Calpain system regulates muscle mass and glucose transporter GLUT4 turnover

The experiments in this study were undertaken to determine whether inhibition of calpain activity in skeletal muscle is associated with alterations in muscle metabolism. Transgenic mice that overexpress human calpastatin, an endogenous calpain inhibitor, in skeletal muscle were produced. Compared with wild type controls, muscle calpastatin mice demonstrated normal glucose tolerance. Levels of the glucose transporter GLUT4 were increased more than 3-fold in the transgenic mice by Western blotting while mRNA levels for GLUT4 and myocyte enhancer factors, MEF 2A and MEF 2D, protein levels were decreased. We found that GLUT4 can be degraded by calpain-2, suggesting that diminished degradation is responsible for the increase in muscle GLUT4 in the calpastatin transgenic mice. Despite the increase in GLUT4, glucose transport into isolated muscles from transgenic mice was not increased in response to insulin. The expression of protein kinase B was decreased by approximately 60% in calpastatin transgenic muscle. This decrease could play a role in accounting for the insulin resistance relative to GLUT4 content of calpastatin transgenic muscle. The muscle weights of transgenic animals were substantially increased compared with controls. These results are consistent with the conclusion that calpain-mediated pathways play an important role in the regulation of GLUT4 degradation in muscle and in the regulation of muscle mass. Inhibition of calpain activity in muscle by overexpression of calpastatin is associated with an increase in GLUT4 protein without a proportional increase in insulin-stimulated glucose transport. These findings provide evidence for a physiological role for calpains in the regulation of muscle glucose metabolism and muscle mass.     

Effects of Calpain on Antioxidant Enzyme Activities

The activities of superoxide dismutase, catalase and glutathione reductase were not affected by in vitro incubation with the intracellular proteinase calpain, suggesting that these enzymes are not in vivo substrates of calpain. In contrast, the activity of another important antioxidant enzyme, glutathione peroxidase, is stimulated in vitro by calpain. This may explain the correlation between elevations in glutathione peroxidase activity and calpain activity which occur in aging, exercised and dystrophic muscle. Calpain treatment in vitro caused a large decrease in the activity of carnosine synthetase which is involved in the synthesis of the putative antioxidant carnosine. This may be the reason for the in vivo correlation between elevated calpain and diminished carnosine levels in aging, hypertensive, denervated and dystrophic muscles.     

Proteomics analysis of starved cells revealed Annexin A1 as an important regulator of autophagic degradation

The peptidyl-proline isomerase, protein never in mitosis gene A interacting-1 (PIN1) binds and isomerizes proteins phosphorylated on serine/threonine before a proline. It was previously found that depletion of PIN1 greatly increased induction of cyclooxygenase-2 and inducible nitric oxide synthase by lowering calpain activity in murine aortic endothelial cells (MAEC). Here we investigated the effect of PIN1 on the endogenous inhibitor of heterodimeric μ- and m-calpains, calpastatin. MAEC were transduced with small hairpin (sh) RNA to knock down PIN1 (KD) or an inactive Control shRNA. Cells were also treated with non-targeted double stranded small inhibitory RNA (siRNA) or siRNA designed to deplete calpastatin. Despite reducing calpain activity, PIN1 KD did not significantly affect the expression of μ- and m-calpains, or calpastatin, compared to Control shRNA. Instead, depletion of PIN1 increased the inhibitory activity of calpastatin. Calpastatin co-immunoprecipitated with endogenous PIN1 and was pulled down with glutathione-S-transferase (GST)–PIN1 fusion protein. Adding GST–PIN1 to KD cell extracts lacking PIN1 reduced calpastatin inhibitory activity. Substrate binding and catalytic domain mutants of PIN1 failed to do so. These results suggest that protein interaction and the proline isomerase functions of PIN1 are required for it to inhibit calpastatin. Furthermore, depletion of calpastatin raised calpain activity and reduced calpain inhibitory activity to similar levels in KD and Control MAEC, indicating that calpastatin is required for PIN1 depletion to lower calpain activity. Thus, PIN1 apparently restrains the ability of calpastatin to inhibit calpain, maintaining calpain activity in endothelial cells. PIN1 may act directly via phosphorylated serine/threonine–proline motifs in calpastatin, or indirectly via other PIN1 substrates that control calpastatin.     

Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3

We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of mu- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.     

