Specific Calpain Inhibitor Research Articles

Brain injury‐induced proteolysis is reduced in a novel calpastatin‐overexpressing transgenic mouse

The calpain family of calcium-dependent proteases has been implicated in a variety of diseases and neurodegenerative pathologies. Prolonged activation of calpains results in proteolysis of numerous cellular substrates including cytoskeletal components and membrane receptors, contributing to cell demise despite coincident expression of calpastatin, the specific inhibitor of calpains. Pharmacological and gene-knockout strategies have targeted calpains to determine their contribution to neurodegenerative pathology; however, limitations associated with treatment paradigms, drug specificity, and genetic disruptions have produced inconsistent results and complicated interpretation. Specific, targeted calpain inhibition achieved by enhancing endogenous calpastatin levels offers unique advantages in studying pathological calpain activation. We have characterized a novel calpastatin-overexpressing transgenic mouse model, demonstrating a substantial increase in calpastatin expression within nervous system and peripheral tissues and associated reduction in protease activity. Experimental activation of calpains via traumatic brain injury resulted in cleavage of α-spectrin, collapsin response mediator protein-2, and voltage-gated sodium channel, critical proteins for the maintenance of neuronal structure and function. Calpastatin overexpression significantly attenuated calpain-mediated proteolysis of these selected substrates acutely following severe controlled cortical impact injury, but with no effect on acute hippocampal neurodegeneration. Augmenting calpastatin levels may be an effective method for calpain inhibition in traumatic brain injury and neurodegenerative disorders.

Calpain cleaves methionine aminopeptidase-2 in a rat model of ischemia/reperfusion

Ischemic stroke results in multiple injurious signals within a cell including dysregulation of calcium homeostasis. Consequently, there is an increase in the enzymatic activity of the calpains, calcium dependent proteases that are thought to contribute to neuronal injury. In addition, cellular stress due to ischemia/reperfusion also triggers a decrease in protein translation through activation of the unfolded protein response (UPR). In the present study we found that methionine aminopeptidase 2 (MetAP2), a critical component of the translation initiation complex, is a calpain substrate. In vitro calpain assays demonstrated that while MetAP2 has autoproteolytic activity, calpain also produces a stable proteolytic fragment at 50kDa using recombinant MetAP2. This 50kDa fragment, in addition to a 57kDa fragment was present in in vitro digestions of rat brain homogenates. Production of these fragments was inhibited by calpastatin, the endogenous and specific inhibitor of calpain. Using an in vivo middle cerebral artery occlusion (MCAO) model only the 57kDa fragment of MetAP2 was observed. These data suggest that calpain activation in stroke may regulate MetAP2-mediated protein translation giving calpains a larger role in the cellular stress response than previously determined.

Development of α-helical calpain probes by mimicking a natural protein-protein interaction.

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs will be useful for future investigation of calpains, while the cross-linking technique will enable exploration of other protein-protein interactions.

μ-CALPAIN MEDIATES MYOCARDIAL APOPTOSIS DURING ISCHAEMIA-REPERFUSION VIA MITOCHONDRIAL PERMEABILITY TRANSITION PORE OPENING

ObjectivesCalpains have been implicated in myocardial ischaemia-reperfusion (I/R) injury. The mitochondrial permeability transition pore (mPTP) subsequently triggers apoptotic cell death during I/R. However, the mechanistic link among calpain activity, mPTP...

RVG-Mediated Calpain2 Gene Silencing in the Brain Impairs Learning and Memory

In the central nervous system, two calpain isoforms are highly expressed: calpain1 and calpain2. Here, we show for the first time that activation of the calpain isoform, calpain2, is a necessary event in hippocampal synaptic plasticity and in learning and memory. We developed a fluorescence resonance energy transfer-based animal model to monitor in vivo calpain activation in single cells and in real time. Additionally, utilizing a novel rabies virus glycoprotein-chimeric peptide, which enabled the transvascular delivery of small interfering RNA to the brain against calpain2, we down-regulated the calpain2 isoform in vivo. Calpain2 gene silencing eliminated long-term potentiation and impaired learning and memory. Our results not only identify the calpain2 isoform as a critical mediator in learning and memory but also highlight an innovative, highly efficient calpain2-targeting peptide capable of isoform-specific gene silencing in the brain. We anticipate these innovative technologies and our better understanding of the calpain machinery, particularly of the calpain2 isoform, will have substantial influence on future translational studies, attracting considerable interest in the use of calpain models and calpain-specific inhibitors in the development of therapeutics.

