Total Calpain Activity Research Articles

Caveolin-3 regulates the activity of Ca2+/calmodulin-dependent protein kinase II in C2C12 cells.

Caveolins, encoded by the Cav gene family, are the main components of caveolae. Caveolin-3 (Cav3) is specifically expressed in muscle cells. Mutations in Cav3 are responsible for a group of muscle diseases called caveolinopathies, and Cav3 deficiency is associated with sarcolemmal membrane alterations, disorganization of T-tubules, and disruption of specific cell-signaling pathways. However, Cav3 overexpression increases the number of sarcolemmal caveolae and muscular dystrophy-like regenerating muscle fibers with central nuclei, suggesting that the alteration of Cav3 expression levels or localization influences muscle cell functions. Here, we used mouse C2C12 myoblasts in which Cav3 expression was suppressed with short hairpin RNA and found that Cav3 suppression impaired myotube differentiation without affecting the expression of MyoD and Myog. We also observed an increase of intracellular Ca2+ levels, total calpain activity, and Ca2+-dependent calmodulin kinase II (CaMKII) levels in Cav3-depleted myoblasts. Importantly, those phenotypes due to Cav3 suppression were caused by the ryanodine receptor activation. Furthermore, pharmacological inhibition of CaMKII rescued the impairment of myoblast differentiation due to Cav3 knockdown. Our results suggest that Cav3 regulates intracellular Ca2+ concentrations by modulating ryanodine receptor activity in muscle cells and that CaMKII suppression in muscle could be a novel therapy for caveolinopathies.

Capn4 aggravates angiotensin II-induced cardiac hypertrophy by activating the IGF-AKT signalling pathway.

Capn4 belongs to a family of calpains that participate in a wide variety of biological functions, but little is known about the role of Capn4 in cardiac disease. Here, we show that the expression of Capn4 was significantly increased in Angiotensin II (Ang II)-treated cardiomyocytes and Ang II-induced cardiac hypertrophic mouse hearts. Importantly, in agreement with the Capn4 expression patterns, the maximal calpain activity measured in heart homogenates was elevated in Ang II-treated mice and oral coadministration of SNJ-1945 (calpain inhibitor) attenuated the total calpain activity measured in vitro. Functional assays indicated that overexpression of Capn4 obviously aggravated Ang II-induced cardiac hypertrophy, whereas Capn4 knockdown resulted in the opposite phenotypes. Further investigation demonstrated that Capn4 maintained the activation of the insulin-like growth factor (IGF)-AKT signalling pathway in cardiomyocytes by increasing c-Jun expression. Mechanistic investigations revealed that Capn4 directly bound and stabilized c-Jun and knockdown of Capn4 increased the ubiquitination level of c-Jun in cardiomyocytes. Additionally, our results demonstrated that the antihypertrophic effect of Capn4 silencing was partially dependent on the inhibition of c-Jun. Overall, these data suggested that Capn4 contributes to cardiac hypertrophy by enhancing the c-Jun-mediated IGF-AKT signalling pathway and could be a potential therapeutic target for hypertrophic cardiomyopathy.

A potential role of calpains in sulfonylureas (SUs) –mediated death of human pancreatic cancer cells (1.2B4)

Sulfonylureas (SUs) are suggested to accelerate the pancreatic β-cells mass loss via apoptosis. However, little is known whether calpains mediate this process. The aim of the present study is to evaluate the involvement of calpains in SUs-induced death of human pancreatic cancer (PC) cell line 1.2B4. The cells were exposed to: glibenclamide, glimepiride and gliclazide for 72 h. The expression analysis of caspase-3 (CASP-3), TP53, calpain 1 (CAPN-1), calpain 2 (CAPN-2) and calpain 10 (CAPN-10) was detected using RT-PCR method. Intracellular Ca2+ concentrations, CASP-3 activity and total calpain activity were also evaluated. Our results have shown that glibenclamide and glimepiride decrease 1.2B4 cells viability with accompanied increase in intracellular Ca2+ concentration and increased expression of apoptosis-related CASP-3 and TP53. Gliclazide did not affect 1.2B4 cell viability and Ca2+ concentration, however, it downregulated CASP-3 and upregulated TP53. Interestingly, 50 μM glimepiride increased expression of CAPN-1, CAPN-2 and CAPN-10 whereas 50 μM glibenclamide solely upregulated CAPN-2 expression. We have shown that 10 μM and 50 μM glibenclamide and glimepiride increased the activity of CASP-3, but decreased total calpain activity. Our results suggest that calpains may be involved in glibenclamide- and glimepiride-induced death of PC cells. However, further investigation is required to confirm the engagement of calpains in SUs-mediated death of PC cells, especially studies on protein level of particular isoforms of calpains should be conducted.