EXTRA-LYSOSOMAL PROTEOLYSIS AND EXPRESSION OF CALPAINS AND CALPASTATIN IN CULTURED THYROID CELLS

Proteolysis at neutral pH in the soluble fraction of cultured pig thyroid epithelial cells was examined using a synthetic calpain substrate, succinyl-Leu-Tyr-7-amino-4-methylcoumarin. The Ca²⁺-independent proteolytic activity was largely inhibited by substances known to affect cysteine- and metalloproteases, whereas no or little effects were obtained with inhibitors affecting serine- and aspartic proteases. Addition of Ca²⁺did not significantly alter the rate of substrate degradation. Biochemical separation via hydrophobic interaction chomatography and Western blotting demonstrated the presence of both m-calpain (40% of total calpain) and μ-calpain (60%) in confluent thyrocytes. Determination of calpastatin activity indicated a 30 times higher level of the inhibitor as compared to total calpain activity. Western blotting showed the presence of a 110kD calpastatin form with additional low mol wt forms possibly representing fragmentation products. In immunofluorescent stainings, m-calpain had a diffuse cytoplasmic distribution whereas μ-calpain was located both in the cytoplasm and at the cell—cell contacts. Calpastatin immunoreactivity was mainly granular and located close to the nucleus, although a fibrillar distribution was also observed. The results show the presence of all components of the calpain/calpastatin system and indicate a strict control of calpain activity in cultured thyrocytes. The different subcellular distributions of calpains and calpastatin suggests that they are compartmentalized and require mobilization to interact.     

Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains

Two forms of calpastatin, differing in their specificity for the homologous calpain isozymes I and II, have been separated from rat skeletal muscle extracts and purified to homogeneity. Calpastatin I, the first form to elute in chromatography on DE32, is more effective against calpain I, while calpastatin II is more effective as an inhibitor of calpain II. Based on their molecular mass (approximately 105 kDa) both calpastatin forms belong to the high molecular mass class found in muscles of other animal species (Murachi, T., 1989, Biochem. Int. 18, 263-294). For calpain I, which is active with low (mu-M) concentrations of Ca2+, maximum inhibition with either calpastatin form was observed over a wide range of Ca2+ concentrations. With calpain II, which requires high (mM) concentrations of Ca2+ for activity, maximum inhibition required Ca2+ concentrations above 1 mM. Both calpastatin forms were found to be highly sensitive to degradation by calpain II, but almost completely resistant to degradation by calpain I. Degradation of calpastatin by calpain II is competitively inhibited by the addition of a calpain substrate. Isovaleryl carnitine (IVC), an intermediate product of L-leucine catabolism, previously demonstrated to be a potent and specific activator of rat skeletal muscle calpain II (Pontremoli, S., Melloni, E., Viotti, P. L., Michetti, M., Di Lisa, F., and Siliprandi, N., 1990. Biochem. Biophys. Res. Commun. 167, 373-380) greatly enhances the rate of degradation of calpastatins by calpain II. IVC, which decreases the Ca2+ requirement for maximal calpain II activity, also decreases the concentration of Ca2+ required for digestion of the inhibitor. For calpain II, regulation by either calpastatins may occur only in the presence of high [Ca2+].     

Calpain and calpastatin in myoblast differentiation and fusion: Effects of inhibitors

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca²⁺-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-β, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-β and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of μ-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.     

Differential regulation of the calpain–calpastatin complex by the L-domain of calpastatin

Here we demonstrate that the presence of the L-domain in calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain–calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca^2 +] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which calpastatin levels are increased. Our findings show that the L-domain of calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.     

Influence of protein nutrition on calpain activity of mouse kidney: Modulation by calpastatin

The effect of protein depletion and refeeding with a normal diet on calpain activity was examined in mouse kidney soluble homogenate. In terms of units per gram of protein, it increased 2.9 times with depletion and decreased upon refeeding. After a DEAE-Sephacel chromatography, the homogenate yielded three enzymatic activities. Their sum, assessed as total calpain activity, was higher than the activity measured before fractionation and did not appreciably change during protein depletion and refeeding. Because the proportion of total activity displayed by the complete homogenate increased with depletion and decreased with refeeding, a low calpastatin content in depleted kidney was envisaged. This was confirmed by direct estimations: depleted kidney had 6 times less calpastatin compared to both normal and 16 h refed tissue. We concluded that a decrease in calpastatin content contributes to an increased calpain activity related to degradable protein in protein depleted kidney. In view of this, it seems not unlikely that the in vivo rate of protein breakdown depicted by kidney during protein depletion and refeeding is in part effected through modulation of the calpain proteolytic system. (Mol Cell Biochem 166: 95-99, 1997)     

The role of ultimate pH in proteolysis and calpain/calpastatin activity in bovine muscle.

