The calpain system originally comprised three molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both mu- and m-calpain are heterodimers containing an identical 28-kDa subunit and an 80-kDa subunit that shares 55-65% sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six "domains" in the 80-kDa subunit: 1). a 19-amino acid NH2-terminal sequence; 2). and 3). two domains that constitute the active site, IIa and IIb; 4). domain III; 5). an 18-amino acid extended sequence linking domain III to domain IV; and 6). domain IV, which resembles the penta EF-hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+ dependent. Since 1989, cDNA cloning has identified 12 additional mRNAs in mammals that encode polypeptides homologous to domains IIa and IIb of the 80-kDa subunit of mu- and m-calpain, and calpain-like mRNAs have been identified in other organisms. The molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma.

The Calpain System

This review suggests that the intracellular functions of calcium are best understood in terms of calcium's functioning as a second messenger. Further, when functioning as a second messenger, calcium completes its mission not by transferring charge nor by binding to lipid but by binding to specific targets, calcium-modulated proteins. This concept is broadly interpreted to include proteins involved in calcium transport. There is strong evidence that many, if not all, of these calcium-modulated proteins are homologs. Their structures and properties are contrasted to those of extracellular calcium-binding proteins which are not homologous to one another or to the intracellular calcium-modulated proteins. Finally, this line of thought leads to a suggestion of the evolutionary reason for the choice of calcium as the sole inorganic second messenger.

Structure and evolution of calcium-modulated proteins.

Mammalian skeletal muscle shows an enormous variability in its functional features such as rate of force production, resistance to fatigue, and energy metabolism, with a wide spectrum from slow aer...

Calcium Ion in Skeletal Muscle: Its Crucial Role for Muscle Function, Plasticity, and Disease

How a cell responds to stress is a central problem in cardiovascular biology. Diverse physiological stresses (eg, heat, hemodynamics, mutant proteins, and oxidative injury) produce multiple changes in a cell that ultimately affect protein structures and function. Cells from different phyla initiate a cascade of events that engage essential proteins, the molecular chaperones, in decisions to repair or degrade damaged proteins as a defense strategy to ensure survival. Accumulative evidence indicates that molecular chaperones such as the heat shock family of stress proteins (HSPs) actively participate in an array of cellular processes, including cytoprotection. The versatility of the ubiquitous HSP family is further enhanced by stress-inducible regulatory networks, both at the transcriptional and posttranscriptional levels. In the present review, we discuss the regulation and function of HSP chaperones and their clinical significance in conditions such as cardiac hypertrophy, vascular wall injury, cardiac surgery, ischemic preconditioning, aging, and, conceivably, mutations in genes encoding contractile proteins and ion channels.

Stress (Heat Shock) Proteins: Molecular Chaperones in Cardiovascular Biology and Disease

Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, has been shown to be a negative regulator of myogenesis. Here we show that myostatin functions by controlling the proliferation of muscle precursor cells. When C2C12 myoblasts were incubated with myostatin, proliferation of myoblasts decreased with increasing levels of myostatin. Fluorescence-activated cell sorting analysis revealed that myostatin prevented the progression of myoblasts from the G1- to S-phase of the cell cycle. Western analysis indicated that myostatin specifically up-regulated p21Waf1, Cip1, a cyclin-dependent kinase inhibitor, and decreased the levels and activity of Cdk2 protein in myoblasts. Furthermore, we also observed that in myoblasts treated with myostatin protein, Rb was predominately present in the hypophosphorylated form. These results suggests that, in response to myostatin signaling, there is an increase in p21 expression and a decrease in Cdk2 protein and activity thus resulting in an accumulation of hypophosphorylated Rb protein. This, in turn, leads to the arrest of myoblasts in G1-phase of cell cycle. Thus, we propose that the generalized muscular hyperplasia phenotype observed in animals that lack functional myostatin could be as a result of deregulated myoblast proliferation.

Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation.

Ca2+-activated Z-disk-removing activity in the P0-40 crude muscle extracts described by Busch et al. (Busch, W. A., Stromer, M. H., Goll, D. E., and Suzuki, A. (1972), J. Cell Biol. 52, 367) was purified from porcine skeletal muscle extracts by using five column chromatographic procedures in succession: (1) 6% agarose; (2) DEAE-cellulose; (3) Sephadex G-200; (4) DEAE-cellulose with a very shallow gradient; (5) Sephadex G-150. All Z-disk-removing activity eluted in a single peak off each column. Z-disk-removing activity always coeluted with Ca2+-activated proteolytic activity, so Z-disk-removing activity in the P0-40 crude muscle extract is due to a single Ca2+-activated protease (CAF). The five column chromatographic procedures produced a 140-fold increase in specific activity of the Ca2+-activated proteolytic enzymic activity; because preparation of the P0-40 crude CAF fraction before chromatography produced a 127-fold increase in specific activity, the entire procedure described here produces a 17 800-fold increase in specific activity of CAF. This increase in specific activity suggests that muscle contains 3.4 mug of CAF per g of muscle fresh weight; this content is in reasonably good agreement with our yields of 0.25-0.76 mug of purified CAF per g of muscle. Purified CAF migrated as a single band during polyacrylamide gel electrophoresis in pH 7.5 Tris-HC1 buffer but migrated as two bands with molecular weights of 80 000 and 30 000 during polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Densitometric scans of sodium dodecyl sulfate-polyacrylamide gels show that the 80 000- and 30 000-dalton subunits make up 85 to 90% of the protein in purified CAF preparations and that these subunits are present in equimolar ratios.

