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Effect of nutrient restriction on calpain and calpastatin content of skeletal muscle from cows and fetuses¹

Department of Animal Science, University of Wyoming, Laramie 82071

	Abstract

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Calpains are crucial for the degradation of myofibrillar proteins in muscle. Calpastatin is a specific inhibitor of calpains. The objective of this study was to elucidate the effect of nutrient restriction on the activity of calpains and calpastatin in the skeletal muscle of both cows and fetuses. Beginning 30 d after conception, 20 cows were fed either a control diet consisting of native grass hay fortified with vitamins and minerals at recommendations for a mature cow to gain 0.72 kg/d or half the vitamins and minerals and millet straw at 68.1% of NE_m requirements. Cows were slaughtered on d 125 of gestation, and the LM was sampled at the 12th rib for calpain and calpastatin measurement. When comparing the muscle samples from nutrient-restricted and control cows, no difference in the activity of calpain I and II was observed; however, there was a significant difference (P < 0.05) in calpastatin activity. Muscle samples from control cows had greater calpastatin content than those of nutrient-restricted cows (P < 0.05); in contrast, the calpastatin content of fetal muscle was greater in fetuses gestated by nutrient-restricted cows than those of control cows (P < 0.05). Further, there were three calpastatin isoforms of 125, 110, and 70 kD detected in fetal muscle, whereas only the110-kD isoform was detected for cow muscle. These results indicate that the activity of the calpain system in skeletal muscle is mainly controlled through the expression of calpastatin. Alternating the calpastatin content in muscle and thereby modulating calpain activity may provide a mechanism for the maintenance of fetal muscle growth during nutrient restriction, whereas skeletal muscle loss in cows is upregulated.

Key Words: Activity • Calpain • Calpastatin • Cow • Fetus • Skeletal Muscle

	Introduction

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Calpains are calcium-dependent cysteine proteases that hydrolyze substrates at selective regions. There are two ubiquitous calpains, termed µ-calpain (calpain I) and m-calpain (calpain II). The prefixes refer to the micromolar and millimolar Ca²⁺ requirement for activation (Goll et al., 2003). Calpastatin is a specific inhibitor of calpains (Goll et al., 2003). Calpains are regulated by Ca²⁺ concentration and by binding to calpastatin. The expression level of calpastatin is regulated by genetics and nutrients (Geesink and Koohmaraie, 1999; Helman et al., 2003). Calpastatin activity was higher (P < 0.05) in the callipyge muscle than that in the normal muscle (Geesink and Koohmaraie, 1999). In dogs, high dietary protein intake increases the content of calpastatin in skeletal muscle (Helman et al., 2003). Currently, the effect of global nutrient restriction on the expression and activity of calpains and calpastatin in muscle is unclear.

Maternal undernutrition due to insufficient food supply influences the physiology and development of both mothers and their fetuses (King, 2003). Repartition of nutrients occurs between mothers and fetuses during maternal nutrient restriction, which may exert long-term effects on offspring in later life (King, 2003; Vonnahme et al., 2003; Kuzawa, 2004). There are currently no reports pertaining to the effects of maternal undernutrition on the development of fetal skeletal muscle. It is quite possible that nutrient restriction influences fetal muscle growth and development. Given that the calpain system regulates the turnover of muscle proteins, the activity of calpains and calpastatin in fetal muscle may be altered as a result of maternal nutrient restriction, which may have long-term effects on the meat quality of offspring. Thus, the objective of the current study was to evaluate calpain and calpastatin levels in skeletal muscle of both cows and fetuses following global nutrient restriction.

	Materials and Methods

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Animals
All animal procedures were approved by the University of Wyoming Animal Care and Use Committee. From d 31 to 125 of gestation, 20 Angus x Gelbvieh rotationally crossed cows (initial BW = 576.8 ± 7.7 kg; BCS = 5.4 ± 0.1) were blocked by BW and fed one of two dietary treatments. Half the cows were allotted to a control diet consisting of native grass hay (12.1% CP, 70.7% in vitro OM disappearance [IVOMD]) fortified with vitamins and minerals fed at NRC (1996) recommendations for a nonlactating, mature cow to gain 0.72 kg/d during the first 120 d of gestation. The other half of the cows were allotted to a nutrient-restricted diet, which consisted of feeding half of the control diet’s vitamins and minerals and millet straw (9.9% CP, 54.5% IVOMD) to provide 68.1% NE_m and 86.7% of metabolizable protein requirements during the first 120 d of gestation (NRC, 1996). The diet formulation resulted in a global nutrient restriction for nutrient-restricted cows, including deficiency in protein, energy, minerals, and vitamins. Cows were housed individually. Similar to our previous report with ewes (Vonnahme et al., 2003), control cows increased in BW by 4.2%, whereas nutrient-restricted cows lost 7.1% of initial BW during the feeding period. Ten pregnant cows from each dietary treatment group were slaughtered at the University of Wyoming Meat Laboratory on 125 d of gestation. Immediately following slaughter, a sample was removed from LM taken from the right side of the cows and fetuses at the 12th rib. Muscle samples were snap-frozen in liquid nitrogen and then stored at –80°C until analysis.

