Research and Reviews: Beef and Sheep

Special Circular 170-99

Relationship of a PCR-SSCP at the Bovine Calpastatin Locus with Calpastatin Activity and Meat Tenderness

H. Y. Chung, M. E. Davis¹, H. C. Hines, and D. M. Wulf
The Ohio State University Department of Animal Sciences

Abstract

This study was designed to investigate the effects of calpastatin activity and myofibril fragmentation index on meat tenderness and the effects of calpastatin genotypes determined using PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) analysis on these variables. Forty-seven purebred Angus bulls were slaughtered at approximately 13 to 15 months of age. Longissimus muscles were prepared to determine myofibril fragmentation index (MFI), Warner-Bratzler Shear (WBS) Force, and calpastatin activity. The PCR primers for the calpastatin (CAST) segments were selected based on exons of the bovine calpastatin cDNA sequences as CAST1 (exon 1C and 1D), CAST5 (exon 5 and 6), and CAST10 (exon 10 and 11). Polymorphisms were detected in all of the calpastatin segments examined. Observed genotypes were AA, AB, and BB for CAST1 and CAST5, and AA, BB, CC, AB, AC, and BC for CAST10 segments. Statistical significance of CAST1, CAST5, and CAST10 genotypes was not detected for calpastatin activity, shear force, or myofibril fragmentation index. A weak positive residual correlation (r = 0.29, P = 0.52) between CAC and WBS was obtained. MFI and WBS were slightly related (r = -0.49, P = 0.26). There was a significant negative correlation between CAC and MFI with r = -0.74 (P = 0.05). PCR-SSCP analysis of the calpastatin locus was not useful for the prediction of calpastatin activity, myofibril fragmentation index, or meat tenderness.

Introduction

Producing meat tenderness that consumers desire is one of the major problems facing the beef industry, because meat tenderization during the postmortem period is highly variable between carcasses. Therefore, studies of biochemical mechanisms for muscle breakdown are essential at the molecular level. It has been reported that the calpain family is mainly responsible for improvements in meat tenderness during postmortem storage (Koohmaraie, 1994) and is also suggested by the results of degradation and weakening of the myofibril proteins near the Z-disks (Kendall et al., 1993; van den Hemel-Grooten et al., 1997). Calpastatin, which is an endogenous inhibitor (EC 3.4.22.17, Ca²⁺ dependent cysteine proteinase), plays a central role in regulation of calpain activity in cells (Murachi et al., 1981; Murachi, 1983; Forsberg et al., 1989) and is considered to be one of the major modulators of the calpains. Therefore, calpastatin may affect proteolysis of myofibrils due to regulation of calpains, which can initiate postmortem degradation of myofibril proteins (Goll et al., 1992; Huff-Lonergan, 1996). This study was carried out to investigate the effects of calpastatin activity and myofibril concentration on meat tenderness, in order to provide information that will increase the accuracy of selection and improve rates of genetic progress for meat tenderness.

Materials and Methods

Sample Preparation

Forty-seven purebred Angus bulls were born in the fall of 1996 at the Eastern Ohio Resource Development Center (EORDC). Bulls were selected for high or low blood-serum insulin-like growth factor I (IGF-I) concentration. Calves were weaned at an average age of approximately 140 days. Following a 140-days postweaning test, bulls were slaughtered at a commercial packing facility in Columbus, Ohio. Longissimus muscles were removed two days postmortem from carcasses of bulls ranging in age from 13 to 15 months. Steaks were cut approximately five cm thick from the longissimus muscle between the 12th and 13th rib. After removing fat, the samples were divided into three parts to measure calpastatin activity (CAC), Warner Bratzler Shear Force (WBS), and myofibril fragmentation index (MFI). The samples for the measurement of shear force were aged at 4°C for seven days, and then transferred to a -70°C freezer. The other samples were stored at -70°C after two days postmortem. For measurement of calpastatin activity, the samples were transferred to the laboratory of Dr. Georganna Whipple at Central Community College in Hastings, Nebraska.