N‐terminal cleavage fragment of focal adhesion kinase is required to activate the survival signalling pathway in cultured myoblasts under oxidative stress

We have previously shown that the cultured L6 myoblasts are susceptible to menadione-induced oxidative stress. Damaged cells were detached from the culture dishes. In the present study, we focused on focal adhesion kinase (FAK), which plays pivotal roles in maintaining focal adhesion function and cell survival. FAK, normally localized at the focal adhesion regions of the myoblasts, was not observed at the regions under oxidative stress induced by menadione and H(2) O(2) . Two cleavage products, 80-kDa N-terminal FAK and 35-kDa C-terminal FAK fragments, as well as full-length FAK (125 kDa) were detected in myoblasts cultured under normal conditions by western blotting with anti-N-terminal FAK or anti-C-terminal FAK sera. Of interest was the finding that the cleavage products of FAK (but not full-length FAK) disappeared under oxidative stress. The cleavage of full-length FAK to N-terminal FAK and C-terminal FAK was inhibited by calpeptin, a specific calpain inhibitor. In addition, pre-incubation of cells with calpeptin resulted in a sharp decrease in survival signals, such as Akt phosphorylation and the ratio of Bcl-2/Bax, under stress conditions. By contrast, not only relative viability, but also Akt phosphorylation and the ratio of Bcl-2/Bax was significantly improved when cells were transfected with a DNA construct of N-terminal FAK-Myc. These results suggest that the N-terminal FAK positively regulates survival signalling in early phases of oxidative stress in the cultured myoblasts.

Advanced glycation end‐products induce calpain‐mediated degradation of ezrin

Advanced glycation end-products (AGEs) are important mediators of diabetic complications via incompletely understood pathways. AGEs bind to intracellular ERM proteins (ezrin, radixin and moesin) that modulate cell shape, motility, adhesion and signal transduction. AGEs bind to the N-terminal domain of ezrin but not full-length ezrin. The AGE binding site may be made accessible either by proteolysis releasing an N-terminal fragment or ezrin activation by phosphorylation. Increased intracellular calcium is a primary event in cell activation by high glucose or AGEs. Calpain activity is increased concomitantly, and ezrin is a calpain substrate. The present study assessed whether glycated proteins affect ezrin cleavage and activation in renal tubule epithelial cells. After 7 days, AGE-BSA decreased ezrin levels in MDCK renal tubular cells to 66 ± 4% of control. AGE-RNAse, ribosylated fetal bovine serum and methylglyoxal-BSA all had similar effects. The AGE-BSA-induced decrease in ezrin was abolished by calpastatin peptide, a specific calpain inhibitor, and 1,2-bis-aminophenoxyethane-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a calcium chelator. Ezrin breakdown products were increased in AGE-BSA-treated cells, with a main fragment of ∼ 43 kDa. In vitro, calpain 1 cleaved recombinant human ezrin, generating breakdown fragments including an N-terminal fragment of ∼ 43 kDa. Studies with ezrin mutants showed that non-phosphorylated ezrin was more susceptible to calpain cleavage. AGE-BSA decreased phosphorylated ERM levels to 31 ± 12% in MDCK cells. Thus, AGE-BSA promotes calpain-mediated proteolysis of ezrin in MDCK cells by both increasing calpain activity and reducing phosphorylation. Therapies targeting both glycated proteins and calpain may provide protection against diabetic complications.