Calpain Activity Assay by Casein Zymography
Frozen muscle sample (0.1 g) was homogenized in 500 µL of extraction buffer containing 20 mM Tris-HCl (pH 7.4 at 4°C), 1% Triton X-100, 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/mL of leupeptin, and 10 of µL/mL pepstatin (Raser et al., 1995; Veiseth et al., 2001). The muscle homogenate was centrifuged at 12,000 x g for 5 min, and the supernatant subjected to casein zymography.

A Hoefer mini-gel system was used to cast gels and to run electrophoresis. For gel preparation, casein sodium salt (0.2% wt/vol) was copolymerized with 10% (wt/vol) acrylamide/bisacrylamide (29:1), 375 mM Tris-HCl (pH 8.8) as the separating gel, and 4% (wt/vol) acrylamide/bisacrylamide (29:1), 330 mM Tris-HCl (pH 6.8) without casein as the stacking gel. Ammonium persulfate (0.04%, wt/vol) and N, N, N', N'-tetramethylethylenediamine (TEMED; 0.028% vol/vol) were used to catalyze the polymerization. The supernatant of each muscle homogenate was mixed with equal amount of sample loading buffer containing 150 mM Tris-HCl (pH 6.8), 20% glycerol (vol/vol), 2 mM 2-mercaptoethanol, 0.004% (wt/vol) bromophenol blue. To determine the optimal quantity of protein for loading, a series of amounts of muscle homogenate was loaded to gel, and it was found that a loading amount between 20 and 200 µg of total protein was ideal for casein zymography. For this assay, 100 µg of total protein was used. The casein gel was prerun with a buffer containing 25 mM Tris-HCl (pH 8.3), 192 mM glycine, 1 mM EGTA, and 0.05% (vol/vol) 2-mercaptoethanol for 15 min at 4°C. After loading samples, the casein gel was run at 150 V until the dye front neared the end of gel. The gel was then removed and incubated in 20 mM Tris-HCl (pH 7.4), 0.05% 2-mercaptoethanol, and 4 mM CaCl₂ with slow shaking for 1 h, followed by a buffer change and incubating at room temperature for 16 h with slow shaking. Then, the gel was stained with Coomassie blue (R-250) for 1 h and then destained with 20% methanol and 7% acetic acid for 2 h (Raser et al., 1995; Veiseth et al., 2001). Calpain I and II appeared as two separated clear bands against a dark blue background. Bands were taken with an Imager Scanner II (Amersham Biosciences, Piscataway, NJ) and the density of bands was quantified by using ImageQuant TL software (Amersham Biosciences).

To decrease variation, an equal number of control and nutrient-restricted samples were loaded to each gel. The gel-to-gel variation was further decreased by quantifying bands against a calpain II standard (prepared from rabbit skeletal muscle, Sigma Chemical Co., St. Louis, MO). Briefly, 0.02 U of calpain II standard was added into a separated well of each gel. The density of the band corresponding to calpain II standard was measured and used to normalize the densities of other bands of different gels.

Calpastatin Content Assay by Immunoblotting
Muscle homogenates were prepared as described above. Each muscle homogenate was mixed with a same amount of 2x standard SDS sample loading buffer. For SDS-PAGE preparation, a separation gel containing 10% (wt/vol) acrylamide/bisacrylamide (29:1), 375 mM Tris-HCl (pH 8.8), 0.1% SDS, 0.04% ammonium persulfate, and 0.028% TEMED, and a stacking gel containing 4% (wt/vol) acrylamide/bisacrylamide (29:1), 330 mM Tris-HCl (pH 6.8), 0.04% ammonium persulfate, and 0.028% TEMED were prepared. Following electrophoresis, the proteins on the gel were transferred to nitrocellulose membrane in a transfer buffer containing 20 mM Tris-base, 192 mM glycine, 0.1% SDS, and 20% methanol.

