Skeletal Muscle Calpastatin Research Articles

Dispersing Clusters with Calpain

A reciprocal relationship exists between agrin and acetylcholine (ACh) in controlling acetylcholine receptor (AChR) clustering at the neuromuscular junction: Agrin promotes clustering, and ACh disperses clusters. Chen et al . provide insight into how these opposing signals may allow stabilization of AChR at the neuromuscular junction by differential activity of the calcium-activated proteases calpains. Exposure of C2C12 myotubes in culture to the cholinergic agonist carbachol triggered a calcium transient, activation of calpain, and stimulation of cyclin-dependent kinase 5 (CDK5) through production of the activator p25 by cleavage of the CDK5 regulatory partner p35. (CDK5 has been previously implicated in AChR dispersal, and p35 is a known substrate for calpain.) Activation of CDK5 by carbachol was blocked by pharmacological inhibition of calpain with calpeptin, and the dispersal of AChR clusters induced by agrin was also prevented if calpain activity was blocked (pharmacologically or by RNAi). In vivo, pharmacologic inhibition of calpain (by injection of pregnant mice) in mice deficient for agrin rescued the formation of AChR clusters, although not all clusters were apposed to the nerve terminals. In transgenic mice overexpressing a calpain-inhibitory peptide calpastatin in skeletal muscle, p25 abundance was lower in the skeletal muscles from the transgenic mice, and AChR clustering was increased compared with control mice. A yeast two-hybrid screen revealed that rapsyn interacted with the large subunit of m-calpain, and mutagenesis analysis identified that the two proteins interacted through the tetratricopeptide repeat (TPR) domain of rapsyn and the regulatory domain (domain III) of calpain. Rapsyn is a protein that interacts with the AChR and is necessary for cluster stabilization. Calpain activity in vitro or in transfected cells was inhibited by rapsyn, and RNAi experiments to knock down rapsyn in C2C12 myotubes showed that p25 was more abundant in control and carbachol-treated cells. Rapsyn, calpain, and the AChR coimmunoprecipitated from C2C12 cells, and agrin stimulated this interaction. Analysis of the synaptic and nonsynaptic regions of diaphragm suggested that m-calpain was relatively evenly distributed between these two regions, but the activity of calpain was substantially less in the synaptic region (based on the abundance of p25). Thus, the authors propose that agrin promotes the interaction of calpain with the inhibitor rapsyn at the synapse, allowing AChR to cluster, and in nonsynaptic regions, calpain is activated in response to the calcium signals induced by ACh and disperses nonsynaptic ACh clusters. F. Chen, L. Qian, Z.-H. Yang, Y. Huang, S. T. Ngo, N.-J. Ruan, J. Wang, C. Schneider, P. G. Noakes, Y.-Q. Ding, L. Mei, Z.-G. Luo, Rapsyn interaction with calpain stabilizes AChR clusters at the neuromuscular junction. Neuron 55 , 247-260 (2007). [PubMed]

Short commuunication: Effect of dietary protein depletion and repletion on skeletal muscle calpastatin during early lactation.

Sixteen multiparous Jersey cows were assigned at calving to one of 4 dietary treatments. An 18% crude protein (CP) diet was fed as a total mixed ration through 30 d in milk (DIM), and beginning at 31 DIM a 9, 12, 15, or 18% CP diet was fed through 58 DIM (depletion). All cows were then fed the 18% CP diet until 84 DIM (repletion). Muscle biopsies were taken under local anesthesia at 49 and 84 DIM from the semitendinosus muscle. Milk production, DMI, and milk component contents were measured. Calpain and calpastatin contents of muscle taken at biopsy were evaluated using Western blotting techniques. Milk production and milk protein content were reduced during the depletion period by decreasing dietary protein. Diet had no effect on milk fat content or DMI. During repletion, DMI was affected by dietary treatment. Western blots of muscle extracts indicated no differences in calpain content at any stage of the experiment or in calpastatin content of muscle at 49 DIM. However, at 84 DIM, calpastatin (135 kDa) was lower or undetectable in cows fed either the 9 or 12% CP diets from 31 to 59 DIM. Bands for a 110-kDa degradation product of calpastatin were present in some cows fed the 9, 12, and 15% CP diets during the depletion period. Results indicate a change in skeletal muscle calpain/calpastatin proteolytic system during protein repletion following depletion with diets of less than 15% CP during early to peak lactation in dairy cows.

Immunoblot analysis of calpastatin degradation: evidence for cleavage by calpain in postmortem muscle.

