calpastatin and Hypertrophy

Hypertrophic scars, the most common complication of burn injuries, are characterized by excessive deposition of fibroblast-derived extracellular matrix proteins. Calpain, a calcium-dependent protease, is involved in the fibroblast proliferation and extracellular matrix production observed in certain fibrotic diseases. However, its role in the formation of post-burn hypertrophic skin scars remains largely unknown. Here, calpain expression and activity were assessed in skin fibroblasts obtained directly from patients with third-degree burns, who consequently developed post-burn hypertrophic scars. Furthermore, the antifibrotic effect of calpastatin, an endogenous calpain inhibitor, was evaluated in human fibroblasts and a murine burn model. The activity, mRNA levels, and protein levels of calpain were markedly higher in fibroblasts from the burn wounds of patients than in normal cells. Selective calpain inhibition by calpastatin markedly reduced not only the proliferation of burn-wound fibroblasts but also the mRNA and protein expression of calpain, transforming growth factor-beta 1, α-smooth muscle actin, type I and type III collagens, fibronectin, and vimentin in burn-wound fibroblasts. The anti-scarring effects of calpastatin were validated using a murine burn model by molecular, histological, and visual analyses. This study demonstrates the pathological role of calpain and the antifibrotic effect of calpastatin via calpain inhibition in post-burn hypertrophic scar formation.

Topics: Adult; Animals; Burns; Calcium-Binding Proteins; Calpain; Cell Proliferation; Cicatrix, Hypertrophic; Collagen Type III; Extracellular Matrix; Extracellular Matrix Proteins; Female; Fibroblasts; Fibronectins; Humans; Hypertrophy; Male; Mice; Middle Aged; RNA, Messenger; Skin; Transforming Growth Factor beta1; Young Adult

Accumulating evidence suggests that the calpain/calpastatin system is involved in skeletal muscle remodelling induced by β(2) -adrenoceptor agonist treatment. In addition to other pathways, the Akt/mammalian target of rapamycin (mTOR) pathway, controlling protein synthesis, and the calcium/calmodulin-dependent protein kinase 2 (CamK2) and AMP-activated protein kinase (AMPK) pathways, recently identified as calpain substrates, could be relevant in β(2) -adrenoceptor agonist-induced skeletal muscle remodelling. In the present study we investigated muscle hypertrophy and phenotypic shifts, as well as the molecular response of components of the Akt/mTOR pathway (i.e. Akt, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), ribosomal protein S6 (rpS6), CamK2 and AMPK), in response to calpastatin overexpression in the skeletal muscle of mice treated with 1 mg/kg per day clenbuterol for 21 days. Using gene electrotransfer of a calpastatin expression vector into the tibialis anterior of adult mice, we found that calpastatin overexpression attenuates muscle hypertrophy and phenotypic shifts induced by clenbuterol treatment. At the molecular level, calpastatin overexpression markedly decreased calpain activity, but was ineffective in altering the phosphorylation of Akt, 4E-BP1 and rpS6. In contrast, calpastatin overexpression increased the protein expression of both total AMPK and total CamK2. In conclusion, the results support the contention that the calpain/calpastatin system plays a crucial role in skeletal muscle hypertrophy and phenotypic shifts under chronic clenbuterol treatment, with AMPK and CamK2 probably playing a minor role. Moreover, the calpastatin-induced inhibition of hypertrophy under clenbuterol treatment was not related to a decreased mTOR-dependent initiation of protein translation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Clenbuterol; Gene Expression Regulation; Hypertrophy; Male; Mice; Mice, Inbred CBA; Muscle, Skeletal; Phenotype