Of a total of three Friesian cows, two of which had been treated with adrenalin before slaughter, Mm longissimus (LO), supraspinatus (SS), triceps brachii (TB) and rectus abdominis (RA) were sampled at different times post mortem (pm). pH, calpain/calpastatin activities and degradation of myofibrillar proteins, as evidenced by SDS-PAGE, were assessed. Contraction characteristics were measured by determining myofibrillar ATPase activities. Adrenalin treatment resulted in a high ultimate pH (6.48 +/- 0.40) and a faster decline pm of calpain I activity. The effect was similar in all four investigated muscles (72.4 +/- 5.4% decline at 24 h pm). The decline in calpain I activity in the control muscles was muscle-dependent and ranged from 22.8-74.3% at 24 h pm. Differences in ultimate pH did not lead to distinct rates of breakdown of proteins with molecular weights lower than that of myosin heavy chain. Calpastatin levels were muscle-dependent and correlated with myofibrillar ATPase activity (r = -0.99). In a second experiment Mm rectus abdominis (RA) and psoas major (PM) of adrenalin-treated (n = 6) and control (n = 6) Friesian-Holstein calves were sampled at 1 and 29 h pm for assessment of calpain activities. At seven days pm the M longissimus (LO) was sampled for tenderness evaluation. pH values were measured at 30 min, 4 h and 29 h pm. Adrenalin treatment resulted in a higher ultimate pH in the three muscles. Higher ultimate pH resulted in lower calpain activities in the RA at 29 h pm (P less than or equal to 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calpain and calpastatin levels in different organs of the rabbit

1. The levels of Ca-independent and Ca-dependent proteolytic activity as well as the activities of calpains and calpastatin in different organs of the rabbit was examined at various developmental stages. 2. Calpain and calpastatin levels were highest in the lung and in the kidney. 3. In all organs examined except the thymus the total level of calpain was higher than that of calpastatin. 4. In the thymus the levels of calpains and calpastatin decreased markedly with age.     

Calpastatin levels affect calpain activation and calpain proteolytic activity in APP transgenic mouse model of Alzheimer's disease

The intracellular Ca(2+)-dependent protease calpain and the specific calpain endogenous inhibitor calpastatin are widely distributed, with the calpastatin/calpain ratio varying among tissues and species. Increased Ca(2+) and calpain activation have been implicated in Alzheimer's disease (AD), with scant data available on calpastatin/calpain ratio in AD. Information is lacking on calpain activation and calpastatin levels in transgenic mice that exhibit AD-like pathology. We studied calpain and calpastatin in Tg2576 mice and in their wild type littermates (control mice). We found that in control mice calpastatin level varies among brain regions; it is significantly higher in the cerebellum than in the hippocampus, frontal and temporal cortex, whereas calpain levels are similar in all these regions. In the Tg2576 mice, calpain is activated, calpastatin is diminished, and calpain-dependent proteolysis is observed in brain regions affected in AD and in transgenic mice (especially hippocampus). In contrast, no differences are observed between the Tg2576 and the control mice in the cerebellum, which does not exhibit AD-like pathology. The results are consistent with the notion that a high level of calpastatin in the cerebellum renders the calpain in this brain region less liable to be activated; in the other brain parts, in which calpastatin is low, calpain is more easily activated in the presence of increased Ca(2+), and in turn the activated calpain leads to further diminution in calpastatin (a known calpain substrate). The results indicate that calpastatin is an important factor in the regulation of calpain-induced protein degradation in the brains of the affected mice, and imply a role for calpastatin in attenuating AD pathology. Promoting calpastatin expression may be used to ameliorate some manifestations of AD.     

Ex vivo measurement of calpain activation in human peripheral blood lymphocytes by detection of immunoreactive products of calpastatin degradation

Limited proteolysis of multiple intracellular proteins by endogenous Ca-dependent cysteine proteases--calpains--is an important regulatory mechanism for cell proliferation, apoptosis etc. Its importance for cellular functions is stressed by existence of endogenous calpain inhibitors--calpastatins. The calpain-calpastatin system within living cells is in a fragile balance, which depends on both partners. The interdependence of calpain--a protease--and calpastatin--an endogenous inhibitor and at the same time a substrate for this enzyme makes any assessment of actual activity of this enzyme in the cells very difficult. In this work we made an attempt to estimate and compare the activity of calpain in human peripheral blood lymphocytes by assessing the levels of limited proteolysis of calpastatin in these cells by western blot, while at the same time the levels of calpain protein inside these cells was measured by flow cytometry. Our results indicate that it is possible to compare (semi-quantitatively) the activities of calpain in peripheral blood CD4+ and CD19+ lymphocytes from various donors that way. Preliminary results showed that calpain activity is increased in the CD4+ T cells isolated from peripheral blood of rheumatoid arthritis patients as compared to control lymphocytes. Extremely high intrinsic activity of calpain was detected in chronic lymphocytic leukemia (CD19+) cells. All this confirms the detection of immunoreactive products of calpastatin as a good maker of endogenous calpain activity.     