CELL MOTILITY PROTEINS IN ENDOMETRIUM CARCINOMA AND ENDOMETRIAL HYPERPLASIA: ASSOCIATION WITH CANCER RISK

Introduction. Proteins associated with cellular motility are known to play an important role in invasion and metastasis of cancer, however there is no evidence of their association with the development of malignant tumors including endometrial cancer (EC).The aim of the present study was to investigate the levels of actin-binding proteins, p45-Ser-β-catenin, and calpain activity in endometrial hyperplasia and in EC.Material and Methods. Total calpain activity, p45-Ser β-catenin, Arp3, gelsolin, cofillin and thymosin β-4 levels were evaluated in 43 postmenopausal patients with stage I–II endometrioid EC and 40 endometrial hyperplasia patients. Flow cytometry and Western blotting were used for expression determination of p45 Ser β-catenin and actin-biding proteins. Total calpain activity was estimated by fluorimetric method.Results. Levels of cofilin-1, thymosin β-4 and calpain activity were higher in cancer tissues than in endometrial hyperplasia. Cofilin-1 and thymosin β-4 levels were associated with the depth of myometrial invasion. The thymosin β-4 expression was correlated with the presence of tumor cervical invasion. Revealed correlations between the actin-binding proteins, p45-Ser-β-catenin and total calpain activity in endometrial hyperplasia tissue, but not in the tissue of cancer, is evidence of the involvement of these proteases in regulation of cell migration in endometrial hyperplasia. Levels of thymosin β-4, cofilin and total calpain activity are independent cancer risk factors in patients with endometrial hyperplasia.Conclusion. The level of actin-binding proteins as well as the total calpain activity were enhanced in endometrium carcinoma tissues compared to endometrial hyperplasia. The levels of thymosinβ-4, cofilin and total calpain activity in endometrial hyperplasia tissues are associated with a hyperplasia transition to cancer and may be considered as predictive biomarkers.

Differences in Titin and Nebulin Gene Expression in Skeletal Muscles of Rats Chronically Alcoholized by Different Methods

This work studied the changes in the levels of the main proteins of the calpain system (μ-calpain, Ca^(2+)-dependent protease, and fragments of its autolysis, inhibitor calpastatin) and μ-calpain substrates (giant proteins of the sarcomere cytoskeleton, titin and nebulin) in skeletal muscle (m. gastrocnemius, m. soleus, m. longissimus dorsi) of rats alcoholized for three months by different methods using agar containing 30% ethanol and nutrient-balanced liquid feed containing 5% ethanol using gel electrophoresis methods under denaturing conditions and immunoblotting. No decrease in the muscle mass/body weight ratio, indicating the development of atrophy, no increase in autolysis of μ-calpain, indicating an increase in the activity of this enzyme, no changes in the content of intact titin (T1), nebulin, μ-calpain and calpastatin, as well as the total calpain activity measured using Calpain Activity Assay Kit were detected in alcoholized rats of both groups. No changes in the total level of titin phosphorylation in the rat muscles of alcoholized groups were detected using Pro-Q Diamond fluorescent dye for phosphate groups of proteins. No statistically significant differences in the content of titin and nebulin mRNA in skeletal muscles of control rats and rats alcoholized using agar were detected. In rats, alcoholized by the method of liquid feed, the levels of titin and nebulin mRNA were increased 1.5-2.5 times possibly due to a higher fat content in such a diet. The presented data may be useful for choosing a chronic alcoholization model for animals.

