Effects of Corticosterone on Connectin Content and Protein Breakdown in Rat Skeletal Muscle

We examined the effects of a glucocorticoid, corticosterone, on calpain activity, connectin content and protein breakdown in rat muscle. The results indicated that calpain activity was increased by corticosterone and thus breakdown of connectin was stimulated followed by increased breakdown of skeletal muscle protein.     

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.     

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.     

Synergism of triiodothyronine and corticosterone on muscle protein breakdown

The concerted effect of triiodothyronine (T3) and corticosterone on muscle protein synthesis and breakdown was studied. Thyroidectomized young male rats were treated with T3 (1.5 microgram/100 g body weight per day), corticosterone (10 mg/100 g body weight per day) and both T3 and corticosterone for 4 days. On the 3rd day of the experiment urine was collected to measure N tau-methylhistidine excretion as an index of muscle protein breakdown. On the last day of the experiment, the rates of protein synthesis in skeletal muscles were measured by the large-dose [3H]phenylalanine method. N tau-Methylhistidine excretion was slightly increased by T3 treatment and it was increased about 3-times by corticosterone treatment. When both T3 and corticosterone were administered, it was increased about 6-fold. The rate of muscle protein breakdown calculated from the difference between the rate of protein synthesis and the growth rate was consistent with these findings. The rate of muscle protein synthesis was increased by T3, and it was decreased by corticosterone. The rate was the same as that of the thyroidectomized control group when the animals were given T3 and corticosterone, showing that T3 restrained the inhibiting effect of corticosterone on muscle protein synthesis. The results indicate that a physiological level of T3 enhances the catabolic action of pharmacological doses of glucocorticoids on muscle protein breakdown.     

Multiple molecular interactions implicate the connectin/titin N2A region as a modulating scaffold for p94/calpain 3 activity in skeletal muscle

p94/calpain 3 is a skeletal muscle-specific Ca(2+)-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.     

Effects of corticosterone on Ca2+ uptake and myofibrillar disassembly in primary muscle cell culture

This study was done to examine the effects of corticosterone, a glucocorticoid, on Ca2+ uptake, proteolysis, and Ca2+ channels in primary cultures of chick muscle cells, to clarify the mechanism of glucocorticoid action on muscle proteolysis. Chick muscle cells were incubated for 24 h in a medium containing corticosterone (30 ng/ml) when the cells were confluent (6 days). To examine the contribution of Ca2+ channels, nifedipine, a Ca2+ channels antagonist, was used. Ca2+ uptake measured with 45CaCl2 was increased three-fold by corticosterone, with a peak at 12 h after the treatment started. The growth of the cells estimated from the protein content and creatine kinase activity was not affected by corticosterone. Proteolysis, evaluated with [3H]tyrosine as a label of the protein and Ntau-methylhistidine release, was unchanged by corticosterone. However, the amount of easily releasable myofilament as a measure of myofibrillar disassembly in the muscle cells was increased by corticosterone, and prevented by nifedipine. These results show that corticosterone increases Ca2+ uptake and starts myofibrillar protein breakdown.     

Effect of beta-agonists on expression of calpain and calpastatin activity in skeletal muscle.

Administration of beta-adrenergic agonists to domestic species can lead to skeletal muscle hypertrophy, probably by reducing the rate of myofibrillar protein breakdown. Myofibrillar breakdown is associated with the calcium-dependent proteinase system (calpains I,II and calpastatin) whose activity also changes during beta-agonist treatment. A number of growth trials using the agonists cimaterol and clenbuterol with cattle, sheep, chicken and rat are reported which suggest a general mechanism whereby beta-agonists reduce calpain I activity, but increase calpain II and calpastatin activity in skeletal muscle. Parallel changes in specific mRNAs indicate that changes in gene expression or stabilisation of mRNA could in part explain the changes in activity.     

Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosaminyl transferase from swine trachea epithelium

It is well established that periods of increased contractile activity result in significant changes in muscle structure and function. Such morphological changes as sarcomeric Z-line disruption and sarcoplasmic reticulum vacuolization are characteristic of exercise-induced muscle injury. While the precise mechanism(s) underlying the perturbations to muscle following exercise remains to be elucidated, it is clear that disturbances in Ca²⁺ homeostasis and changes in the rate of protein degradation occur. The resulting elevation in intracellular [Ca²⁺] activates the non-lysosomal cysteine protease, calpain. Because calpain cleaves a variety of protein substrates including cytoskeletal and myofibrillar proteins, calpain-mediated degradation is thought to contribute to the changes in muscle structure and function that occur immediately following exercise. In addition, calpain activation may trigger the adaptation response to muscle injury. The purpose of this paper is to: (i) review the chemistry of the calpain-calpastatin system; (ii) provide evidence for the involvement of the non-lysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to exercise (calpain hypothesis); and (iii) describe the possible involvement of calpain in the inflammatory and regeneration response to exercise.     