Membranes were incubated in a blocking solution consisting of 5% nonfat dry milk in TBS/T (0.1% Tween-20, 50 mM Tris-HCl [pH 7.6], and 150 mM NaCl) for 1 h. Then, membranes were incubated overnight in primary antibody (mouse-anti-calpastatin, Affinity Bioreagents, Golden, CO) with 1:5,000 dilution in TBS/T with 1% nonfat dry milk. At the end of the primary antibody incubation, the membranes were washed three times for 5 min each with 20 mL of TBS/T. After that, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody raised in monkeys against mouse IgG (1:2,000) for 1 h in TBS/T with gentle agitation. After three 10-min washes, membranes were visualized using ECL Western blotting reagents (Amersham Bioscience) and exposure to film (MR, Kodak, Rochester, NY). The density of bands was quantified by using an Imager Scanner II and ImageQuant TL software (Helman et al., 2003; Du et al., 2004).

Measurement of Carcass Weight, Cross-Section Area of Longissimus Muscle, and Fetal Carcass Weight
Carcass weight for cows was obtained after removing head, skin, and internal organs; fetal carcass weight was obtained after removing internal organs (n = 10).

Statistical Analyses
Data were analyzed as a completely randomized design using the GLM procedure of the SAS (SAS Inst., Inc., Cary, NC). The relative density of bands, carcass weight, and LM area were analyzed, and individual animal was used as the source of variation. The differences in the mean values were compared by Tukey’s multiple comparison, and mean values and standard deviation were reported (P < 0.05).

	Results

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The calpain system controls the turnover of myofibrillar proteins, the largest protein reservoir in the body. During nutrient restriction, insufficient protein supply may impair protein synthesis and accelerate the turnover of myofibrillar proteins. This may be achieved by an alternation in the activity of the calpain system. Thus, the activities of calpain I and II and calpastatin were analyzed. For the calpain activity measurement, casein zymography was used. One problem associated with the zymography is that it is difficult to calculate the actual enzyme activity, and thus only relative activities were reported.

When comparing the muscle samples from control and nutrient-restricted animals, no difference in the calpain I or II activity was detected (Figures 1 and 2). Because calpastatin is a specific inhibitor of calpains and its presence in muscle directly controls the calpain activity, the content of calpastatin was further analyzed. There was significant difference in the content of calpastatin between muscle samples from control cows and nutrient-restricted cows (Figure 3). As shown in Figure 3A, the calpastatin content was higher (P < 0.05) in the muscles from control cows than in the muscles of nutrient-restricted cows. In contrast, the calpastatin content was higher (P < 0.05) in fetuses from nutrient-restricted cows (Figure 3B) than in fetus from control cows. Further, in cow muscle, only one immunoreactive band of calpastatin was detected, whereas in fetuses, three bands of calpastatin were detected (Figure 3). These three bands were present in all fetal samples. Because the fetal muscle was snap-frozen immediately after sampling and kept at –80°C until analysis, and because several protease inhibitors were used during sample homogenization, it is unlikely that these bands were due to hydrolysis of calpastatin, but rather they were isoforms of calpastatin existing in vivo. Using the same preparation method, only one band was detected in the cow muscle sample (Figure 3A), which further supports the existence of more than one isoform of calpastatin existing in fetal muscle. When the relative density of individual bands was compared (Table 1), the 125-kD band was the same for both dietary treatments; however, the expression of the 110- and 70-kD bands was greater (P < 0.01 and P < 0.05, respectively) in fetuses from nutrient-restricted cows.

View larger version (13K): [in this window] [in a new window]   Figure 1. Calpain I activity in skeletal muscle of cows (A) and fetuses (B). Casein zymography was used to determine the calpain I activity in the skeletal muscle of 10 cows and 10 fetuses from control and nutrient-restricted diets, respectively. Each sample was measured twice in two different gels, and the averaged data were used for statistical analysis. The density of bands in different gels was normalized by referring to a calpain II standard (n = 10).