Design of Primers

In order to supply useful information associated with markers for the whole population, identification of regions of the genome that contribute to variation in meat tenderness is necessary. Therefore, primers were selected for coding regions of calpastatin domain L and 1. A primer was designed using the exon and intron sequence that was published for ovine calpastatin exon 1C and 1D (CAST1) of domain L (CAST1 : 5’- CTTGTCATCC GACTTCACCT, and 3’- TCTTCTTTTC TCTTTGGGTG GA) by Roberts et al. (1996). Two primers were designed using sequences based on the bovine calpastatin cDNA (Killefer and Koohmaraie, 1993) exon 5 and 6 (CAST5 : 5’- ATGAGAAAAA AACCCAAGAA GTAA, and 3’- TACCTTTCCT TTTGTTGATT TCTC) of domain L (CAST5), and exon 10 and 11 (CAST10 : 5’- AGAGGAACTG GGTAAAAGAG AATC, and 3’- TCAAGGAGTC TGGAGGAGGC) of domain 1 (CAST10). The primer for CAST10 from domain 1 included repetitive sequences also found in domains 2, 3, and 4.

Measurement of Warner-Bratzler Shear (WBS) Force

Frozen samples were thawed at 4°C for one day, and cut into 2.54-cm-thick pieces. Samples were cooked to a final internal temperature of 71°C for 12 minutes on a cooking grill. After cooking, samples were standardized at room temperature for three hours, and cores were prepared to be 1.27 cm in diameter. A minimum of six and a maximum of 10 cores were obtained from each steak. Shear force was measured using the Warner-Bratzler Shear equipment, which had crosshead speed force set at 200 mm/min, and was reported in kilograms.

Measurement of MFI
(Myofibril Fragmentation Index)

After thawing at 4°C overnight, five grams of muscle were minced. Myofibril isolation was performed using a modification of the method described by Uytterhaegen et al. (1994). Briefly, minced muscle samples were homogenized with 25 mL of MFI buffer (0.25 M sucrose, 0.5M Tris, pH 7.6, 1 mM EDTA, and 1 mM NaN₃). The pellet was collected by centrifugation at 1,000 x G for 10 minutes (4°C). The pellet was resuspended in 10 mL of cold MFI buffer and vortexed until well mixed. After centrifugation, the pellet was resuspended with 25 mL of 0.15 M KCl. To dissolve myofibrils, 30 mL of sample buffer (0.01 M Imidazole, 1% SDS, and 2% 2-MCE, pH 7.0) was added and placed at room temperature overnight. After filtration, protein content was measured by adding 100 uL of 0.1% Coomassie Brilliant Blue R-250 per 5 mL of sample, and placed at 37°C for two hours. Absorbance was read at 590 nm with a spectrophotometer, and MFI was calculated using 200 x absorbency.

Measurement of Calpastatin Activity

Five grams of muscle tissue were extracted in 25 mL of extraction buffer (150 mM Tris-HCl, 5 mM EDTA, pH 8.3, 0.2% beta-MCE). Homogenization was carried out two times at 500 rpm for 30 seconds. The homogenate was centrifuged for 30 min at 12,000 x G. After filtration, the volume was recorded. A portion of the supernatant was heated for 15 minutes at 98°C, then cooled on ice for 15 minutes. Cooled aliquots were centrifuged for one hour at 1,500 x G. The heated samples were then assayed for activity with a volume needed for 50 to 80% inhibition of known m-calpain activity. The sample volume was brought up to 1 mL using elution buffer (20 mM Tris-HCl, pH 7.35, 0.5 mM EDTA, 0.2% MCE). Twenty microliters of partially purified lung m-calpain was added along with 1 mL of assay media (100 mM Tris, pH 7.5 with 1 N acetic acid, 1 mM NaN₃, 0.5% casein and 0.2% MCE), followed by 50 uL of 200 mM CaCl₂. The assay reaction took place for one hour at 25°C. The reaction was stopped by adding 2 mL of 5% TCA. The reaction mixture was centrifuged at 1,500 x G for 30 minutes. The A₂₇₈ was determined on the supernatant, and the activity was calculated.