In hypertension, angiotensin (Ang) II is a critical mediator of cardiovascular remodeling, whose prominent features include myocardial and vascular media hypertrophy, perivascular inflammation, and fibrosis. The signaling pathways responsible for these alterations are not completely understood. Here, we investigated the importance of calpains, calcium-dependent cysteine proteases. We generated transgenic mice constitutively expressing high levels of calpastatin, a calpain-specific inhibitor. Chronic infusion of Ang II led to similar increases in systolic blood pressure in wild-type and transgenic mice. In contrast, compared with wild-type mice, transgenic mice displayed a marked blunting of Ang II-induced hypertrophy of left ventricle. Ang II-dependent vascular remodeling, ie, media hypertrophy and perivascular inflammation and fibrosis, was also limited in both large arteries (aorta) and small kidney arteries from transgenic mice as compared with wild type. In vitro experiments using vascular smooth muscle cells showed that calpastatin transgene expression blunted calpain activation by Ang II through epidermal growth factor receptor transactivation. In vivo and in vitro models of inflammation showed that impaired recruitment of mononuclear cells in transgenic mice was attributable to a decrease in both the release of and the chemotactic response to monocyte chemoattractant protein-1. Finally, results from collagen synthesis assay and zymography suggested that limited fibrogenesis was attributable to a decrease in collagen deposition rather than an increase in collagen degradation. These results indicate a critical role for calpains as downstream mediators in Ang II-induced cardiovascular remodeling and, thus, highlight an attractive therapeutic target.

Topics: Angiotensin II; Animals; Aorta; Blood Pressure; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Disease Models, Animal; Fibrosis; Genetic Therapy; Hypertension; Hypertrophy; Hypertrophy, Left Ventricular; Inflammation; Infusion Pumps, Implantable; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Myocardium; NF-kappa B; NFATC Transcription Factors; Renal Artery; Time Factors; Ventricular Remodeling

Changes in muscle growth, calpastatin activity, and tenderness of three muscles were assessed in 20 callipyge and 20 normal wether lambs slaughtered at live weights (LW) of 7, 20, 36, 52, and 69 kg. At 24 h postmortem, the longissimus (LM), semimembranosus (SM), and supraspinatus (SS) muscles were removed and weighed and samples were obtained for calpastatin activity (CA; 24 h) and Warner-Bratzler shear force (WBS; aged 6 d). For muscle weights and calpastatin activity, the weight group x muscle x phenotype interaction was significant (P < 0.05). Muscle weights were similar (P > 0.05) between phenotypes for all three muscles at 7 kg LW. At 20 kg LW, the LM and SM muscles from the callipyge lambs were heavier (P < 0.05) than those from normal lambs; however, the SS did not differ (P > 0.05) between phenotypes at 7, 20, or 52 kg. From 20 to 69 kg LW, the LM and SM weights were 42 and 49% heavier (P < 0.05) for callipyge than for normal lambs. Calpastatin activity of the callipyge LM was greater (P < 0.05) than that of normal LM at 36, 52, and 69 kg. In the callipyge LM, CA was similar (P > 0.05) at 20, 36, and 52 kg LW and did not differ (P > 0.05) from 7-kg or 69-kg values. Calpastatin activity declined (P < 0.05) across the growth curve for the SM and SS, but values were higher (P < 0.05) in the SM in callipyge than in normal lambs. Shear force values of the LM were lower (P < 0.05) for normal lambs at 36, 52, and 69 kg LW than for callipyge lambs. In the SM and SS, WBS values decreased (P < 0.05) across the growth curve, but values were higher (P < 0.05) for callipyge lambs in the SM only. These data indicate that the selective muscular hypertrophy of the callipyge phenotype develops during the postnatal growth period between 7 and 20 kg LW (19 and 100 d of age). Longissimus and semimembranosus muscles in the callipyge lambs were over 40% heavier from 20 to 69 kg LW; however, they also had higher levels of calpastatin activity and Warner-Bratzler shear force during this time period, indicating the need for postmortem tenderization treatments to improve palatability.

Topics: Age Factors; Animals; Body Composition; Calcium-Binding Proteins; Hypertrophy; Male; Meat; Muscle Development; Muscular Diseases; Organ Size; Phenotype; Sheep; Sheep Diseases