A negative correlation exists between calpastatin activity and meat tenderness. Therefore, it is important to determine the mechanism of calpastatin inactivation in postmortem skeletal muscle. Western immunoblot analysis was performed to determine the protease(s) responsible for degradation of muscle calpastatin during postmortem storage. To accomplish this, purified calpastatin was digested with different proteases in vitro, and their pattern of calpastatin degradation was compared with that of calpastatin degradation in postmortem muscle. Polyclonal antibodies raised in mice against recombinant bovine skeletal muscle calpastatin were used to monitor calpastatin degradation. Lamb longissimus was stored at 4 degrees C and sampled at 0, 6, 12, 24, 72, 168, and 336 h postmortem. Postmortem storage produced a discrete pattern of calpastatin degradation products that included immunoreactive bands at approximately 100, 80, 65, 54, 32, and 29 kDa. Undegraded calpastatin (130 kDa) was barely detectable after 72 h of postmortem storage at 4 degrees C, and no immunoreactive calpastatin was observed by 336 h postmortem. For in vitro proteolysis, lamb longissimus calpastatin (0 h postmortem) was purified using Affi-Gel Blue chromatography. Calpastatin was digested with m-calpain, mu-calpain, cathepsin B, proteasome, trypsin, or chymotrypsin. Each of these enzymes degraded calpastatin. Immunoreactive fragments resulting from digestion of calpastatin with m- and mu-calpain were similar to each other and closely resembled those observed during postmortem aging of lamb longissimus at 4 degrees C. Digestion of calpastatin with mu-calpain reduced calpastatin activity. Degradation of calpastatin by other proteases resulted in unique patterns of immunoreactive fragments, distinct from that observed in longissimus. Thus, m- and(or) mu-calpain seem to be responsible for calpastatin degradation during postmortem storage of meat.

The calpain system and skeletal muscle growth

The first protein of a group of proteins now identified as belonging to the calpain system was purified in 1976. The calpain system presently is known to be constituted of three well-characterized proteins; several lesser studied proteins that have been isolated from invertebrates; and 10 mRNAs, two each in Drosophila and C. elegans and six in vertebrates, that encode proteins, which, based on sequence homology, belong to the calpain family. The three well-characterized proteins in the calpain family include two Ca2+-dependent proteolytic enzymes, µ-calpain and m-calpain, and a protein, calpastatin, that has no known activity other than to inhibit the two calpains. A substantial amount of experimental evidence accumulated during the past 25 yr has shown that the calpain system has an important role both in rate of skeletal muscle growth and in rate and extent of postmortem tenderization. Calpastatin seems to be the variable component of the calpain system, and skeletal muscle calpastatin activity is highly related to rate of muscle protein turnover and rate of postmortem tenderization. The current paradigm is that high calpastatin activity: 1) decreases rate of muscle protein turnover and hence is associated with an increased rate of skeletal muscle growth; and 2) decreases calpain activity in postmortem muscle and hence is associated with a lower rate of postmortem tenderization. This article summarizes some of the known properties of the calpain system and discusses the potential importance of the calpain system to animal science. Key words: Calpain, calpastatin, postmortem tenderization, skeletal muscle growth

An Improved Purification Protocol for Heart and Skeletal Muscle Calpastatin Reveals Two Isoforms Resulting from Alternative Splicing

Employment of a new protocol for efficient purification of calpastatin revealed the presence of two forms of calpastatin in porcine heart with apparent molecular weights of 125 and 145 kDa. Purification from bovine muscle resulted in a single species with an apparent molecular weight of 125 kDa. The presence of multiple species of calpastatin in porcine heart does not appear to be an artifact of the purification procedure since Western blotting revealed the presence of two types of calpastatin in ovine and porcine heart, but only one type in bovine heart and bovine, ovine, and porcine muscle. The origin of the two species in porcine heart was examined by RT-PCR and direct sequencing of calpastatin cDNA. This analysis revealed that porcine skeletal muscle exclusively produces transcripts lacking exon 3, while porcine heart produces transcripts that include or lack exon 3, consistent with the presence of two isoforms of the protein. The 125-kDa form of porcine calpastatin therefore appears to be a result of alternative splicing of the calpastatin transcript. The biological significance of the heart specific isoform is not clear; however, its ability to inhibit μ- or m-calpain does not differ considerably. The present purification protocol yielded 4.9 and 1.8 mg calpastatin per kg tissue from porcine heart and bovine skeletal muscle, respectively.

Development of an enzyme-linked immunosorbent assay (ELISA) for quantification of skeletal muscle calpastatin.