PCR Procedure

Three microliters of 10 X reaction buffer (10 mM Tris, pH 8.3, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl₂ ), 10 uM dNTP, 10 pmol of each primer, 50 ng genomic DNA, and two units of Taq DNA polymerase (Gibco BRL, Grand Island, N.Y.) in a final volume of 30 uL were used. PCR conditions were 95°C for two minutes for the first cycle and 94°C for one minute for denaturation, 57°C for one minute for annealing, and 72°C for 1.5 minutes for polymerization, with a total of 35 cycles (Perkin Elmer Cetus, Norwalk, Conn.). For the genotyping of all loci, the sample was diluted with 16 uL of distilled water and 8 uL of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% Glycerol). After heating at 95°C for five minutes, amplification products were immediately placed on ice. Polymorphisms were detected by SSCP with 0.5 and 1.0 X mutation detection enhancement (MDE, FMC, Rockland, ME) gels. The mixture was electrophoresed for 20 hours at 250 V and 12°C. DNA fragments were visualized using ethidium bromide for CAST1 and silver staining for CAST5 and CAST10 loci.

Statistical Analysis

Allele frequencies were calculated and compared between lines. Least squares means and standard errors were determined for WBS, CAC, and MFI. Fixed effects in the model included calpastatin genotypes, age of dam, and IGF-I selection line, and age of bull as a covariate. Data were analyzed by general linear models produces and Fisher’s least significant difference test (SAS, 1985), and a comparison error rate of 0.05 was conducted to compare least squares means.

Results and Discussion

Two alleles (A and B) were observed for the CAST1 and CAST5 segments, and three alleles (A, B, and C) were observed for the CAST10 segment. Within the CAST1 and CAST5 segments, a monomorphic fragment was detected in the middle (Figures 1 and 2), but there were no monomorphic bands within the CAST10 segment (Figure 3). Allele frequencies within the two IGF-I selection lines were estimated for the calpastatin segments (Table 1). Allele frequencies within the two lines were similar for all gene segments.

AA AB BB AA BB AB AA BB BB BB BB BB BB		1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Figure 1. PCR-SSCP patterns of 13 bulls for CAST1 segment (exon 1C and 1D) from cDNA coding for a unique domain L. Fast and slow fragments were designated as A and B alleles, respectively. Each lane is labeled with the assigned genotypes.		Figure 2. PCR-SSCP patterns of 18 bulls for CAST5 segment (exon 5 and 6) from cDNA coding for a unique domain L. Fast and slow fragments were designated as A and B alleles, respectively. Lane 1, 7, 10, 13, 14, 15, and 17 : AA, 4, 6, 8, 11, 16, and 18 : AB, and 2, 3, 5, and 9 : BB.
AA BC AC AB BB BC BC AA CC AC BC AA AC CC BC AC BB
Figure 3. PCR-SSCP patterns of 17 bulls for CAST10 segment (exon 10 and 11) from cDNA encoding for repetitive domain 1. A, B, and C alleles were assigned to fragments from fast to slow, and each lane was labeled with the assigned genotypes.

The calpastatin locus has been identified using restriction fragment length polymorphism in bovine (Bishop et al., 1993) and human (Maki et al., 1989; Takashi et al., 1990). The bovine calpastatin gene has been sequenced for skeletal muscle with five different domains identified (Killefer and Koohmaraie, 1993). The polymorphisms we found were detected in the coding region of a unique domain L and repetitive domain I. However, Killefer and Koohmaraie (1993) did not detect polymorphism using a probe generated from the cDNA coding for domains L and 1 of bovine calpastatin. Lonergan et al. (1995) reported BamHI and EcoRI RFLPs using a 2.2 kb cDNA probe coding for calpastatin domains 2, 3, 4, and 3’ UTR.