High Molecular Weight Calmodulin-Binding Protein: 20 Years Onwards—A Potential Therapeutic Calpain Inhibitor

Apoptosis in cardiovascular diseases is considered to be a major reason for heart failure. Caspase-independent apoptosis due to calpains and other proteases occurs due to increase in intracellular Ca(2+) levels which act on a feed-forward mechanism. Calpains are Ca(2+)-activated cysteine proteases present in the cytosol as inactive proenzymes. Calpastatin is most efficient and specific calpain inhibitor present in vivo. Earlier, we had reported the expression of novel high molecular weight calmodulin-binding protein (HMWCaMBP) in human and animal cardiac tissue and in very minute quantities in brains and lungs. HMWCaMBP showed calpastatin activity and was also found to be highly homologous to calpastatin I and calpastatin II. Decreased expression of HMWCaMBP was observed during ischemia as it is susceptible to proteolysis by calpains during ischemia-reperfusion. In normal myocardium, HMWCaMBP may protect its substrate from calpains. However, during an early stage of ischemia/reperfusion due to increased Ca(2+) influx, calpain activity often exceeds HMWCaMBP activity. This leads to proteolysis of HMWCaMBP and other protein substrates, resulting in cellular damage. The role of HMWCaMBP in ischemia/reperfusion is yet to be elucidated. The present review summarizes the developments in area of HMWCaMBP from the authors' laboratory and its potential for therapy.

Calpastatin overexpression limits calpain-mediated proteolysis and behavioral deficits following traumatic brain injury

Traumatic brain injury (TBI) results in abrupt, initial cell damage leading to delayed neuronal death. The calcium-activated proteases, calpains, are known to contribute to this secondary neurodegenerative cascade. Although the specific inhibitor of calpains, calpastatin, is present within neurons, normal levels of calpastatin are unable to fully prevent the damaging proteolytic activity of calpains after injury. In this study, increased calpastatin expression was achieved using transgenic mice that overexpress the human calpastatin (hCAST) construct under control of a calcium–calmodulin-dependent kinase II α promoter. Naïve hCAST transgenic mice exhibited enhanced neuronal calpastatin expression and significantly reduced protease activity. Acute calpain-mediated spectrin proteolysis in the cortex and hippocampus induced by controlled cortical impact brain injury was significantly attenuated in calpastatin overexpressing mice. Aspects of posttraumatic motor and cognitive behavioral deficits were also lessened in hCAST transgenic mice compared to their wildtype littermates. However, volumetric analyses of neocortical contusion revealed no histological neuroprotection at either acute or long-term time points. Partial hippocampal neuroprotection observed at a moderate injury severity was lost after severe TBI. This study underscores the effectiveness of calpastatin overexpression in reducing calpain-mediated proteolysis and behavioral impairment after TBI, supporting the therapeutic potential for calpain inhibition. In addition, the reduction in spectrin proteolysis without accompanied neocortical neuroprotection suggests the involvement of other factors that are critical for neuronal survival after contusion brain injury.

The tyrosine phosphatase HD-PTP (PTPN23) is degraded by calpains in a calcium-dependent manner

HD-PTP (PTPN23) is a non-transmembrane protein tyrosine phosphatase which contributes to the signal transduction pathways involved in the regulation of cell migration and invasion. We here demonstrate in T24 bladder carcinoma cells that HD-PTP undergoes calcium-dependent degradation which can be prevented by specific calpain inhibitors. In addition, treatment of the cells with the calpain inhibitor calpeptin results in the redistribution of endogenous HD-PTP to the periphery of the cells.Since (i) calpains are overexpressed in some tumors and (ii) the downregulation of HD-PTP enhances cell migration and invasion, we propose that HD-PTP degradation by calpains might result in the acquisition of a more aggressive phenotype in neoplastic cells.

English

Calpastatin (CAST) is a specific inhibitor of calpain (calcium-dependent cysteine protease). This study investigated the potential for variation in yak (Bos grennies) CAST. PCR-SSCP analysis of exon 6 of yak CAST revealed three unique patterns (named A-C). Se- quencing of the amplicons revealed two nucleotide substitutions. One substitution (c.398G/C) would nominally change the amino acid se- quence (p.S133T) of yak calpastatin. The variant sequence A which carried c.398C was the most common in the yaks tested (95.1%). This is the first report that found yak CAST is variable, and as in pigs, sheep and cattle, this variation may affect animal production traits.