An indirect antibody ELISA was developed for rapid and sensitive quantification of skeletal muscle calpastatin. Polyclonal antibodies were raised in rabbits against recombinant calpastatin, corresponding to domains 2, 3, and 4 of bovine skeletal muscle calpastatin. Western blot analysis revealed that these antibodies specifically recognize an immunoreactive calpastatin protein of approximately 130 kDa in prerigor skeletal muscle extracts. The intensity of the immunoreactive bands corresponds qualitatively with assayable calpastatin activity. For ELISA development, optimum dilutions of sample, primary anti-calpastatin antibody, and peroxidase-conjugated secondary antibody were determined by titration. A dilution optimum for coating of Immulon 4 (Dynatech) plates was observed when heated muscle extracts were diluted to 2 to 4 micrograms of protein/mL and incubated for 2 h at 37 degrees C. Optimum primary (30 micrograms IgG/mL) and secondary (Sigma A-6154; 1:1000 dilution) antibody incubations were for 1 h at 37 degrees C. Tetramethylbenzidine was used as substrate and A450 of the stopped reaction product was recorded in an automated plate reader. Calpastatin ELISA results were linearly related to calpastatin activity (calpain inhibitory activity) of heated longissimus muscle homogenates from prerigor lamb (r2 = .89; n = 40) and beef aged for 24 or 48 h (r2 = .90; n = 47). Intra-assay CV was < 5% (n = 8) and inter-assay CV was < 6% (n = 5). This assay offers advantages of speed, simplicity, and sensitivity over conventional methodology for calpastatin quantification.

Relationship of restriction fragment length polymorphisms (RFLP) at the bovine calpastatin locus to calpastatin activity and meat tenderness.

Restriction fragment length polymorphisms (RFLP) have been identified at the bovine calpastatin locus. The objective of the present study was to determine whether these polymorphisms are related to variations in calpastatin activity or beef tenderness in unrelated animals of mixed breeding. A sample of 83 crossbred steers from sires representing eight different breeds was examined to determine this relationship. A 2.2-kb cDNA coding for domains 2 through 4 plus a 3' untranslated region of bovine skeletal muscle calpastatin was used as a probe for calpastatin RFLP. Polymorphisms were found using the restriction enzymes BamHI and EcoRI. Polymorphic restriction fragments for BamHI were 9.0 and 5.0 kb and for EcoRI were 6.0 and 4.0 kb. Allelic frequencies for BamHI restriction fragments were .53 for the 9.0-kb allele and .47 for the 5.0-kb allele. Allelic frequencies for EcoRI restriction fragments were .43 for the 6.0-kb allele and >57 for the 4.0-kb allele. No polymorphisms were identified using the restriction enzymes BglII, DraI, or PstI. No associations between EcoRI and BamHI RFLP and 24-h calpastatin activity of Warner-Bratzler shear force at 14 d postmortem were detected. Therefore, the polymorphic EcoRI and BamHI restriction sites within the bovine calpastatin locus do not detect DNA sequence differences responsible for variation in calpastatin activity or tenderness of aged beef. Therefore, these polymorphisms cannot be used to predict tenderness of aged beef from unrelated animals of mixed breeding. These results do not exclude the possibility that other DNA sequences in or near the bovine calpastatin gene are responsible for variation in calpastatin activity or meat tenderness. The lack of a relationship between these calpastatin RFLP and meat tenderness must be distinguished from the well-documented relationship between calpastatin activity and meat tenderness. Therefore, further development of calpastatin-based methods for predicting beef tenderness in unrelated animals of mixed breeding should focus on basic factors influencing calpastatin activity at the molecular and cellular level.

A comparative study on gene expression of calpains, calpastatin, cathepsin D, and proteasome in tissues of fed and fasted rabbits

