THE ROLE OF THE CALPAIN/CALPASTATIN SYSTEM IN MUSCLE HYPERTROPHY ASSOCIATED WITH DOUBLE-MUSCLING IN BEEF

T. D. Pringle, R. L. West [1], S. E. Williams, and D. D. Johnson [1]

Summary

Five normal-muscled (NM; Angus) steers and five double-muscled (DM; Piedmontese) bulls were used to study the role of the calpain/calpastatin system in the muscle hypertrophy and meat tenderness associated with double-muscling. DM cattle had significantly heavier carcasses and higher dressing percentages. DM carcasses also had less backfat, larger ribeye areas, lower yield grades (2.8 vs 0.4), and lower marbling scores (Sm48 vs Tr76) than NM carcasses. µ-Calpain activity was higher in DM cattle than in NM cattle, while calpastatin was not affected by breed type. Shear force was also not affected by breed type. These data show the typical differences in carcass characteristics between NM and DM cattle. Furthermore, it does not appear that the muscle hypertrophy associated with double-muscling in beef is related to the calpain system, as has been shown in other hypertrophic models, specifically with ß-adrenergic, agonist-treated cattle.

Introduction

The calpain/calpastatin system is an endogenous, Ca2+-dependent proteinase system, theorized to initiate in-vivo muscle protein degradation. This system, especially the calpastatin component, appears to be related to meat tenderness through the regulation of postmortem proteolysis. An animal model that exogenously elevates calpastatin activity, dietary inclusion of ß-agonists, has been shown to cause muscle hypertrophy and decrease meat tenderness in lambs and cattle.

An alternative model for the study of muscle growth and meat tenderness regulation examined double-muscling in cattle. Double-muscling, a recessive trait in cattle, results in extreme muscularity. These cattle produce heavier muscled, lower fat carcasses that have higher muscle:bone ratios than normal-muscled cattle. Meat from double-muscled cattle has been reported to be more tender than meat from normal-muscled cattle. Based on the hypothesis that the calpain/calpastatin system is related to muscle growth and meat tenderness, this study investigated postnatal muscle growth in double-muscled cattle, focusing on potential regulation by the calpain/calpastatin system.

Experimental Methods

Five normal-muscled (NM) steers and five double-muscled (DM) bulls were pen fed, by muscling type, a high concentrate diet from weaning until slaughter. NM steers were slaughtered at a backfat constant end point of .4 inches, determined by ultrasound. As a NM steer reached the end point, the heaviest DM bull was pair slaughtered. The cattle were slaughtered at the University of Florida abattoir. Within 1 hour of slaughter, samples were removed from the muscles of the loin (longissimus dorsi, LD) and round (biceps femoris, BF) for measurement of calpains and calpastatin activities. The samples were homogenized and shipped overnight on ice to the University of Georgia. To minimize any loss of activity during shipping, proteinase inhibitors were included in the extraction buffer. Postmortem pH and temperature decline were measured in the LD and BF, with measure-ments taken every 3 hours for 24 hours.

Carcass yield and quality grade data were collected by trained personnel at the University of Florida after a 24-hour chill. Following carcass data collection, three steaks (1 inch) were removed from each muscle, vacuum packaged, and aged for 1, 7, or 14 days. The steaks were then cooked to 158°F on electric grills and shear force was measured using a Warner-Bratzler shear device.

Data were analyzed using analysis of variance procedures. The model for carcass data included muscling type (NM vs DM); the model for pH, temperature, and calpains and calpastatin activities included muscling type and muscle (LD vs BF); and the model for shear force included muscling type, muscle, and aging time (1 vs 7 vs 14). Correlations were calculated across muscling types and muscles.

Results and Discussion

The DM cattle were not significantly heavier than the NM cattle at slaughter, but they produced heavier carcasses and higher dressing percentages (Table 1). The increased yield of carcass weight was in part due to the increased amount of muscling in the carcasses. This was evident in measures of both ribeye area and ribeye area per hundredweight of carcass (REACWT). Measures of fatness were significantly lower in the DM carcasses, including external and internal fat depots. As a result of increased muscling and decreased fatness, the DM carcasses had two full lower yield grades than the NM carcasses. As the meat packing industry moves to a value-based system for purchasing cattle, muscling will impact price. Several packers are already utilizing REACWT in their pricing formulas, and thus carcasses from these animals may offer the advantages of more red meat yield and less waste fat.

The NM and DM carcasses were not different in maturity (Table 1). However, the NM carcasses had significantly higher marbling scores and quality grades than did DM carcasses. Quality grades for the NM cattle averaged low choice, while the DM carcasses averaged high standard. This again emphasizes the overall reduction in fat content of carcasses from DM cattle.

