Research and Reviews: Beef and Sheep

Special Circular 170-99

Effects of Calpain Proteolysis and Calpain Genotypes on Meat Tenderness of Angus Bulls

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

Abstract

Relationships of the calpain system with meat tenderness and carcass traits were examined in 47 purebred Angus bulls from two lines divergently selected for blood serum IGF-I concentration. Genotyping was performed by PCR-SSCP (single strand conformation polymorphism) and RFLP (restriction fragment length polymorphism) analysis. The primers were selected from the calpain I large subunit (CANP1L4) and calpain IV small subunit (CANP4S). Bulls from 13 to 15 months of age were slaughtered, and carcass traits, including fat thickness (FAT); longissimus muscle area (LMA); percentage of kidney, pelvic, and heart fat (KPH); hot carcass weight (HCW); marbling score (MAR); and quality grade (QUL), were measured. Activities of calpastatin (CAC), u-calpain (UCL), and m-calpain (MCL) were measured, and Warner-Bratzler Shear Force (WBS) and myofibril fragmentation index (MFI) were determined. Differences in CANP4S genotypes (BB > AB > AA) were found for KPH (P < 0.05), and IGF-I selection line influenced MFI and CAC (P < 0.05). CAC was higher in the low line than in the high line, but MFI was higher in the high line. Strong positive residual correlations were detected between CAC and UCL, MCL and MAR, MCL and QUL, and MAR and QUL (P < 0.05). Negative relationships were detected between CAC and FAT with r = -0.56 (P < 0.05), and between UCL and MFI with r = -0.99 (P < 0.05). It may be possible to use calpain genotypes classified by PCR-SSCP and RFLP procedures in marker-assisted selection programs to improve meat tenderness and carcass traits of beef cattle.

Introduction

Meat tenderness is one of the main subjects of interest in the beef industry because of consumer requirements for tenderness. Physiological change in muscle structure during the postmortem period is very complex. Calpain has been shown to initiate postmortem degradation of myofibril proteins (Goll et al., 1992a,b; Huff-Lonergan et al., 1996). Calpain is a non-lysosomal protease and has two forms (u- and m-calpain). Calpain is responsible for breakdown of myofibril protein, which is closely related to meat tenderness. Therefore, study of the calpain systems may help explain changes in the meat tenderness.

Carcass traits are often used to evaluate meat quality. Marbling, which is one of the major traits used in evaluation of meat quality, is highly correlated with meat tenderness. Higher levels of marbling are associated with improved tenderness, juiciness, and flavor, and with reduced variation in tenderness of cooked beef (Wulf et al., 1996). Significant relationships among carcass traits (O’Mara et al., 1996), and between the calpain system and meat tenderness (Shackelford et al., 1994) have been reported. Carcass traits are influenced by genetic variations, and genetic differences in tenderness, marbling and calpastatin activity have been reported among and within breeds of cattle (Shackelford et al., 1994; Wulf et al., 1996). Therefore, this study was designed to investigate effects of activities of the calpain system and carcass traits on meat tenderness, as well as the influence of calpain genotypes on these traits.

Materials and Methods

Animals

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, OH. Longissimus muscles were removed two days postmortem, and the carcasses were split between the 12th and 13th ribs. Traits measured included calpastatin activity (CAC); u-calpain activity (UCL); m-calpain activity (MCL); marbling score (MAR); fat thickness (FAT); quality grade (QUL); hot carcass weight (HCW); longissimus muscle area (LMA); kidney, pelvic, and heart fat percent (KPH); myofibril fragmentation index (MFI); and Warner Bratzler shear force (WBS).

Sample Preparation

Steaks were cut approximately 5-cm thick from the longissimus muscle between the 12th and 13th ribs. After removing all external fat and peripheral connective tissue, the samples were divided into several parts for measurement of CAC, UCL, MCL, WBS, and MFI. The samples for shear-force determination were aged at 4°C for seven days, and then transferred to a -70°C freezer. For measurement of calpain and calpastatin activity, the samples were stored at -70°C two days after slaughter and transferred to the laboratory of Dr. Georganna Whipple at Central Community College in Hastings, Neb., 20 days after slaughter.