Elevated calpain activity in acute myelogenous leukemia correlates with decreased calpastatin expression

Calpains are intracellular cysteine proteases that have crucial roles in many physiological and pathological processes. Elevated calpain activity has been associated with many pathological states. Calpain inhibition can be protective or lethal depending on the context. Previous work has shown that c-myc transformation regulates calpain activity by suppressing calpastatin, the endogenous negative regulator of calpain. Here, we have investigated calpain activity in primary acute myelogenous leukemia (AML) blast cells. Calpain activity was heterogeneous and greatly elevated over a wide range in AML blast cells, with no correlation to FAB classification. Activity was particularly elevated in the CD34+CD38− enriched fraction compared with the CD34+CD38+ fraction. Treatment of the cells with the specific calpain inhibitor, PD150606, induced significant apoptosis in AML blast cells but not in normal equivalent cells. Sensitivity to calpain inhibition correlated with calpain activity and preferentially targeted CD34+CD38− cells. There was no correlation between calpain activity and p-ERK levels, suggesting the ras pathway may not be a major contributor to calpain activity in AML. A significant negative correlation existed between calpain activity and calpastatin, suggesting calpastatin is the major regulator of activity in these cells. Analysis of previously published microarray data from a variety of AML patients demonstrated a significant negative correlation between calpastatin and c-myc expression. Patients who achieved a complete remission had significantly lower calpain activity than those who had no response to treatment. Taken together, these results demonstrate elevated calpain activity in AML, anti-leukemic activity of calpain inhibition and prognostic potential of calpain activity measurement.

Calpain Inhibition Attenuates Angiotensin II–induced Abdominal Aortic Aneurysms and Atherosclerosis in Low-density Lipoprotein Receptor–deficient Mice

Chronic infusion of angiotensin II (AngII) augments atherosclerosis and abdominal aortic aneurysm (AAA) formation in hypercholesterolemic mice. AngII-induced AAAs are associated with medial macrophage accumulation and matrix metalloproteinase (MMP) activation. Inhibition of calpain, a calcium-activated neutral cysteine protease, by overexpression of its endogenous inhibitor, calpastatin, attenuates AngII-induced leukocyte infiltration, perivascular inflammation, and MMP activation in mice. The purpose of this study was to define whether pharmacological inhibition of calpain influences AngII-induced AAAs in hypercholesterolemic mice. Male low-density lipoprotein receptor-/- mice were fed a fat-enriched diet and administered with either vehicle or a calpain-specific inhibitor, BDA-410 (30 mg/kg per day) for 5 weeks. After 1 week of feeding, mice were infused with AngII (1000 ng/kg per minute) for 4 weeks. AngII-infusion profoundly increased aortic calpain protein and activity. BDA-410 administration had no effect on plasma cholesterol concentrations or AngII-increased systolic blood pressure. Calpain inhibition significantly attenuated AngII-induced AAA formation and atherosclerosis development. BDA-410 administration attenuated activation of MMP12, proinflammatory cytokines (IL-6, monocyte chemoattractant protein-1), and macrophage infiltration into the aorta. BDA-410 administration significantly attenuated thioglycolate-elicited macrophage accumulation in the peritoneal cavity. We conclude that calpain inhibition using BDA-410 attenuated AngII-induced AAA formation and atherosclerosis development in low-density lipoprotein receptor-/- mice.

Mechanistic involvement of the calpain‐calpastatin system in Alzheimer neuropathology

The mechanism by which amyloid-β peptide (Aβ) accumulation causes neurodegeneration in Alzheimer's disease (AD) remains unresolved. Given that Aβ perturbs calcium homeostasis in neurons, we investigated the possible involvement of calpain, a calcium-activated neutral protease. We first demonstrated close postsynaptic association of calpain activation with Aβ plaque formation in brains from both patients with AD and transgenic (Tg) mice overexpressing amyloid precursor protein (APP). Using a viral vector-based tracer, we then showed that axonal termini were dynamically misdirected to calpain activation-positive Aβ plaques. Consistently, cerebrospinal fluid from patients with AD contained a higher level of calpain-cleaved spectrin than that of controls. Genetic deficiency of calpastatin (CS), a calpain-specific inhibitor protein, augmented Aβ amyloidosis, tau phosphorylation, microgliosis, and somatodendritic dystrophy, and increased mortality in APP-Tg mice. In contrast, brain-specific CS overexpression had the opposite effect. These findings implicate that calpain activation plays a pivotal role in the Aβ-triggered pathological cascade, highlighting a target for pharmacological intervention in the treatment of AD.