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Purification from porcine muscle.

The purified Ca2+-activated protease (CAF) isolated from porcine skeletal muscle and capable of removing Z-disks from intact myofibrils is optimally active on either myofibril or casein substrates at pH 7.5 and in the presence of 1 mM Ca2+ and at least 2 mM 2-mercaptoethanol. No CAF activity is detected when 1 mM Mg2+, Mn2+, Ba2+, Co2+, Ni2+, and Fe2+ are added singly. When added with 1 mM Ca2+, Co2+, Cu2+, Ni2+, and Fe2+ inhibit, whereas Mg2+, Mn2+, and Ba2+ have no effect on CAF activity. CAF is irreversibly inhibited by iodoacetate but is unaffected by soybean trypsin inhibitor. S0/20,W=5.90 S, and sedimentation equilibrium molecular weight - 112 000 for purified CAF. Because purified CAF migrates as two polypeptide chains with molecular weights of 80 000 and 30 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CAF molecule must consist of one each of these two polypeptide chains. Approximate molecular dimensions of 38 X 220 A can be calculated for CAF from calibrated gel permeation column data or from S0/20,W and the molecular weight. Amino acid composition and physical properties of purified CAF distinguish it from the known catheptic enzymes and from other proteases found in blood or in granulocytes. Purified CAF removes Z-disks the 400-A periodicity associated with troponin in the I band and partly degrades M lines but causes no other ultrastructurally detectable effects when incubated with myofibrils. These results agree with the earlier finding that purified CAF degrades troponin, tropomyosin, and C-protein but has no effect on myosin, actin, or alpha-actinin, and suggest that CAF may have a physiological role in disassembly of intact myofibrils during metabolic turnover of myofibrillar proteins.

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme.

A calcium-activated protease possibly involved in myofibrillar protein turnover. Isolation of a low-calcium-requiring form of the protease.

Objectives of this study were to determine the influence of trenbolone acetate (TBA) and estradiol (E2) in a combined implant on feedlot performance, carcass characteristics, and carcass composition in finishing steers. Sixty-four large-framed (394.1 kg) crossbred steers were randomly assigned to one of four pens. Subsequently, pens were randomly assigned to one of two treatments, implanted (120 mg of TBA and 24 mg of E2) and nonimplanted. Eight steers/treatment were slaughtered for initial carcass composition. Remaining steers were assigned to one of three serial slaughter dates (d 40, 115, or 143). Implantation increased circulating trenbolone (TBOH) and E2 concentrations throughout the trial. Implantation increased ADG 18% (P .05) on dry matter intake. Feed efficiency was improved 13% during d 0 to 40 (P < .01) and from d 41 to 115 (P = .07). Longissimus muscle area was larger (P < .05) in implanted steers than in nonimplanted steers on d 115. Carcasses from implanted steers had a smaller (P < .05) percentage of kidney, pelvic, and heart (KPH) fat on d 143 than those from nonimplanted steers. Carcasses from implanted steers possessed more carcass protein (P < .05) on d 40. Implanted steers had an 82% increase (P < .05) in daily carcass protein accretion during the first 40 d. Implantation increased (P < .01) carcass water but did not affect carcass fat accumulation throughout the feeding period. The combined TBA+E2 implant improved feedlot performance and stimulated carcass protein accretion in feedlot steers.

Effect of a Combined Trenbolone Acetate and Estradiol Implant on Feedlot Performance, Carcass Characteristics, and Carcass Composition of Feedlot Steers

Muscle satellite cells were isolated from seven yearling steers implanted for 31 d with a combined implant that contained 120 mg of trenbolone acetate (TBA) and 24 mg of estradiol (E2) and from seven nonimplanted, control steers. Implanted steers had a 28% greater ADG and a 23% greater feed efficiency than did nonimplanted steers. Implanted steers had increased (P<.001) circulating IGF-I concentrations on d 6, 14, and 31 after implantation, and circulating IGF-I concentrations in control steers remained constant or decreased (P<.05) at these times. Maximum fusion percentage was greater (P< .005) in satellite cell cultures isolated from implanted steers (ISC cultures) than in satellite cell cultures isolated from control steers (NSC cultures) (72.8% vs 54.8%, respectively). Satellite cell cultures isolated from implanted steers (ISC cultures) also contained a greater (P<.001) number of myotube nuclei than did NSC cultures (7,998 nuclei/cm2 vs 5,150 nuclei/cm2, respectively). After 72 h in culture, the number of cells (corrected for plating density) was 43% greater (P<.05) in ISC cultures than in NSC cultures. [3H]Thymidine incorporation rates per 10(5) cells at 24 and 34 h after plating were greater (P<.05) in ISC cultures than in NSC cultures; however, incorporation rates did not differ at 72 h. These data indicate that TBA + E2 implantation may result in an in vivo activation of muscle satellite cell proliferation that can be detected in cell culture. This activation may play an important role in TBA + E2-enhanced muscle growth.

William R Dayton

Papers

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Purification from porcine muscle.

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme.

A calcium-activated protease possibly involved in myofibrillar protein turnover. Isolation of a low-calcium-requiring form of the protease.

Effect of a Combined Trenbolone Acetate and Estradiol Implant on Feedlot Performance, Carcass Characteristics, and Carcass Composition of Feedlot Steers

Activation state of muscle satellite cells isolated from steers implanted with a combined trenbolone acetate and estradiol implant