Superoxide-forming NADPH oxidase preparation of pig polymorphonuclear leucocyte.

The effect of corticosterone on myofibrillar protein breakdown in diabetic rats was investigated in order to assess the possible counteracting effects of the secondary rise in plasma insulin concentrations which normally accompanies such treatment. N^τ-Methylhistidine excretion, an index of myofibrillar protein breakdown, was compared before and after corticosterone treatment (4.0 mg/100 g body wt. per day) of normal control, adrenalectomized, 10-day-streptozotocin-diabetic and adrenalectomized diabetic rats. Diabetic rats received 1.5 units of insulin/100 g body wt. per day throughout the experiment and showed marked hyperglycaemia and glucosuria during corticosterone treatment, whereas non-diabetic rats had only mild hyperglycaemia but elevated insulin concentrations. Corticosterone treatment increased the average rate of myofibrillar protein breakdown by 68% and 95% respectively in non-diabetic and diabetic rats. Net loss of muscle non-collagen protein for the same 7-day period was greater in diabetic than in non-diabetic animals (4.15 versus 2.84% per day), and the calculated average synthesis rates were lowest in diabetic rats. Adrenalectomy had little effect except to decrease slightly the rate of muscle protein breakdown. These results show that the rise in plasma insulin concentrations that accompanies exogenous corticosterone administration to non-diabetic rats diminishes the catabolic effect of this glucocorticoid on muscle. Insulin appears to antagonize the effects of the glucocorticoid by attenuating the increased rates of myofibrillar protein breakdown and, to a lesser extent, by limiting the decrease in synthesis rates.     

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)     

Interactions of muscle beta-connectin with myosin, actin, and actomyosin at low ionic strengths

The interaction of the muscle elastic protein connectin with myosin and actin filaments was investigated by turbidimetry, viscosity, flow birefringence measurements, and electron microscopic observations. In KCl concentrations lower than 0.15 M at pH 7.0 at 25 degrees C, both myosin and actin filaments were aggregated by connectin. Myosin filaments were entangled with each other in the presence of connectin. Actin filaments were assembled into bundles under the influence of connectin just as under that of alpha-actinin. The physiological significance of the interactions of connectin with myosin and actin filaments is discussed in relation to the localization of connectin in myofibrils. The Mg2+-activated ATPase activity of actomyosin was appreciably enhanced by connectin in the presence of KCl concentrations lower than 0.1 M. The extent of activation by connectin was smaller than by alpha-actinin. The enhancement of the ATPase activity may be due to acceleration of the onset of superprecipitation of actomyosin.     

Complex Interactions Between Polyamines and Calpain-Mediated Proteolysis in Rat Brain

Polyamine synthesis is induced by various extracellular signals, and it is widely held that this biochemical response participates in cell growth and differentiation. Certain of the triggers for synthesis in brain tissues also increase the breakdown of high-molecular-weight structural proteins, apparently by activating calcium-dependent proteases (calpains). The present experiments tested the possibility that calpain activity is modulated by polyamines. Spermine, spermidine, and putrescine all increased calcium-dependent proteolysis of [14C]casein by soluble fractions of rat brain. The order of potency was spermine greater than spermidine greater than putrescine, with apparent affinities of 30, 300, and 6,000 microM, respectively. Each of the three polyamines at physiological concentrations also potentiated the calcium-dependent breakdown of two endogenous high-molecular-weight structural proteins known to be substrates of calpain, in both supernatant and membrane fractions. The thiol protease inhibitor leupeptin, a known calpain inhibitor, also inhibited calcium-dependent proteolysis in the presence and absence of polyamines. The polyamines did not increase the activity of purified calpain I or calpain II determined with either [14C]casein or purified spectrin as the substrate, nor did they interfere with the inhibitory effects of calpastatin, an endogenous inhibitor of calpain. However, polyamines potentiated the stimulation of endogenous but not purified calpain activity produced by an endogenous calpain activator. These results suggest a role for polyamines in protein degradation as well as protein synthesis.     