View larger version (17K): [in this window] [in a new window]   Figure 2. Calpain II activity in skeletal muscle of cows (A) and fetuses (B). Casein zymography was used to determine the calpain II activity in the skeletal muscle of 10 cows and 10 fetuses from control and nutrient-restricted diets respectively. Each sample was measured twice in two different gels, and the averaged data were used for statistical analysis. The density of bands in different gels was normalized by referring to a calpain II standard (n = 10).

View larger version (23K): [in this window] [in a new window]   Figure 3. Calpastatin content in the skeletal muscle of cows (A) and fetuses (B). Western blot was used to measure the calpastatin content in the skeletal muscle of 10 cows and 10 fetuses from control and nutrient-restricted diets respectively. The density of the calpastatin immunoreactive band at 110 kb was measured for cattle muscle, whereas for fetal muscle, three calpastatin immunoreactive bands were measured and summed. Asterisks indicate that treatment means differ, P < 0.05 (n = 10).

	Discussion

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The results of the current study show that calpastatin is altered under conditions where the calpains are not changed. Previous studies have also found that protein depletion followed by feeding a normal diet does not change calpain I and II activities in mouse kidneys (Goicoechea and Conde, 1997). And ß-agonist administration did not alter calpain I and II activities, even though it did induce muscle hypertrophy (Speck et al., 1993). In both these instances, however, calpastatin activity was increased following refeeding or ß-agonist administration (Speck et al., 1993; Goicoechea and Conde, 1997). The content and/or phosphorylation of calpastatin likely has an important role in regulating calpain activity in cells (Averna et al., 2003).

Because the calpastatin content of nutrient-restricted cows was significantly lower, the lower calpastatin content should result in a higher calpain activity in the muscle of nutrient-restricted cows, which might contribute to the loss in muscle mass in nutrient-restricted cows. The LM area at the 12th rib for the control cow was 72 ± 9 cm² vs. 60 ± 7 cm² for the nutrient-restricted cow (Table 2). This decreased muscle area was due to increased myofibrillar protein turnover, as well as to decreased protein synthesis following nutrient restriction. In mice, calpain activity increased 2.9 times during depletion and decreased on refeeding, and the change in calpain activity was due to the change in calpastatin content (Goicoechea and Conde, 1997). On the other hand, providing dogs with a high-quality protein source in diet upregulated the expression level of calpastatin in muscle (Helman et al., 2003).

In contrast to the data from cow muscle, the calpastatin content was higher in the muscle of fetuses gestated by cows on the nutrient-restricted diet. The increased calpastatin content in fetal muscle in the nutrient-restricted group might be a protective mechanism for maintaining fetal muscle during nutrient deprivation. Indeed, there was no difference in fetal weight detected between nutrient-restricted and control cows (Table 2). The reason for the increased calpastatin content in fetal muscle is unclear, but it might be related to an increased catecholamine level in the fetuses of nutrient-restricted cows.

It has been reported that catecholamine levels were increased in the rat fetuses of nutrient-restricted dams (Petry et al., 2000). For adult rats, however, catecholamines are only elevated at the initial stage of dietary restriction (Hilderman et al., 1996). The ß-adrenergic agonist, cimaterol, increased calpastatin activity, which induced muscle hypertrophy (Speck et al., 1993; Liu et al., 1994; Parr et al., 2000). For pigs continuously infused with epinephrine, the basal levels of extractable calpastatin activity were increased in skeletal muscle (Parr et al., 2000). These changes in the expression of calpastatin might be partially due to the structure of the promoter region of calpastatin gene. A cAMP-responsive cis element was identified in the promoter region of calpastatin gene, which explains the responsiveness of calpastatin expression to the cAMP level in cells (Cong et al., 1998a,b). Because cAMP is a universal starvation signal, and numerous cell-signaling pathways can induce the production of cAMP in muscle cells, the expression of calpastatin may change due to the changes in cell signaling (Cong et al., 1998a; Averna et al., 2003). Furthermore, several reports show that calpastatin is also regulated at the level of translation (Barnoy et al., 2000; Parr et al., 2000).