Aberrant cyclin A expression and centrosome overduplication induced by hepatitis B virus Pre-S2 mutants and its implication in hepatocarcinogenesis

Ground glass hepatocytes harboring hepatitis B virus (HBV) pre-S2 mutants have been recognized as pre-neoplastic lesions of hepatocellular carcinoma (HCC). The pre-S2 mutants accumulated in endoplasmic reticulum (ER) can induce ER stress, upregulate cyclin A and promote hepatocyte proliferation. Notably, cyclin A was aberrantly detected in the cytoplasm, instead of nucleus, of pre-S2 mutant-transgenic mice livers, thereby raising the potential role of cytoplasmic cyclin A in HBV hepatocarcinogenesis. In this study, we confirmed that cyclin A was detected in the cytoplasm in the majority of HBV-related HCC tissues. In vitro, the pre-S2 mutant-initiated ER stress could induce cytoplasmic cyclin A mediated via cleavage by the calcium-dependent protease μ-calpain, resulting in an N-terminal truncated product which was preferentially located in the cytoplasm. The aberrant cyclin A expression subsequently induced centrosome overduplication, and this effect was abolished by calpain-specific inhibitors or RNA interference targeting to cyclin A. Overall, our data indicate that HBV pre-S2 mutant may elicit aberrant cyclin A expression and centrosome overduplication through ER stress induction and thereby represent a potential mechanism for the chromosome instability in HBV hepatocarcinogenesis.

Calpains Contribute to Vascular Repair in Rapidly Progressive Form of Glomerulonephritis: Potential Role of Their Externalization

Calpains, calcium-activated proteases, mediate the angiogenic signals of vascular endothelial growth factor. However, their involvement in vascular repair has not been investigated and the underlying mechanisms remain to be fully elucidated. A rapidly progressive form of glomerulonephritis in wild type and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor, was studied. Calpastatin transgene expression prevented the repair of peritubular capillaries and the recovery of renal function, limiting mouse survival. In vitro analysis detected a significant reduction of both intracellular and extracellular calpain activities in transgene expressing cells, whereas Western blotting revealed that proangiogenic factors vascular endothelial growth factor and norepinephrine increased calpain exteriorization. In vitro, extracellular calpains increased endothelial cell proliferation, migration and capillary tube formation. In vivo, delivery of nonpermeable extracellular calpastatin was sufficient to blunt angiogenesis and vascular repair. Endothelial cell response to extracellular calpains was associated with fibronectin cleavage, generating fibronectin fragments with proangiogenic capacity. In vivo, fibronectin cleavage was limited in the kidney of calpastatin transgenic mice with nephritis. This study demonstrates that externalized calpains participate in angiogenesis and vascular repair, partly by promoting fibronectin cleavage and thereby amplifying vascular endothelial growth factor efficiency. Thus, manipulation of calpain externalization may have therapeutic implications to control angiogenesis.

Overexpression of a minimal domain of calpastatin suppresses IL-6 production and Th17 development via reduced NF-κB and increased STAT5 signals.

Calpain, a calcium-dependent cysteine protease, is reportedly involved in the pathophysiology of autoimmune diseases such as rheumatoid arthritis (RA). In addition, autoantibodies against calpastatin, a natural and specific inhibitor of calpain, are widely observed in RA. We previously reported that E-64-d, a membrane-permeable cysteine protease inhibitor, is effective in treating experimental arthritis. However, the exact role of the calpastatin-calpain balance in primary inflammatory cells remains unclear. Here we investigated the effect of calpain-specific inhibition by overexpressing a minimal functional domain of calpastatin in primary helper T (Th) cells, primary fibroblasts from RA patients, and fibroblast cell lines. We found that the calpastatin-calpain balance varied during Th1, Th2, and Th17 development, and that overexpression of a minimal domain of calpastatin (by retroviral gene transduction) or the inhibition of calpain by E-64-d suppressed the production of IL-6 and IL-17 by Th cells and the production of IL-6 by fibroblasts. These suppressions were associated with reductions in RORγt expression and STAT3 phosphorylation. Furthermore, inhibiting calpain by silencing its small regulatory subunit (CPNS) suppressed Th17 development. We also confirmed that overexpressing a minimal domain of calpastatin suppressed IL-6 by reducing NF-κB signaling via the stabilization of IκBα, without affecting the upstream signal. Moreover, our findings indicated that calpastatin overexpression suppressed IL-17 production by Th cells by up-regulating the STAT5 signal. Finally, overexpression of a minimal domain of calpastatin suppressed IL-6 production efficiently in primary fibroblasts derived from the RA synovium. These findings suggest that inhibiting calpain by overexpressing a minimal domain of calpastatin could coordinately suppress proinflammatory activities, not only those of Th cells but also of synovial fibroblasts. Thus, this strategy may prove viable as a candidate treatment for inflammatory diseases such as RA.

