Fibre type-specific expression of the calpain proteolytic system in skeletal muscle.

The calpain proteolytic system has an important role in all animal cells, responding to fluctuating levels of Ca” to initiate irreversible limited proteolysis of specific target proteins. The enzyme system has been implicated in a number of physiological and pathological processes’ involving cytoskeletal reorganisation, and in muscle is thought to be involved in fibre differentiation, growth and myofibrillar turnover. We have investigated the fibre typespecific expression of the components of this system, namely pand m-calpain, muscle-specific p94-calpain and their inhibitor calpastatin. In mammalian skeletal muscle, classical histochemistry based on the myofibrillar ATPase has identified three major fibre types, namely ‘fast’ IIA and IIB and ‘slow’ type I. Each of these major types expresses a single myosin heavy chain (MHC) isoform, MHCIIA, MHCIIB and MHCI respectively, which can be discriminated immunochemically and used for identification purposes. In addition, a third fast type MHCIIX (or MHCIID) has been discovered in small mammals and a continuum of hybrid fibre types exists as a result of some fibres co-expressing two MHC isoforms2. Porcine longissirnus dorsi (LD) and trapezius (TZ) muscle samples were characterized by SDS PAGE of myosin extracts and immunoblotting using anti MHC monoclonal antibodies (mAbs) specific to MHC-fast (types IIA and IIB) and MHCslow (type I) (Novacastra). On this basis porcine LD contained mainly fast fibre types and TZ was found to be a slower muscle consisting of 40% slow type I fibres. Snapfrozen samples were pulverised in liquid nitrogen and extracted in 20mM Tris-HC1, 5mM EDTA, pH 7.5 including the proteinase inhibitor AEBSF (Melford) at 10pg/ml. After separation from calpastatin by phenyl sepharose, pand mcalpain were resolved on a Iml SOURCE l5Q ion exchange column (Pharmacia), and assayed by a caseinolytic procedure3. Micro-calpain activity was 3925 26ukg (mean sem, n=6) and 286k 46 (n=6) in LD and TZ respectively and was eluted as a double peak in both cases. In contrast mcalpain appeared as a single peak with greater activity @<0.05) in TZ of 5982 58u/kg(n=6) compared to 3815 39(n=6) in LD. After SDS PAGE, calpain column fractions were electrotransferred to nitrocellulose and probed with antiporcine pand m-calpain 8OkDa subunit antisera raised against GST fusion proteins. Blots were scanned and analysed by OPTIMAS 5.1. Both pand m-calpain patterns mirrored their activity profiles with the ratio of the m-calpain immunoreactivity in LD and TZ being 1 : 1.6. Since the single 8OkDa pand mbands in both muscle types had identical mobility, the differential m-calpain staining suggested a different level of expression of this isoform in the two types, rather than posttranslational modifications such as autolytic activation or phosphorylation. Similar analysis was made of extractable calpastatin in these two muscle types. After separation from pand m-calpain using phenyl sepharose chromatograph?, total extractable calpastatin inhibitory activity quantified against m-calpain in a caseinolytic assay was 4,500+400u/kg(n=3) and 4,800t 200(n=3) in LD and TZ respectively. Extracts were prepared as for calpain but including also lpg/ml leupeptin and analysed by SDS PAGE prior to probing with an anticalpastatin antibody4. The immunoblot revealed major bands of approximate molecular weight 135 and IOOkDa, with an identical pattern in the two muscle types. After scanning and quantification, the immunostaining material was approximately equal in the two muscle types. Calpastatin mRNA has been reported to be alternatively spliced in skeletal and cardiac muscle, which may explain the differing electro horetic mobility of immunostaining bands in these tissueg5. However this tissue-specific differential expression does not seem to extend to muscle of different fibre types such as LD and TZ. Whole muscle samples of LD and TZ were homogenised, subjected to SDS PAGE, blotted and probed with an anti p94calpain antibody, revealing a single 94kDa band.of identical mobility in the two muscle types. Preliminary analysis of the scanned immunoblots showed p94 immunoreactivity to be higher @<0.05) in LD at 2251.7 arbitrary absorbance units (n=3) than in TZ, 1052.3(n=3) Overall, the 60% increase in m-calpain and the unchanged calpastatin in the slower TZ muscle may reflect capacity for a higher rate of protein turnover in slow fibres. Since TZ was shown to contain 40% slow type I fibres, such fibres can be predicted to contain between two and three times as much mcalpain activity as fast IIA and IIB types. In contrast, whole muscle homogenates from LD indicated a 100% higher level of p94 polypeptide than in TZ, suggesting that slow fibre types contain very little, if any, p94, at least in TZ. Bundles of approximately 20 fibres have been dissected from LD and adductor longus muscle (AL), which we have shown using anti MHC mAbs to have an even greater proportion of slow fibres than TZ. Preliminary studies indicate that p94 can readily be detected in bundles from both types, albeit at a lower level in AL bundles. To explain these observations, studies are being undertaken at single-fibre level. If the relative quantity of mand p94-calpain in isolated slow fibres differs from that predicted from the whole muscle study, this would indicate that there may be muscle-specific differences in the calpain system which are unrelated to fibre type.