Reduced SNAP25 Protein Fragmentation Contributes to SNARE Complex Dysregulation in Schizophrenia Postmortem Brain

Recent studies associated schizophrenia with enhanced functionality of the presynaptic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. Altered degradation pathways of the three core SNARE proteins: synaptosomal-associated protein 25 (SNAP25), syntaxin-1 and vesicle-associated membrane protein (VAMP) could contribute to enhanced complex function. To investigate these pathways, we first identified a 15-kDa SNAP25 fragment (f-S25) in human and rat brains, highly enriched in synaptosomal extractions, and mainly attached to cytosolic membranes with low hydrophobicity. The presence of f-S25 is consistent with reports of calpain-mediated SNAP25 cleavage. Co-immunoprecipitation assays showed that f-S25 retains the ability to bind syntaxin-1, which might prevent VAMP and/or Munc18-1 assembly into the complex. Quantitative analyses in postmortem human orbitofrontal cortex (OFC) revealed that schizophrenia (n = 35), but not major depression (n = 15), is associated with lower amounts of f-S25 (−37%, P = 0.027), and greater SNARE protein–protein interactions (35%, P < 0.001), compared with healthy matched controls (n = 28). Enhanced SNARE complex formation was strongly correlated with lower SNAP25 fragmentation rates (R = 0.563, P < 0.001). Statistical mediation analyses supported the hypothesis that reduced f-S25 density could upregulate SNARE fusion events in schizophrenia. Cortical calpain activity in schizophrenia did not differ from controls. f-S25 levels did not correlate with total calpain activity, indicating that if present, schizophrenia-related calpain dysfunction might occur locally at the presynaptic terminals. Overall, the present findings suggest the existence of an endogenous SNARE complex inhibitor related to SNAP25 proteolysis, associated with enhanced SNARE activity in schizophrenia.

Chronic alcohol intoxication is not accompanied by an increase in calpain proteolytic activity in cardiac muscle of rats

Enzymatic activity of Ca2+-dependent calpain proteases as well as the content and gene expression ofμ-calpain (activated by micromolar calcium ion concentrations), calpastatin (inhibitor of calpains), and titin (substrate for calpains) were investigated in cardiac muscles of rats subjected to chronic alcoholization for 3 and 6 months. There was no increase in the "heart weight/body weight" parameter indicating development of heart hypertrophy in the alcoholized rats, while a decreasing trend was observed for this parameter in the rats after 6-month modeling of alcoholic cardiomyopathy, which indicated development of atrophic changes in the myocardium. Fluorometric measurements conducted using the Calpain Activity Assay Kit did not reveal any changes in total calpain activity in protein extracts of cardiac muscles of the rats alcoholized for 3 and 6 months. Western blot analysis did not show reliable changes in the contents ofμ-calpain and calpastatin, and SDS-PAGE did not reveal any decrease in the titin content in the myocardium of rats after the chronic alcohol intoxication. Autolysis ofμ-calpain was also not verified, which could indicate that proteolytic activity of this enzyme in myocardium of chronically alcoholized rats is not enhanced. Using Pro-Q Diamond staining, changes in phosphorylation level of titin were not detected in cardiac muscle of rats after chronic alcoholization during three and six months.A decrease inµ-calpain and calpastatin mRNA content (~1.3-fold, p ≤ 0.01 and ~1.9-fold, p ≤ 0.01, respectively) in the myocardium of rats alcoholized for 3 months and decrease in calpastatin mRNA (~1.4-fold, p ≤ 0.01) in animals alcoholized for 6 months was demonstrated using real-time PCR. These results indicate negative effect of chronic alcohol intoxication on expression of the abovementioned genes.