Effect of glucocorticoid administration on the rate of muscle protein breakdown in vivo in rats, as measured by urinary excretion of Nτ-methylhistidine

The role of glucocorticoids in regulating the rate of muscle protein breakdown was evaluated by measuring excretion of N(tau)-methylhistidine during administration of various doses of corticosterone to adrenalectomized rats. Groups of rats received daily subcutaneous injections of 0, 0.2, 0.5, 1.0, 5.0 or 10.0mg of corticosterone/day per 100g body wt. for 7 days, followed by 3 days without hormone treatment, after which they were killed. A group with intact adrenal glands served as an additional control. All animals were pair-fed with the untreated adrenalectomized group. No significant differences were noted in growth rate or N(tau)-methylhistidine excretion between the intact or adrenalectomized control groups, or those given 0.2, 0.5 and 1.0mg of corticosterone, whereas growth ceased and N(tau)-methylhistidine excretion rose markedly in the groups receiving 5 and 10mg of corticosterone. After these two high doses of corticosterone, but not after lower doses, there was a loss of weight of the gastrocnemius muscle per 100g of final body wt., but not of the soleus and extensor digitorum longus muscles. The two highest doses of corticosterone also resulted in an increase in liver weight per 100g of final body wt. Lower doses of corticosterone did not cause these changes. Plasma corticosterone concentrations, measured on the final day of injection and again at the time of killing, were decreased to near zero by adrenalectomy and were little raised by doses of 0.2 and 0.5mg daily, but were increased to within the normal range by the 1mg dose. At 5 and 10mg doses, plasma corticosterone concentrations were sustained at 2-3 times those of intact rats, and thus in the range reported for rats exposed to severe stress. Rats given 5 and 10mg doses of corticosterone had glycosuria, and showed considerably elevated concentrations of insulin in the plasma. It is concluded that plasma concentrations of glucocorticoids within the normal range do not regulate the rate of muscle protein breakdown, whereas excessive plasma concentrations of corticosteroids, equivalent to those observed in severe stress, can accelerate muscle protein breakdown.     

Phosphorylation Modulates Calpain-Mediated Proteolysis and Calmodulin Binding of the 200-kDa and 160-kDa Neurofilament Proteins

Abstract: The effects of enzymatic dephosphorylation on neurofilament interaction with two calcium-binding proteins, calpain and calmodulin, were examined. Dephosphorylation increased the rate and extent of 200-kDa neurofilament protein proteolysis by calpain. In contrast, dephosphorylation of the 160-kDa neurofilament protein did not alter the rate or extent of calpain proteolysis. However, the calpain-induced breakdown products of native and dephosphorylated 160-kDa neurofilament protein were different. Dephosphorylation did not change the proteolytic rate, extent, or breakdown products of the 68-kDa neurofilament protein. Calmodulin binding to the purified individual 160- and 200-kDa neurofilament proteins was increased following dephosphorylation. These results suggest that phosphorylation may regulate the metabolism and function of neurofilaments by modulating interactions with the calcium-activated proteins calpain and calmodulin.     

Experimental hyperthyroidism in rats increases the expression of the ubiquitin ligases atrogin‐1 and MuRF1 and stimulates multiple proteolytic pathways in skeletal muscle

Muscle wasting is commonly seen in patients with hyperthyroidism and is mainly caused by stimulated muscle proteolysis. Loss of muscle mass in several catabolic conditions is associated with increased expression of the muscle‐specific ubiquitin ligases atrogin‐1 and MuRF1 but it is not known if atrogin‐1 and MuRF1 are upregulated in hyperthyroidism. In addition, it is not known if thyroid hormone increases the activity of proteolytic mechanisms other than the ubiquitin–proteasome pathway. We tested the hypotheses that experimental hyperthyroidism in rats, induced by daily intraperitoneal injections of 100 µg/100 g body weight of triiodothyronine (T3), upregulates the expression of atrogin‐1 and MuRF1 in skeletal muscle and stimulates lysosomal, including cathepsin L, calpain‐, and caspase‐3‐dependent protein breakdown in addition to proteasome‐dependent protein breakdown. Treatment of rats with T3 for 3 days resulted in an approximately twofold increase in atrogin‐1 and MuRF1 mRNA levels. The same treatment increased proteasome‐, cathepsin L‐, and calpain‐dependent proteolytic rates by approximately 40% but did not influence caspase‐3‐dependent proteolysis. The expression of atrogin‐1 and MuRF1 remained elevated during a more prolonged period (7 days) of T3 treatment. The results provide support for a role of the ubiquitin–proteasome pathway in muscle wasting during hyperthyroidism and suggest that other proteolytic pathways as well may be activated in the hyperthyroid state. J. Cell. Biochem. 108: 963–973, 2009. © 2009 Wiley‐Liss, Inc.     