Design of Primers

The primer for the calpain I large subunit of domain 4 (CANP1L4) was designed based on the human calpain I cDNA sequence (Emori et al., 1986; Genbank accession number, M13363). The primer sequences were TTC AGG CCA ATC TCC CCG ACG (forward) and GAT GTT GAA CTC CAC CAG GCC CAG (reverse). The primer for the calpain II small subunit (CANP4S) was published by Zhang et al. (1996). The PCR primer sequences were CCC CTC GCA CAC ATT ACT CCA AC (forward) and ATA CGG CCT GCC ACT TTT TGA TG (reverse).

Measurement of Warner-Bratzler Shear (WBS) Force

Frozen samples were thawed at 4°C for one day and then 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 parallel to the muscle fiber orientation. An average shear force was calculated and recorded for each steak and was reported in kilograms. WBS was scored from 1 to 10 (1 = tender and 10 = tough).

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 min (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) were 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 Calpain Activity

Extraction and assay of calpain were performed using the method described by Koohmaraie (1992). Ten grams of lean muscle tissue were extracted in 30 mL of extraction buffer (150 mM Tris-HCl, 5 mM EDTA, pH 8.3, 0.2% MCE), including inhibitors (100 mg/mL ovomucoid, 6 mg/liter leupeptin, and 2 mM PMSF). The homogenate was centrifuged for two hours at 12,000 x G. After filtration, samples were dialyzed against dialysis buffer (40 mM Tris, 5 mM EDTA, pH to 7.35, and .01% MCE) for approximately 24 hours. The supernatant was saved after centrifugation (12,000 x G) for 30 minutes at 4°C and loaded onto a small column (1.5 x 20 cm) packed with DEAE-sephacel (Pharmacia) and equilibrated with elution buffer (40 mM Tris, 0.5 mM EDTA, pH 7.35, and 0.01% MCE). Unbound proteins were rinsed by washing the column with elution buffer until absorbance at 278 nm was less than 0.2. Bound proteins were eluted with an increasing gradient of 400 mL of linear gradient containing elution buffer with 25 to 325 mM of NaCl, and 140 fractions were collected for 3 mL. Then m-calpain and u-calpain were collected in fractions 55-85 and 101-120, respectively. Assay of 1 mL of each fraction was conducted by adding 1 mL of assay media (100 mM Tris, 1 mM NaN3, 7 mg/mL of Casein, pH 7.5, and 2 ul/mL of MCE) and 100 uL of 100 mM CaCl2. Incubation was carried out at 25 °C for 60 minutes. Reaction was stopped by adding 2 mL of 5% TCA (Trichloroacetic acid). The assay mixture was centrifuged for 30 minutes at 2,000 rpm. Absorbance was read at A₂₇₈, compared with pooled fractions, and the activity was calculated.

Measurement of Calpastatin Activity

Assay of calpastatin was performed using the method described by Whipple and Koohmaraie (1992). 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 sec. The homogenate was centrifuged for 30 minutes 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.