Chymotrypsin-Like Activity of Proteasomes and Total Calpain Activity in Gastric and Colorectal Cancer

Chymotrypsin-like activity of proteasomes and total calpain activity were studied in patients with stage T2-4N0-3M0 gastric cancer and stage T2-4N0-2M0-1 colorectal cancer. Activities of proteasomes and calpains in gastric and colorectal cancer tissues were higher than in the corresponding normal tissues. Changes in activities of proteasomes and calpains were mutually related. The appearance of lymphogenic metastases in gastric cancer was associated with the increase in calpain activity. The progress of colorectal cancer and development of lymphogenic and hematogenic metastases were associated with elevated chymotrypsin-like activity of proteasomes.

Expression and activity of proteases in metastasis of ovarian cancer

The total chymotrypsin-like activity of proteasornes, the activity of 20S- and 26S-proteasome pools and calpains, and the expression of metalloproteinase PAPP-A in primary tumors and metastasized tissues were studied in 13 patients with epithelial ovarian cancer. It was shown that initiation of the process of tumor dissemination occurs against the background of active proteolytic processes; A decrease in activity of 26S-proteasomes and total calpain activity and increased expression ofmetalloproteinase PAPP-A in the primary tu mors were found in patients with ascites as compared with patients without ascites. The disease progression after treatment and achieved stabilization were found in patients with decreased activity of intracellular proteases and a high content of PAPP-A in the primary tumors.

Calpain-2 Compensation Promotes Angiotensin II-Induced Ascending and Abdominal Aortic Aneurysms in Calpain-1 Deficient Mice

Background and ObjectiveRecently, we demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development.Methodology/ResultsTo investigate the relative contribution of calpain-1 and -2 in development of AngII-induced AAs, male LDLr −/− mice that were either calpain-1 +/+ or −/− were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no significant effect on body weight or blood pressure during AngII infusion. Moreover, calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AAs. Interestingly, AngII infusion induced increased expression of calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410, a calpain inhibitor, along with AngII-infusion significantly attenuated AngII-induced ascending and abdominal AA formation in both calpain-1 +/+ and −/− mice as compared to vehicle administered mice. Furthermore, BDA-410 administration attenuated AngII-induced aortic medial hypertrophy and macrophage accumulation. Western blot and immunostaining analyses revealed BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A, an actin binding cytoskeletal protein in aorta.ConclusionCalpain-2 compensates for loss of calpain-1, and both calpain isoforms are involved in AngII-induced aortic aneurysm formation in mice.

Abstract 516: Calpain-2 Compensation Promotes Angiotensin II-induced Ascending and Abdominal Aortic Aneurysms in Calpain-1 Deficient Mice

Background and Objective We demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development. Methods and Results Male LDLr-/- mice that were either calpain-1 +/+ or -/- were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no effect on body weight or blood pressure during AngII infusion. Intimal areas of ascending aorta and maximum external width of the suprarenal abdominal aorta were measured by en face. Calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AA development (P = NS). Interestingly, AngII infusion induced increased expression of calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410 along with AngII infusion significantly attenuated AngII-induced ascending (Vehicle - +/+: 15.0 ± 1.0, -/-: 14.9 ± 0.8; BDA: +/+: 11.5 ± 0.6, -/-: 12.1 ± 0.6 mm 2 ; P&lt;0.05) and abdominal (Vehicle - +/+: 1.7 ± 0.1, -/-: 1.6 ± 0.1; BDA: +/+: 1.0 ± 0.1, -/-: 1.1 ± 0.1 mm; P&lt;0.05) AA formation in both calpain-1 +/+ and -/- mice as compared to vehicle administered mice. Western blot and immunostaining analyses revealed BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A (P&lt;0.05), an actin binding cytoskeletal protein, and medial macrophage accumulation in aorta. Conclusion Calpain-2 compensates for loss of calpain-1, and promotes AngII-induced ascending and abdominal AAs in calpain-1 deficient mice.