Changes in Brain Protease Activity in Aging

Abstract: We measured changes in protease activity with aging, conducting assays of cathepsin D and calpain II activities and the rate of degradation of cytoskeletal proteins, preparing the enzymes and substrates from young and aged brains. Calpain preparations added to the young and to the aged substrates were standardized with casein as substrate so that age-related changes in calpain specificity and substrate susceptibility were measured. Several age-related differences were observed in substrate susceptibility and in enzyme activity. With respect to substrate, the neurofilament protein from young animals was somewhat more susceptible to calpain action than that from older animals. With respect to enzyme activity, calpain from aged brain cleaved neurofilament protein at a faster rate than did calpain from young. With neurofilaments, the most rapid breakdown usually occurred when enzyme from aged tissue was incubated with substrate from young. Kidney enzyme of aged rats incubated with neurofilament substrate of aged rats resulted in a more rapid breakdown than enzyme of young kidney incubated with substrate of young. The age dependence of tubulin breakdown was somewhat different from that of neurofilament breakdown. The most rapid breakdown usually occurred when using enzyme from young with tubulin from young. Incubation of neurofilament protein or tubulin with cathepsin D did not reveal any differences with aging. These studies suggest that an increase in enzyme activity observed previously during aging may also include changes in the properties of the enzyme (substrate specificity) and/or in the properties of their endogenous substrates (susceptibility to breakdown).     

Aerobic exercise training upregulates skeletal muscle calpain and ubiquitin-proteasome systems in healthy mice

Aerobic exercise training (AET) is an important mechanical stimulus that modulates skeletal muscle protein turnover, leading to structural rearrangement. Since the ubiquitin-proteasome system (UPS) and calpain system are major proteolytic pathways involved in protein turnover, we aimed to investigate the effects of intensity-controlled AET on the skeletal muscle UPS and calpain system and their association to training-induced structural adaptations. Long-lasting effects of AET were studied in C57BL/6J mice after 2 or 8 wk of AET. Plantaris cross-sectional area (CSA) and capillarization were assessed by myosin ATPase staining. mRNA and protein expression levels of main components of the UPS and calpain system were evaluated in plantaris by real-time PCR and Western immunoblotting, respectively. No proteolytic system activation was observed after 2 wk of AET. Eight weeks of AET resulted in improved running capacity, plantaris capillarization, and CSA. Muscle RING finger-1 mRNA expression was increased in 8-wk-trained mice. Accordingly, elevated 26S proteasome activity was observed in the 8-wk-trained group, without accumulation of ubiquitinated or carbonylated proteins. In addition, calpain abundance was increased by 8 wk of AET, whereas no difference was observed in its endogenous inhibitor calpastatin. Taken together, our findings indicate that skeletal muscle enhancements, as evidenced by increased running capacity, plantaris capillarization, and CSA, occurred in spite of the upregulated UPS and calpain system, suggesting that overactivation of skeletal muscle proteolytic systems is not restricted to atrophying states. Our data provide evidence for the contribution of the UPS and calpain system to metabolic turnover of myofibrillar proteins and skeletal muscle adaptations to AET.     

In vivo effect of a beta-adrenergic agonist on activity of calcium-dependent proteinases, their specific inhibitor, and cathepsins B and H in skeletal muscle

DEAE-Sephacel and phenyl-Sepharose chromatography were compared as methods for separating and quantitatively isolating calpain I, calpain II, and calpastatin from lamb muscle extracts. DEAE-Sephacel chromatography gave greater than 90% recovery of all three proteins, while phenyl-Sepharose gave only 70, 66, and 48% of the DEAE recovery of calpain I, calpain II, and calpastatin, respectively. Additionally, DEAE-Sephacel chromatography was shown to effectively separate calpastatin and calpain I. Consequently DEAE-Sephacel appears to be superior to phenyl-Sepharose for quantitative isolation of the components of the calcium-dependent proteinase system from muscle extracts. Dietary administration of beta-agonist (L-644, 969; Merck Sharpe & Dohme Research Laboratories) decreases extractable calpain I activity in lamb longissimus dorsi (LD) muscle by 10-14% (P less than 0.05), increases calpain II activity by 34-42% (P less than 0.001), and increases calpastatin activity by 59-75% (P less than 0.001). Additionally, net cathepsin B activity is reduced by 30% (P less than 0.05) in the LD of beta-agonist-treated lambs. Reduced activity of the calcium-dependent or catheptic proteinase systems may contribute to the increased protein accretion in muscles of beta-agonist-treated lambs.