Genotyping

Calpain genotyping was conducted using PCR-SSCP (polymerase chain reaction-single-strand conformational polymorphism) methods for the CANP1L4 locus and RFLP (restriction fragment length polymorphism) analysis for the CANP4S locus. PCR was performed with a final volume of 30 uL, including 3 uL 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 of genomic DNA, and two units of Taq DNA polymerase (Gibco BRL, Gelthersburg, Md). After denaturation for two minutes at 95°C, a total of 35 cycles for CANP1L4 were adapted to 94°C/1 min, 57°C/1 min, and 72°C/1 min (MJ Research, Inc., PT-200, Watertown, Mass.). PCR cycles for CANP4S were 94°C/1 min, 57°C/1 min, and 72°C/2 min (Perkin Elmer Cetus, Norwalk, Conn.). For the genotyping of CANP1L4, 8 uL of PCR products were diluted with 16 uL of distilled water and 8 uL of loading buffer (0.25% Bromophenol blue, 0.25% Xylene cyanol FF, 70% Glycerol). After heating at 95°C for five minutes, samples were placed on ice. Polymorphisms for the CANP1L4 locus were detected by SSCP with 8% polyacrylamide and 10% formamide gels. The mixture was electrophoresed for 14 hours at 300 V and 24°C. For the CANP4S locus, RFLP was conducted with Hha I restriction enzyme (GCG/C) at 37 °C for two hours. Restriction fragments were separated on 1.2% agarose gels. DNA fragments for both loci were visualized using ethidium bromide.

Statistical Analysis

Forty-seven bulls were classified by calpain genotypes, and gene frequencies for calpain loci were calculated. Least squares means and standard errors were determined for CAC, UCL, MCL, WBS, MFI, FAT, LMA, KPH, MAR, HCW, and QUL using a model that included fixed effects for age of dam, calpain genotype and line, and age of bull as a covariate. Data were analyzed using general linear models. Least squares means were compared using Fisher’s least significant difference test (SAS, 1985) with a comparison error rate of 0.05. Residual correlations among dependent variables were also estimated using the previous model.

Results and Discussion

Polymorphisms in the bovine calpain I large subunit for the coding region of domain 4 (CANP1L4) and calpain IV small subunit (CANP4S) were detected. Frequencies of the A and B allele at the CANP4S locus were calculated as 0.44 and 0.56, respectively. A higher A allele (0.87) than B allele (0.13) frequency was observed at the CANP1L4 locus. PCR generated an approximately 1,800 bp fragment at the CANP4S locus. Allele frequencies were tested for chi-square goodness of fit for Mendelian inheritance with no significant departures from expected values. After the PCR product was cut by Hha I restriction enzyme, three genotypes were observed on a 1.2% agarose gel (Figure 1): AA (280, 600, and 920 bp), BB (1,800 bp), and AB (280, 600, 920, and 1,800 bp). The frequency of AA (0.44) at the CANP4S locus for Angus was higher than that reported (0.20) in Angus by Zhang et al. (1996). The PCR-SSCP pattern for the CANP1L4 locus is shown in Figure 2. Genetic variants for calpain I domain 4, which is the calcium binding region, have not been reported in the bovine.

M 1 2 3 4 5 6 7		1 2 3 4 5 6 7 8 9 10 11 12 13
Figure 1. Bovine PCR-RFLP pattern for calpain gene (CANP4S) with Hha I (GCG/C) digestion. Fast and slow bands were designated as AA and BB, respectively, and each genotype was assigned. M is a 100 bp size marker. Lanes 1 and 4: AA, lanes 2 and 5: BB, and lanes 3, 6, and 7 : AB.		Figure 2. Bovine PCR-SSCP gel electrophoresis of PCR product amplified for CANP1L4. Fast and slow bands were designated as AA and BB, respectively, and each genotype was assigned. Lanes 2 and 8: AA, lanes 1, 4, 7, 10, 11, 12 and 13: AB, and lanes 3, 5,6, and 9: BB.

The least squares means for CAC, WBS, and MFI classified by calpain genotypes are shown in Table 1. Calpain genotypes did not explain significant variation in CAC, WBS, or MFI. CANP1L4 genotypes explained some variation in WBS (P = 0.17), and CANP4S genotypes explained some variation in MFI (P = 0.15). Lonergan et al. (1995) reported that calpastatin genotypes did not explain significant variation in CAC or meat tenderness. No reports are available to explain the variation in meat tenderness or MFI using calpain genotypes. However, calpain is considered to be a major candidate protease for breakdown of myofibril proteins (Lonergan et al., 1995; Wulf et al., 1996).