New allelic variant of the ovine calpastatin gene

Calpastatin, a specific inhibitor of the calpains, participates in muscle growth and tenderization. Amplified fragment of 12 intron of CAST was used; the gene encoding calpastatin, from 164 lambs from synthetic lines for restriction fragment length polymorphism analysis. Although, CAST has previously been investigated in sheep, the observed polymorphism incidence in this population was higher. Furthermore, three previously identified alleles as well as one new allelic variant (allele “e”) have been detected. Eight genotypes were observed in this population. The results of this study may be useful in further research supporting associations between polymorphism in the ovine CAST gene on both the function and expression gene. Key words: Polymorphism, sheep, calpastatin, RFLP, meat quality.

Calpain activation is involved in acute manganese neurotoxicity in the rat striatum in vivo

Manganese is essential for life, yet chronic exposure to this metal can cause a neurodegenerative disease named manganism that affects motor function. In the present study we have evaluated Mn neurotoxicity after its administration in the rat striatum. The participation of the calcium-dependent protease calpain and the apoptosis-related protease caspase-3, in Mn-induced cell death was monitored in the striatum and globus pallidus. Mn induced the activation of both proteases, although calpain activation seems to be an earlier event. Moreover, while the broad-spectrum caspase inhibitor QVD did not significantly prevent Mn-induced cell death, the specific calpain inhibitor MDL-28170 did. The role of NMDA glutamate receptors on calpain activity was also investigated; blockage of these receptors by MK-801 and memantine did not prevent calpain activation, nor Mn-induced cell death. Finally, studies in striatal homogenates suggest a direct activation of calpain by Mn ions. Altogether the present study suggests that additional mechanisms to excitotoxicity are involved in Mn-induced cell death, placing calpain as an important mediator of acute Mn neurotoxicity in vivo.

Calpain-mediated dystrophin disruption may be a potential structural culprit behind chronic doxorubicin-induced cardiomyopathy

The critical importance of dystrophin to cardiomyocyte contraction and sarcolemmal and myofibers integrity, led us to test the hypothesis that dystrophin reduction/loss could be involved in the pathogenesis of doxorubicin-induced cardiomyopathy, in order to determine a possible specific structural culprit behind heart failure. Rats received total cumulative doses of doxorubicin during 2 weeks: 3.75, 7.5, and 15 mg/kg. Controls rats received saline. Fourteen days after the last injection, hearts were collected for light and electron microscopy, immunofluorescence and western blot. The cardiac function was evaluated 7 and 14 days after drug or saline. Additionally, dantrolene (5 mg/kg), a calcium-blocking agent that binds to cardiac ryanodine receptors, was administered to controls and doxorubicin-treated rats (15 mg/kg). This study offers novel and mechanistic data to clarify molecular events that occur in the myocardium in doxorubicin-induced chronic cardiomyopathy. Doxorubicin led to a marked reduction/loss in dystrophin membrane localization in cardiomyocytes and left ventricular dysfunction, which might constitute, in association with sarcomeric actin/myosin proteins disruption, the structural basis of doxorubicin-induced cardiac depression. Moreover, increased sarcolemmal permeability suggests functional impairment of the dystrophin-glycoprotein complex in cardiac myofibers and/or oxidative damage. Increased expression of calpain, a calcium-dependent protease, was markedly increased in cardiomyocytes of doxorubicin-treated rats. Dantrolene improved survival rate and preserved myocardial dystrophin, calpain levels and cardiac function, which supports the opinion that calpain mediates dystrophin loss and myofibrils degradation in doxorubicin-treated rats. Studies are needed to further elucidate this mechanism, mainly regarding specific calpain inhibitors, which may provide new interventional pathways to prevent doxorubicin-induced cardiomyopathy.