Effects of Medium‐Voltage Electrical Stimulation on Postmortem Changes in Fat‐Tailed Sheep

Effects of different medium-voltage electrical stimulation (ES) and ageing on postmortem changes in longissimus dorsi muscle of the fat-tailed sheep were studied. Fifteen male animals were divided into 5 equal groups (n= 3) including: T₁ (control, without ES), T₂ (100 V/30 s), T₃ (100 V/60 s), T₄ (150 V/30 s), and T₅ (150 V/60 s) with fixed frequency of 50 Hz. Five minutes after sticking, the carcasses were stimulated in order of the treatments. After normal processing, they were kept at 6 °C for 14 d. ES accelerated the glycolytic rate resulting in a significant fast fall in pH (P < 0.05) during the 1st 6-h postmortem (PM) with a gradual decline until 24-h PM, and a simultaneous significant reduction in adenosine triphosphate (ATP) content (P < 0.05). There was a significant (P < 0.05) decline in the total calpain activity during the 1st 6-h PM. The muscles from ES carcasses had significantly (P < 0.05) lower water holding capacity (WHC) than those from nonstimulated ones. Ageing revealed a significant (P < 0.05) effect on the reduction of WHC. No significant difference was found for the mean value of the muscle color (L*, a*, and b*) in all treatment groups during ageing (P > 0.05). The results of free amino acid (FAA) content and myofibrillar fragmentation index (MFI) revealed a significant improvement of proteolysis and tenderness by ES and ageing (P < 0.05). In the present study, higher voltage/duration (150 V/60 s) showed greater effects and significantly accelerated glycolysis, pH decline, and ATP depletion and thus decreased the time for rigor completion and improved the tenderness.

Time course in calpain activity and autolysis in slow and fast skeletal muscle during clenbuterol treatment

Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that β2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4mg·kg-1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9days of treatment, while hypertrophy was observed only in EDL after 9days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.

Calpain Inhibitor SNJ-1945 Attenuates Events Prior to Angiogenesis in Cultured Human Retinal Endothelial Cells

Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and microvascular permeability. VEGF-induced cytoskeletal reorganization plays a crucial role in angiogenesis. Cytoskeletal organization in endothelial cells is regulated by calpain proteases (EC 3.4.22.17). Calpains are a family of 14 calcium-regulated, intracellular cysteine proteases, which modulate cellular functions by limited, specific proteolysis. Calpain 1 (mu-calpain) and calpain 2 (m-calpain) are the 2 major typical calpain isoforms and are responsible for most calpain activity in endothelial cells. The purpose of the present study was to determine if an orally available form of calpain inhibitor, SNJ-1945, prevented angiogenesis induced by VEGF in cultured retinal endothelial cells. Human retinal microvascular endothelial cells (HRMEC) were incubated with VEGF (60-100 ng/mL) for 24 h. Calcium uptake was measured with Fluo8. Total calpain activity was measured using fluorescent-labeled casein substrate, and separate activities for calpains 1 and 2 were assessed by casein zymography. Proteolysis of endogenous calpain substrate alpha-spectrin in situ was analyzed by immunoblotting. Angiogenesis in vitro was evaluated by measuring cell migration and tube formation into Matrigel. Incubation of HRMEC with VEGF resulted in calcium uptake, increased activity of mainly calpain 2, and increased calpain proteolysis of alpha-spectrin. Treatment of endothelial cells with calpain inhibitor SNJ-1945 reversed VEGF-mediated tube formation and cell motility. Inhibition of angiogenesis by specific calpain inhibitor in the presence of VEGF supported our hypothesis that calpains may be involved in VEGF-mediated angiogenesis in retinal endothelial cells. Therefore, manipulating calpain activity by calpain inhibitor SNJ-1945 might provide a promising therapy for management of pathological angiogenesis, such as that occurring in proliferative retinopathy and age-related macular degeneration with neovascularization.