Molecular mechanisms underlying changes in muscle protein turnover are not fully understood. In this study, effects of fasting on mRNA concentrations encoding several proteinases in skeletal muscle were investigated. Proteinases included calpains I and II, cathepsin D, and proteasome. We also examined effects of fasting on the calpain small subunit, calpastatin, and on β-actin mRNAs for comparative purposes. Fasting increased mRNAs encoding all proteinases, calpain small subunit, and calpastatin in skeletal muscle but reduced β-actin mRNA. This effect was most pronounced for cathepsin D. To determine whether the changes observed in skeletal muscle occurred in other tissues, we examined effects of fasting on proteinase mRNA concentrations in liver, lung, and kidney. Fasting either had no effect or reduced proteinase, calpastatin, and calpain small subunit mRNA concentrations in these tissues. Fasting reduced β-actin mRNA concentration in all tissues. Therefore, proteinase gene expression in skeletal muscle differs from other tissues during fasting. Despite the changes in calpain mRNA concentrations in muscle, changes in calpain activities were not detected. This suggests that either calpain synthesis was concomitantly reduced or calpain turnover was increased during fasting. Differences in calpain mRNA concentrations were detected among tissues and these were related to differences in calpain concentrations and activities among tissues. We conclude that calpain and other proteinase genes are co-regulated in muscle in a manner that differs from other tissues, and that fasting-dependent changes in muscle calpain mRNA serve to maintain calpain concentrations at fixed levels at a time when muscle protein synthesis is reduced or calpain stability is reduced. Finally, we conclude that differences in calpain mRNAs among tissues underlie the differences in tissue calpain concentrations and activities.

Bovine skeletal muscle calpastatin: cloning, sequence analysis, and steady-state mRNA expression

Calpastatin is a specific inhibitor of the calpains. Calpains play a key role in postmortem tenderization of meat and have been hypothesized to be involved in muscle protein degradation in living tissue. Isolation, cloning of complementary DNA, and nucleotide sequencing of bovine calpastatin from the longissimus muscle have been completed. Two clones were identified that encompass the entire coding sequence. Clone pCR41, derived by reverse transcription-PCR, covers domains L and 1; clone pBSA1, obtained from cDNA library screening, covers domains 2 through 4 in addition to the 3'-nontranslated region. Nucleotide sequence analysis of the cDNA for bovine calpastatin revealed an average nucleotide sequence identity of approximately 70 to 80% compared with published calpastatin nucleotide sequences of human, rabbit, and pig. Exon 3, corresponding to a highly conserved 22-amino acid region, was deleted from bovine calpastatin domain L. The calculated molecular weight of bovine skeletal muscle calpastatin of 706 amino acid residues (M(r) 75,842) corresponds to the value of purified bovine skeletal muscle calpastatin as determined by SDS-PAGE (M(r) 68,000). Northern blot analysis revealed the presence of multiple calpastatin mRNA transcripts having estimated sizes of 3.8, 3.0, and 1.5 kb in beef and 3.8, 3.0, 2.5, and 1.5 kb in sheep. Calpastatin mRNA expression was increased with beta-adrenergic agonist-induced muscle hypertrophy.

Modulation of inhibitory efficiency of rat skeletal muscle calpastatin by phosphorylation

Rat skeletal muscle calpastatin form is markedly modified in its inhibitory properties by means of a reverse reaction which involves both phosphorylation and dephosphorylation. Dephospho-calpastatin shows greater inhibitory efficiency versus μ-calpain, whereas phospho-calpastatin shows maximal inhibition versus m-calpain. Both forms are present in fresh rat muscle. Phosphorylation has been reproduced “in vitro” using a homologous Ca2+ independent protein kinase and found to result in the incorporation of approximately one mole of 32P per mole of protein. Dephosphorylation was induced by treatment with alkaline phosphatase and 32P release shown found to correlate with modifications of the inhibitory properties. This reversible covalent modification of calpastatin is considered an important advancement in the understanding of how different calpain isoforms can be more efficiently controlled by a single inhibitor isozyme form.

Calpain inhibitor in rabbit skeletal muscle: an immunochemical and histochemical study.

Calpastatin, the endogenous inhibitor of calcium-activated neutral proteases (calpains; EC 3.4.22.17), was studied in the rabbit vastus lateralis muscle by means of immunochemical and immunohistochemical techniques. Immunoaffinity chromatography using an antibody raised against the 34-kDa monomer of the 68-kDa dimeric inhibitor allowed us to isolate three main proteins (130-, 100- and 80-kDa). These proteins strongly inhibited calpain activity in muscle homogenate (I50 at about 50 micrograms/ml). Immunohistochemical experiments showed that calpastatin-related immunoreactivity was present in all fibre types (oxidative, glycolytic, oxidative-glycolytic) at both surface and cytoplasmic level. However, a few (20%) of the slow-twitch, oxidative fibres (5% of the total fibres), did not contain the cytoplasmic inhibitor. Specific immunoreactivity for calpastatin was also associated with the interstitial connective tissue. These results suggest that (i) calpastatin in skeletal muscle, as in other tissues, is present as a mixture of proteins of various molecular weights and (ii) the inhibitor may act not only in the cytoplasm but also at the surface or extracellular level.