Least squares means and standard errors were determined for Warner Bratzler Shear force (WBS), calpastatin activity (CAC), and myofibril fragmentation index (MFI) by calpastatin genotypes (Table2). Genotypes based on calpastatin fragments did not explain significant variation in calpastatin activity, myofibril fragmentation index, or shear force. Lonergan et al. (1995) also reported no significant differences for calpastatin activity or WBS using their calpastatin cDNA RFLP. However, Wulf et al. (1996) stated that meat tenderness will differ between breeds and between individuals within breeds because of genetic differences, and Koohmaraie (1992) reported that calpastatin activity is highly heritable. Therefore, genetic components do influence calpastatin activity and meat tenderness. There was no evidence that allelic frequencies differed between IGF-I selection lines (Table 1) and thus lines did not significantly affect the frequency of calpastatin genotypes. Calpastatin activity and WBS tended to be higher for both homozygotes than for the heterozygote at the CAST5 segment. In addition, slightly higher calpastatin activity was observed for genotypes containing the A allele at the CAST10 segment. These results indicate that genetic differences may be useful for future study. Further experiments to develop methods to predict meat tenderness in unrelated animals need to focus either on variations in calpastatin at the protein level or on identifying the source of genetic variation at the molecular level.

Meat tenderness is a complicated trait because there are many sources of variation that affect postmortem meat tenderization, such as non-genetic effects of time on feed, stress, carcass chilling, postmortem aging time, cooking methods, etc., as well as genetic effects. Koohmaraie (1994) suggested that approximately 30% of the variation in tenderness can be explained by additive gene effects within a single breed, and that approximately 70% of the variation is explained by environmental or non-additive gene effects. Consequently, because of the high relationship (Uytterhaegen et al., 1994; Shackelford et al., 1994) between calpastatin activity and meat tenderness, meat tenderness may be controlled by genetic markers.

Residual correlations among traits are shown in Table 3. The correlation between WBS and CAC was 0.29 (P = 0.52). This result was similar to the correlation of 0.31 reported by Lonergan et al. (1995). Several studies have reported a positive correlation between calpastatin activity and WBS because calpastatin inhibits calpain, which can initiate postmortem muscle breakdown (Uytterhaegen et al., 1992; Shackelford et al., 1994).

Furthermore, Koohmaraie (1994) reported that the genetic correlation between postrigor calpastatin activity and Warner Bratzler shear force exceeds 0.5; this finding demonstrated that selection against calpastatin activity could result in improved meat tenderness. Shackelford et al. (1994) observed that decreasing tenderness associated with Bos indicus breeding seems to be highly related to increasing calpastatin activity at 24 hours postmortem. In addition, Wulf et al. (1996) found a correlation of 0.42 between shear force and calpastatin activity in the Limousine and Charolais breeds. The principal reason for observed differences in rates of postmortem tenderization is differences in rate of degradation of myofibril proteins, which is probably mediated by the calpain proteolytic system. Therefore, one would logically expect MFI to be negatively correlated with WBS or CAC. The present study showed a significant negative correlation between CAC and MFI. The correlation between WBS and MFI, though relatively strong, was not significant with this small number of samples.

Conclusion

PCR single-strand conformation polymorphism analysis of the calpastatin gene was not useful for prediction of calpastatin activity, myofibril fragmentation index, or meat tenderness. However, other studies have shown calpastatin activity to be highly related to meat tenderness. It is possible that other variations at the calpastatin locus could be used to predict calpastatin activity and meat tenderness in marker-assisted selection programs. In addition, at the molecular level, studies should focus on gene expression and marker typing in unrelated populations to evaluate candidate genes.

Acknowledgment

The authors wish to thank Dr. Georganna Whipple for performing the calpastatin assay and Judy Riggenbach for her technical assistance.

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¹ For more information, contact at: The Ohio State University, 221 Plumb Hall, 2027 Coffey Road, Columbus, OH 43210; (614) 292-4984, Fax (614) 292-7116; email:davis.28@osu.edu