Comparative studies on calpain activity of different muscles of cattle, camel, sheep and goat

Summary Tenderness is the single most important factor influencing consumer acceptance of meat. The calpain proteolytic system is known to be responsible for the post-mortem tenderization of meat. The purpose of this study was to determine and compare the tensile strength and total calpain activities in different muscles of camel, cattle, sheep and goat. In camels, the effect of age and sex of animal was also studied. Twenty-four animals (camel, cattle, sheep and goat) were sampled randomly after slaughtering. Samples from biceps femoris, longissimus dorsi and triceps brachii and heart were obtained from each animal. The tensile strength was calculated using an Instron Universal testing machine. After homogenization of samples in 0.1 M NaCl and centrifugation, total calpain activity was determined by fluorometric method. Despite significant differences in tensile strength, no significant difference (P>0.05) was observed among calpain activities of different muscles in each species. Inter-species differences however, were significant (P 0.05). In camel, adult animals had higher calpain activity and tensile strength than youngs; sex had no significant effects. Correlation coefficients of calpain activity and tensile strength were negative and not significant in all species. In conclusion, meats from different species might show different degrees of tenderness, partly due to the difference in their calpain activity.

Early activation and redistribution of calpain activity in skeletal muscle during hindlimb unweighting and reweighting

The aims of this study were the following: (i) to determine whether activation of the Ca2+-activated protease, calpain, is an early event during hindlimb unweighting (HU) in skeletal muscle; and (ii) to assess whether calpain activity is greater during reweighting compared with HU alone. Rats were exposed to 12, 24, and 72 h, or 9 d of HU, followed by reweighting for 0, 12, or 24 h. Calpain activities were assayed for total, soluble, and particulate fractions. Total calpain activity was increased in the soleus at all HU time points, whereas activities were elevated in the gastrocnemius only after 9 d of HU. With reweighting, calpain activity remained elevated at all time points for both muscles. In general, reweighting the gastrocnemius increased its calpain activity more than during HU only, whereas reweighting the soleus did not produce additional increases in its calpain activity. The increases in calpain activity were associated with a proportional increase in activity of the particulate (membrane- and protein-associated) fraction. The results suggest that calpain activation is an early event during HU in the soleus, and that the increases in calpain activity in both muscles are associated with a redistribution of activity from cytosolic to particulate fractions.

40. Increased Transgene Expression of Dystrophin in mdx Muscle by RNAi-Mediated Silencing of Calpain Expression

Top of pageAbstract Plasmid DNA-based dystrophin gene transfer can be a way to cure Duchenne muscular dystrophy (DMD) at the molecular level. However, there are several technical difficulties in DMD gene therapy. Although intravascular delivery of naked plasmid DNA solves some of the problems associated with the delivery of dystrophin gene to widespread muscle cells, a marked increase in the expression level of dystrophin may be required for effective DMD gene therapy. Several studies have reported that the cellular free calcium is increased in dystrophic muscle and this increase activates calcium-dependent proteases, calpains. Because calpains degrade dystrophin, the silencing of calpain expression in dystrophic muscle cells may improve transgene expression of dystrophin. In this study, we used RNA interference (RNAi) to silence calpain expression in muscle cells. To this end, we constructed plasmid DNA expressing small interfering RNA (siRNA) targeting one of three isoforms of calpain, under the control of U6 promoter (psiCalp). Cationic liposome-mediated transfection of psiCalp targeting |[mu]|- calpain, m-calpain or p94 (calpain 3) resulted in significant reduction in the corresponding mRNA level in C2C12 myoblasts. The reduction in the mRNA level was associated with the reduced calpain activity in the cells. Total calpain activity in the gastrocnemius muscle of mdx mice (3- to 8-weeks-old) was found to be significantly greater than that of control (C57BL/10) mice. Intramuscular injection of 10|[sim]|100 |[mu]|g psiCalp into mdx muscle reduced the calpain activity. Then, plasmid DNA encoding a human micro-dystrophin cDNA (CS1, 4.9 kb), pCAG-CS1, was injected into the gastrocnemius muscle of 3-weeks-old mdx mice. Intramuscular injection of naked pCAG-CS1 resulted in significant, but low expression of the microdystrophin. On the other hand, co-injection of psiCalp greatly increased the expression of the micro-dystrophin, which was confirmed by immunostaining and immunoblot analysis. These results indicate that RNAi-mediated silencing of calpain expression is effective in increasing transgene expression of micro-dystrophin in mdx muscle. Although the silencing might be transient, the restoration of (micro-)dystrophin expression could inhibit aberrant activation of calpains in muscle cells.