Mammaliam calpastatins cDNA can be classified into four types that differ according to the N-terminal sequences. Type II, which encodes a high-molecular-weight isoform, is mainly expressed in mature skeletal and cardiac muscle in rats, cattle, and humans (Takano et al., 2000). In this study, only one band at 110 kD was detected in cow muscle, which is in agreement with former reports (Takano et al., 2000). In pigs, a 135-/145-kD doublet was detected in the skeletal muscle (Parr et al., 2000). For canine skeletal muscle, however, three calpastatin isoforms were detected with a molecular weight of approximately 125, 110, and 70 kD (Helman et al., 2003). There is nothing in the literature regarding the expression of calpastatin in the skeletal muscle of early gestation fetuses. In the current study, we found that three isoforms of calpastatin expressed simultaneously in fetal tissue. The biological functions of individual calpastatin isoforms in fetal muscle remain to be determined. Further, the ratios of individual isoforms were different between muscle from nutrient-restricted and control fetuses. The 110- and 70-kD bands were higher (P < 0.05) in muscle samples from nutrient-restricted cows, but not for the 125-kD band. In agreement with this observation, a former study reported that dietary proteins influenced the relative ratio of calpastatin isoforms in canine muscle (Helman et al., 2003). In pigs, infusion of ß-adrenoceptor agonist increased the skeletal calpastatin 135-kDa band intensity (P < 0.01; Parr et al., 2001).

	Implications

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These data suggest that the cows experiencing restricted nutrient intake during early gestation elicited a change in calpastatin, but not calpains. The calpastatin was down-regulated in the skeletal muscle of nutrient-restricted cows but up-regulated in the fetal muscle due to nutrient restriction. This differential regulation of calpastatin in cow muscle and fetal muscle during nutrient restriction implies that the calpain system might play an important role in the control of muscle growth and degradation. It is unclear what mechanisms are associated with this differential calpastatin expression, but it may be a protective mechanism to ensure the survival of fetuses during nutrient restriction. These data show the complexity in the expression of calpastatin in muscle and further studies will be needed to elucidate mechanisms associated with these observations.

	Footnotes

 
¹ This work was supported by National Research Initiative Competitive Grant 2003-35206-12814 from the USDA Cooperative State Research, Education, and Extension Service.

² Correspondence—phone: 307-766-3429; fax: 307-766-2355; e-mail: mindu{at}uwyo.edu.

Received for publication March 25, 2004. Accepted for publication May 27, 2004.

	Literature Cited

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Averna, M., R. De Tullio, P. Capini, F. Salamino, S. Pontremoli, and E. Melloni. 2003. Changes in calpastatin localization and expression during calpain activation: A new mechanism for the regulation of intracellular ca(2+)-dependent proteolysis. Cell. Mol. Life Sci. 60:2669–2678.[Medline]

Barnoy, S., L. Supino-Rosin, and N. S. Kosower. 2000. Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability. Biochem. J. 351:413–420.

Carragher, N. O., M. A. Westhoff, D. Riley, D. A. Potter, P. Dutt, J. S. Elce, P. A. Greer, and M. C. Frame. 2002. V-src-induced modulation of the calpain-calpastatin proteolytic system regulates transformation. Mol. Cell. Biol. 22:257–269.[Abstract/Free Full Text]

Cong, M., D. E. Goll, and P. B. Antin. 1998a. cAMP responsiveness of the bovine calpastatin gene promoter. Biochim. Biophys. Acta 1443:186–192.[Medline]

Cong, M., V. F. Thompson, D. E. Goll, and P. B. Antin. 1998b. The bovine calpastatin gene promoter and a new n-terminal region of the protein are targets for cAMP-dependent protein kinase activity. J. Biol. Chem. 273:660–666.[Abstract/Free Full Text]

Du, M., L. Hao, A. Lopez-Campistrous, and C. Fernandez-Patron. 2004. Agonist-induced activation of matrix metalloproteinase-7 promotes vasoconstriction through the epidermal growth factor-receptor pathway. Circ. Res. 94:68–76.[Abstract/Free Full Text]

Fritz, J. D., and M. L. Greaser. 1991. Changes in titin and nebulin in postmortem bovine muscle revealed by gel-electrophoresis, western blotting and immunofluorescence microscopy. J. Food Sci. 56:607–610, 615.