The present experiment was conducted to determine the effect of the callipyge phenotype on traits affecting muscle growth and meat tenderness. Dorset wethers (N = 40) that were either carriers or non-carriers were fed grain and slaughtered at 169 d of age. Callipyge phenotype did not affect (P > .05) slaughter weight, hot carcass weight, or weights of the heart, spleen, viscera, kidney-pelvic fat, head, and pelt; however, callipyge lambs had a higher dressing percentage and lighter lungs, liver, and kidneys (P < .01). Callipyge lambs had reduced fat thickness and marbling score and higher leg scores and longissimus area (34%). Adductor (30%), biceps femoris (42%), gluteus group (31%), longissimus (32%), psoas group (20%), quadriceps femoris (18%), semimembranosus (38%), and semitendinosus (26%) weights were higher in the callipyge phenotype (P < .01); however, phenotype did not affect (P > .05) weights of infraspinatus or supraspinatus. Longissimus pH and temperature declines were not affected (P > .05) by phenotype. Longissimus myofibril fragmentation index was lower at 1 (27%), 7 (35%), and 21 (37%) d postmortem and Warner-Bratzler shear force was higher at 1, 7, and 21 d postmortem in the callipyge phenotype (P < .01). Shear force values of callipyge lambs at 21 d postmortem tended to be greater (P = .12) than shear force values of non-carriers at 1 d postmortem . Activities of calpastatin (83%) and m-calpain (45%) were higher in the callipyge (P < .01); however mu-calpain activity was not affected (P > .05). Longissimus and semitendinosus RNA concentration, DNA content, RNA content, protein content, and the RNA:DNA ratio were higher (P < .05), but DNA concentration, protein concentration, and protein:DNA were not affected in the callipyge phenotype. The higher calpastatin activity associated with callipyge suggests that protein degradation may be reduced in the live animal. Additionally, the increased muscle DNA content associated with the callipyge phenotype suggests an increase in satellite cell proliferation, and results in an increased capacity of skeletal muscle to accumulate and maintain myofibrillar protein. These results suggests that both reduced rate of protein degradation and higher capacity for protein synthesis are consequences of the callipyge condition.

Topics: Actinin; Animals; Blotting, Western; Body Weight; Calcium-Binding Proteins; Calpain; Connectin; Desmin; Hydrogen-Ion Concentration; Hypertrophy; Male; Meat; Muscle Development; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Nucleic Acids; Phenotype; Protein Kinases; Sheep; Sheep Diseases; Temperature; Troponin

To examine the effect of a beta-adrenergic agonist (BAA) on muscle growth, proteinase activities, and postmortem proteolysis, 16 wether lambs were randomly assigned to receive 0 or 4 ppm of L644,969 in a completely mixed high-concentrate diet for 6 wk. Weight of the biceps femoris was 18.6% heavier in treated lambs. At 0 h after slaughter, treated lambs had higher cathepsin B (35.6%), cathepsins B + L (19.1%), calpastatin (62.8%), and m-calpain (24.6%) than control lambs, but both groups had similar mu-calpain activities. In both longissimus and biceps femoris muscles, treated lambs had higher protein and RNA and lower DNA concentrations. However, total DNA was not affected, indicating that the increase in muscle mass was probably due to muscle hypertrophy rather than to hyperplasia. The pattern of postmortem proteolysis was significantly altered by BAA feeding. In treated lambs, postmortem storage had no effect on the myofibril fragmentation index and degradation of desmin and troponin-T. These results indicate that the ability of the muscle to undergo postmortem proteolysis has been dramatically reduced with BAA feeding. Similar proteolytic systems are thought to be involved in antemortem and postmortem degradation of myofibrillar proteins, so BAA-mediated protein accretion is probably due, at least in part, to reduced protein degradation. To examine whether protein synthesis was altered with BAA feeding, the level of skeletal muscle alpha-actin mRNA was quantified. Longissimus muscle alpha-actin mRNA abundance was 30% greater in BAA-fed lambs. Collectively, these results indicate that dietary administration of BAA increases muscle mass through hypertrophy and that the increase in muscle protein accretion is due to reduced degradation and possibly to increased synthesis of muscle proteins.

Topics: Actins; Adrenergic beta-Agonists; Animals; Calcium-Binding Proteins; Calpain; Cathepsins; DNA; Endopeptidases; Hypertrophy; Least-Squares Analysis; Male; Meat; Muscle Development; Muscle Proteins; Muscles; Postmortem Changes; Pyridines; Random Allocation; RNA; RNA, Messenger; Sheep; Weight Gain