 1992. J. Anim. Sci. 70(1): 208.
Sorimachi, H., S. Imajoh-Ohmi, Y. Emori, H. Kawasaki, S. Ohno, Y. Minami and K. Suzuki. 1989. J. Biol. Chem. 264,20106.
Sorimachi, H., N. Toyama-Sorimachi, T. Saido, H. Kawasaki, H. Sugita, M. Miyasaka, K. Arahata, S. Ishiura, and K. Suzuki. 1993. J. Biol. Chem. 268,10539
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