Geesink, G. H., and M. Koohmaraie. 1999. Postmortem proteolysis and calpain/calpastatin activity in callipyge and normal lamb biceps femoris during extended postmortem storage. J. Anim. Sci. 77:1490–1501.[Abstract/Free Full Text]

Goicoechea, S. M., and R. D. Conde. 1997. Influence of protein nutrition on calpain activity of mouse kidney: Modulation by calpastatin. Mol. Cell. Biochem. 166:95–99.[Medline]

Goll, D. E., V. F. Thompson, H. Li, W. Wei, and J. Cong. 2003. The calpain system. Physiol. Rev. 83:731–801.[Abstract/Free Full Text]

Goll, D. E., V. F. Thompson, R. G. Taylor, and J. A. Christiansen. 1992. Role of the calpain system in muscle growth. Biochimie 74:225–237.[Medline]

Helman, E. E., E. Huff-Lonergan, G. M. Davenport, and S. M. Lonergan. 2003. Effect of dietary protein on calpastatin in canine skeletal muscle. J. Anim. Sci. 81:2199–2205.[Abstract/Free Full Text]

Hilderman, T., K. McKnight, K. S. Dhalla, H. Rupp, and N. S. Dhalla. 1996. Effects of long-term dietary restriction on cardiovascular function and plasma catecholamines in the rat. Cardiovasc. Drugs Ther. 10:247–250.

King, J. C. 2003. The risk of maternal nutritional depletion and poor outcomes increases in early or closely spaced pregnancies. J. Nutr. 133:1732S–1736S.[Abstract/Free Full Text]

Koohmaraie, M., M. P. Kent, S. D. Shackelford, E. Veiseth, and T. L. Wheeler. 2002. Meat tenderness and muscle growth: Is there any relationship? Meat Sci. 62:345–352.

Kuzawa, C. W. 2004. Modeling fetal adaptation to nutrient restriction: Testing the fetal origins hypothesis with a supply-demand model. J. Nutr. 134:194–200.[Abstract/Free Full Text]

Liu, C., J. Y. Yeh, B. R. Ou, and N. E. Forsberg. 1994. Actions of a beta-adrenergic agonist on muscle protein metabolism in intact, adrenalectomized, and dexamethasone-supplemented adrenalectomized rats. J. Nutr. Biochem. 5:43–49.

Locker, R. H., and D. J. C. Wild. 1984. The fate of the large proteins of the myofibril during tenderizing treatments. Meat Sci. 11:89–108.

NRC. 1996. Nutrient Requirements of Beef Cattle. 7th ed. Natl. Acad. Press, Washington, DC.

Parr, T., P. L. Sensky, M. K. Arnold, R. G. Bardsley, and P. J. Buttery. 2000. Effects of epinephrine infusion on expression of calpastatin in porcine cardiac and skeletal muscle. Arch. Biochem. Biophys. 374:299–305.[Medline]

Parr, T., P. L. Sensky, R. G. Bardsley, and P. J. Buttery. 2001. Calpastatin expression in porcine cardiac and skeletal muscle and partial gene structure. Arch. Biochem. Biophys. 395:1–13.[Medline]

Petry, C. J., M. W. Dorling, C. L. Wang, D. B. Pawlak, and S. E. Ozanne. 2000. Catecholamine levels and receptor expression in low protein rat offspring. Diabet. Med. 17:848–853.[Medline]

Raser, K. J., A. Posner, and K. K. Wang. 1995. Casein zymography: A method to study mu-calpain, m-calpain, and their inhibitory agents. Arch. Biochem. Biophys. 319:211–216.[Medline]

Speck, P. A., K. M. Collingwood, R. G. Bardsley, G. A. Tucker, R. S. Gilmour, and P. J. Buttery. 1993. Transient changes in growth and in calpain and calpastatin expression in ovine skeletal muscle after short-term dietary inclusion of cimaterol. Biochimie 75:917–923.[Medline]

Takano, J., M. Watanabe, K. Hitomi, and M. Maki. 2000. Four types of calpastatin isoforms with distinct amino-terminal sequences are specified by alternative first exons and differentially expressed in mouse tissues. J. Biochem. (Tokyo) 128:83–92.[Abstract/Free Full Text]

Veiseth, E., S. D. Shackelford, T. L. Wheeler, and M. Koohmaraie. 2001. Effect of postmortem storage on mu-calpain and m-calpain in ovine skeletal muscle. J. Anim. Sci. 79:1502–1508.[Abstract/Free Full Text]

Vonnahme, K. A., B. W. Hess, T. R. Hansen, R. J. McCormick, D. C. Rule, G. E. Moss, W. J. Murdoch, M. J. Nijland, D. C. Skinner, P. W. Nathanielsz, and S. P. Ford. 2003. Maternal undernutrition from early- to mid-gestation leads to growth retardation, cardiac ventricular hypertrophy, and increased liver weight in the fetal sheep. Biol. Reprod. 69:133–140.[Abstract/Free Full Text]

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