Table 1. USDA Carcass Yield and Quality Grade Factors for Normal-Muscled and Double-Muscled Cattle
	Muscling Type
Traits	Normal-Muscled	Double-Muscled	P-valuea
Yield Components
Slaughter weight, lbs.	1044.0	1100.0	NS
Hot carcass weight, lbs.	617.1	723.1	**
Dressing percentage, %	59.1	65.7	**
Fat over the ribeye, in.	.41	.11	**
Ribeye area, in2	11.1	17.1	**
Ribeye area/hundred weight of hot carcass, in2	1.80	2.36	**
Kidney, pelvic and heart fat, %	2.1	1.5	**
USDA yield grade	2.8	0.4	**
Quality Components
Overall maturityb	A32	A36	NS
Marbling score	Sm48	Tr76	**
USDA quality grade	Ch-	St+	**
aNS, Means not different (p>.10); **Means differ, (p<.01). bThe average of lean and bone maturities.

Postmortem temperature and pH decline were monitored to determine how the additional muscle mass might influence these factors related to tenderness (Table 2). There was a general trend for both temperature (p<.10) and pH (p<.05) to be higher in DM carcasses as compared with NM carcasses. Temperature was probably higher because of the additional muscle mass, since the temperature was recorded in the center of the loin (LD) and round (BF) muscles. Given the higher temperature, muscle metabolism should continue at a faster rate in DM carcasses, resulting in a lower muscle pH. The reason for the higher pH in DM carcasses when compared with NM carcasses was not apparent.

In the calpain/calpastatin system, only µ-calpain activity was influenced by muscling type (DM > NM; Table 3). Other work relating the calpain/calpastatin system to muscle growth and meat tenderness has suggested a strong relationship between calpastatin activity, muscle weight and meat tenderness. However, in this study of genetically enhanced muscling, calpastatin activity was not affected. It is also interesting to note the differences in sex class between the DM and NM cattle, with the DM cattle being intact males and the NM cattle being castrates. Previous work comparing the calpain/calpastatin system in intact males and castrates has reported that the intact males had higher calpastatin activity; therefore, differences in calpastatin activity across the muscling types may have been obscured by the sex class differences. The remaining factor related to tenderness, sarcomere length, did not differ across muscling types. This indicates that there was no cold shortening in the DM carcasses, even though there was a very limited amount of external carcass fat.

Table 2. Postmortem Temperature and pH Decline in Normal-Muscled and Double-Muscled Cattlea
Postmortem Time, h	Temperatureb		pHc
	Normal-Muscled	Double-Muscled	Normal-Muscled	Double-Muscled
1	98.4	97.7	6.78	6.92
3	90.9	93.0	6.25	6.38
6	79.5	82.6	5.72	5.85
9	69.8	72.7	5.68	5.70
12	62.2	64.4	5.47	5.54
15	58.5	59.9	5.51	5.50
18	53.2	54.5	5.55	5.52
21	50.2	51.1	5.43	5.51
24	47.5	48.9	5.44	5.48
aData averaged across LD and BF muscles. b°F. c-log[H+].

Table 3. Factors Associated with Meat Tenderness in Normal-Muscled and Double-Muscled Cattle
	Muscling Type
Traits	Normal-Muscled	Double-Muscled	P-valuea
µ-Calpainb	65.1	77.4	**
m-Calpainb	39.5	40.7	NS
Calpastatinb	149.8	154.0	NS
Sarcomere length, µm	1.79	1.80	NS
Shear force, lbs.c
1 day	13.0	12.4	NS
7 days	10.8	10.3	NS
14 days	9.7	10.0	NS
aNS, means not different, p>.10; ** Means differ, p<.01. bCaseinolytic activity (Abs278) in 50 grams of tissue. cData averaged across the LD and BF muscles.

Based on sex class differences, as well as differences in marbling, one would expect the NM meat to be more tender. However, neither meat tenderness nor the response to aging was affected by muscling type (Table 3). There was a significant correlation between µ-calpain activity and tenderness after 1 day of aging (Table 4), but calpastatin activity was not correlated to any measures of muscle growth or meat tenderness in this group of cattle.

Table 4. Correlations of the Calpain/Calpastatin System With Muscle Growth and Meat Tenderness Parameters in Normal-Muscled and Double-Muscled Cattlea
Traits	µ-Calpain	Calpastatin
Dressing percentage	.47	-.30
Ribeye area	.26	-.22
Ribeye area/hundred weight of hot carcass	.28	-.14
Shear force, 1 day	-.58**	.24
Shear force, 7 days	-.26	-.25
Shear force, 14 days	-.13	-.03
**Correlation significant (p<.01). aData averaged across the LD and BF muscles.

Conclusions

Carcasses from DM cattle had a much higher yield of muscle and less fat than their NM counterparts. DM carcasses also had less intramuscular fat and lower quality grades; however, this did not appear to influence meat tenderness. Unlike previous results, no apparent relationship was found between calpastatin activity and muscle growth or meat tenderness in the DM cattle. In fact, the only significant relationship between the calpain/calpastatin system and measures of muscle growth and tenderness was a correlation between µ-calpain activity and early tenderness (WBS, 1 day).

Thus, it appears possible to produce acceptably tender beef without excess fat and that these types of cattle may have merit in breeding systems designed to genetically reduce carcass fat. However, careful selection of complementary breeds must be made to overcome some of the quality defects (particularly low marbling scores) in the carcasses from DM cattle.