Evaluation of conditions for calpain inhibition in the rat spinal cord: effective postinjury inhibition with intraspinal MDL28170 microinjection.

Calpains (calcium-activated cysteine proteases) are strongly implicated in the secondary damage that follows contusion injury to the spinal cord. Calpains are activated within a few minutes following injury and their elevated activity persists for 24 h, thereby providing a reasonable window of opportunity for postinjury inhibition. Previous studies demonstrated decreased axonal damage and neurofilament proteolysis with postinjury intravenous administration of relatively low concentrations of the calpain inhibitors leupeptin, E-64-D, and calpeptin. We sought to determine if conditions under which calpain inhibitors were administered in previous studies resulted in effective calpain inhibition, and to identify conditions that result in significant calpain inhibition following spinal cord injury. Contusive spinal cord injury was produced in female Long-Evans rats using the NYU impactor at the 12.5-25-mm height setting. The results demonstrate that intravenous administration of 1 mg/kg E-64-D or 250 micro g/kg calpeptin does not inhibit total calpain activity in the rat spinal cord, measured using a BODIPY-FL labeled casein assay. Intravenous administration of MDL28170 (20 mg/kg) resulted in mild calpain inhibition and a modest decrease in the proteolysis of calpain substrates alpha-spectrin and MAP2. Intraspinal microinjection of 50 nmoles/19 micro g MDL28170, either 30 min prior to or 20 min following contusion injury, resulted in a more robust inhibition of total calpain activity and greater attenuation of alpha-spectrin breakdown and MAP2 proteolysis. The decreased proteolysis persisted 24 h postinjury. Together, the results demonstrate that direct microinjection of the calpain inhibitor MDL28170 is more effective than intravenous infusion in reducing calpain activity and decreasing the injury-induced proteolysis of calpain substrates.

Activation of Ca(2+)-dependent proteolysis in skeletal muscle and heart in cancer cachexia.

Cachexia is a syndrome characterized by profound tissue wasting that frequently complicates malignancies. In a cancer cachexia model we have shown that protein depletion in the skeletal muscle, which is a prominent feature of the syndrome, is mostly due to enhanced proteolysis. There is consensus on the views that the ubiquitin/proteasome pathway plays an important role in such metabolic response and that cytotoxic cytokines such as TNFα are involved in its triggering (Costelli and Baccino, 2000), yet the mechanisms by which the relevant extracellular signals are transduced into protein hypercatabolism are largely unknown. Moreover, little information is presently available as to the possible involvement in muscle protein waste of the Ca2+-dependent proteolysis, which may provide a rapidly activated system in response to the extracellular signals. In the present work we have evaluated the status of the Ca2+-dependent proteolytic system in the gastrocnemius muscle of AH-130 tumour-bearing rats by assaying the activity of calpain as well as the levels of calpastatin, the natural calpain inhibitor, and of the 130 kDa Ca2+-ATPase, both of which are known calpain substrates. After tumour transplantation, total calpastatin activity progressively declined, while total calpain activity remained unchanged, resulting in a progressively increasing unbalance in the calpain/calpastatin ratio. A decrease was also observed for the 130 kDa plasma membrane form of Ca2+-ATPase, while there was no change in the level of the 90 kDa sarcoplasmic Ca2+-ATPase, which is resistant to the action of calpain. Decreased levels of both calpastatin and 130 kDa Ca2+-ATPase have been also detected in the heart of the tumour-bearers. These observations strongly suggest that Ca2+-dependent proteolysis was activated in the skeletal muscle and heart of tumour-bearing animals and raise the possibility that such activation may play a role in sparking off the muscle protein hypercatabolic response that characterizes cancer cachexia. © 2001 Cancer Research Campaignhttp://www.bjcancer.com

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.