Animals with the AA genotype for both loci appeared to be slightly more tender than those with AB and BB genotypes (Table 1). This result may have been due to the small number of observations for the AA homozygous genotype, and it is necessary to increase sample size. At the CANP4S locus, animals with AA and AB genotypes had twice as high as MFI values as the animals with BB genotypes. IGF-I selection line was a significant source of variation for CAC (P < 0.05) and MFI (P < 0.05). The mean calpastatin activity for the low line was higher (4.23) than for the high line (4.10), and the least squares mean of MFI was higher in the high line (83.15) than in the low line (80.34). Different selection criteria in the high and low lines may have caused the phenotypic differences in CAC and MFI. Cottin et al. (1994) reported that calpain is highly correlated with IGF-I during embryo development. This result may indicate a possible genetic relationship between calpain and IGF-I.

There were no significant differences among calpain genotypes for carcass traits other than KPH (Tables 2 and 3). CANP4S genotypes explained significant variation in KPH (BB > AB > AA). Animals with AB and BB genotypes tended to have higher values than animals with AA genotypes at the CANP1L4 locus for most of the carcass traits. This result also may have been due to the small number of observations for the AA genotype. A similar result was observed for KPH, MAR, and QUL at CANP4S locus. However, homozygous genotypes tended to have higher values than heterozygous genotypes for FAT, LMA, and HCW. We suggest that calpain loci may have significant influences on carcass traits when the sample size is expanded.

At the beginning of the postmortem period, u-calpain disappears very quickly and is affected by calpastatin activity and concentration. It might be concluded that the principal reason for the 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. UCL was highly correlated with CAC (r = 0.92, P < 0.05) and MFI (r = -0.99, P < 0.05). This finding supports the hypothesis that calpastatin is one of the main inhibitors of u-calpain, which initiates breakdown of myofibril proteins (Goll et al., 1992a, b; Koohmaraie, 1992; Shackelford et al., 1994; Uytterhaegen et al., 1994; Lonergan et al., 1995; Huff-Lonergan et al., 1996). Goll et al. (1992a) also reported a high correlation between UCL and CAC (r = 0.80).

Because m-calpain is secondarily responsible for postmortem breakdown of muscle proteins (Goll et al., 1992a; Koohmaraie, 1992), the MCL may be lowly correlated with CAC, MFI, and WBS. A strong correlation was not found between UCL and MCL, or between CAC and MCL. MCL was highly correlated with MAR and QUL. These results may indicate that MCL is involved in breakdown of myofibril proteins after breakdown of myofibril tissues by UCL. Due to the large amount of MCL during the postmortem period, MCL activity can be related to MAR and QUL during meat aging. MAR and QUL are also highly related to pH during the postmortem period, and therefore, pH is under the influence of the large amount of MCL because of the rapid disappearance of UCL. A strong positive correlation was observed between QUL and MAR, but none of the carcass traits were significantly correlated with WBS (Table 4). However, WBS had slightly strong relationships with UCL and MCL. This may be a good indication that calpain activity influences meat tenderness.

Morgan et al. (1993) stated that decreased 12th rib fat thickness could result in greater chilling rate, and thus, a decreased rate of decline in calpastatin activity in meat from bulls. Our strong negative correlation between FAT and CAC (r = -0.57, P < 0.05) was consistent with this hypothesis. FAT was also negatively correlated with WBS and MFI, but the correlations were not significant.

Conclusion

Our findings indicate that calpain loci may be a good source of candidate genes to predict KPH. Differences in enzymatic activity of calpain and myofibril fragmentation index between IGF-I selection lines may be explained by different selection criteria used in the two lines. Consequently, results of the present study, and future genotypic data from these animals, based on variation in the calpain loci, will provide critical information for establishing calpain as a candidate gene.

Acknowledgment

The authors wish to thank Dr. Georganna Whipple for performing calpastatin, u-calpain, and m-calpain assays, 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