The calpastatin/calpain system in trout Salmo trutta trutta muscle

Many recent reports suggest that the calpastatin/calpain system plays a role in cellular growth and differentiation. Defects of the calpastatin/calpain system have been linked to cellular dysfunctions, apoptosis, myocardial infarct, and dystrophies. The calpastatin/calpain system has also been implicated in post‐mortem tenderization of skeletal muscle through degradation of key myofibrillar and associated proteins, a process of key importance to meat quality. In the present study we investigate the presence and activity of the calpastatin/calpain system in trout muscle samples, collected at 0, 3, 18 and 28 h post‐mortem, by immunohistochemistry method. Calpastatin is a specific endogenous enzyme of cytosol, modulating the ubiquitous calpains. Calpastatin was found in samples obtained in vivo and immediately post‐mortem, but its concentration declined rapidly in samples obtained 3, 18 and 28 h post‐mortem. The ubiquitous m e m‐calpains, which are localized on Z line proteins and activated by intracellular Ca²⁺ increase, showed a rapid decline within 3 h post‐mortem. By contrast p94 calpain, which is specific to skeletal muscle, showed a slow decrease post‐mortem which was independent of intracellular Ca²⁺ increase. Our results suggest that the mechanism of activation and activity of the calpastatin/calpain system in trout is similar to that described in mammals.     

Dysregulation of the calpain-calpastatin system plays a role in the development of cerulein-induced acute pancreatitis in the rat

Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed micro-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg i.p.), pancreatitis was induced by cerulein injections (10 microg/kg i.p.; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both micro-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity (P < 0.01), pancreatic edema formation (P < 0.05), histological score for eight parameters (P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.     

Association of calpain (Ca(2+)-dependent thiol protease) with its endogenous inhibitor calpastatin in myoblasts.

Calpain isozymes (intracellular, Ca(2+)-dependent thiol proteases) are present in the cytoplasm of many cells, along with their endogenous specific inhibitor, calpastatin. Previously, we found that the levels of mu-calpain and m-calpain (activated by microM and mM Ca(2+), respectively) remain about the same during myoblast differentiation and fusion. By contrast, the calpastatin level, which is high during the initial stages of differentiation, diminishes markedly before myoblast fusion, allowing the proteolysis that is required for myotube formation. In the present study, we used immunoprecipitation to investigate the molecular association between calpain and calpastatin in dividing myoblasts and in the initial stages of myoblast differentiation. Immunoprecipitation (IP) was performed in two ways: (1) IP of calpain, using an anti-calpain antibody that recognized both isozymes; and (2) IP of calpastatin (using anti-calpastatin). Calpastatin was co-precipitated when calpain was immunoprecipitated; calpain was co-precipitated when calpastatin was immunoprecipitated. The results indicate that calpastatin is associated with calpain in dividing myoblasts and in myoblasts during the initial stages of differentiation, thereby preventing calpain activation at this stage. Prior studies carried out in vitro have shown a Ca(2+)-dependent interaction of calpain with calpastatin. The results described here suggest that an association between calpain and calpastatin could occur within cells in the presence of physiological Ca(2+)levels. It is proposed that the status of cellular calpain-calpastatin association is modulated by cell constituents, for which some possibilities are suggested.     

Age-dependent degradation of calpastatin in kidney of hypertensive rats

Hypertensive rats from the Milan strain show a significant decrease in calpastatin activity as compared with normotensive control animals. Calpastatin deficiency is age-related and highly relevant in kidney, heart, and erythrocytes and of minor entity in brain tissue. In normotensives the changes during aging in the levels of calpastatin activity and mRNA are consistent with an increase of calpastatin protein. In hypertensive rats such a relationship during aging is not observed, because a progressive accumulation of mRNA is accompanied by a lower amount of calpastatin protein as compared with control rats. Together with the low level of calpastatin in kidney of hypertensive rats, a progressive accumulation of an active 15-kDa calpastatin fragment, previously shown to represent a typical product of calpain-mediated calpastatin degradation, is also observed. Evidence for such intracellular proteolysis by Ca(2+)-activated calpain is provided by the normalization of the calpastatin level, up to that of control animals, in hypertensive rats treated with drugs known to reduce both blood pressure and intracellular Ca(2+) influx. Further evidence is provided by the disappearance, in these conditions, of the 15-kDa calpastatin fragment. These data allow the conclusion that calpastatin degradation is a relevant part of the overall mechanism for regulating calpain activity.     

Treatment of rats with calpain inhibitors prevents sepsis-induced muscle proteolysis independent of atrogin-1/MAFbx and MuRF1 expression

Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting.