calpain and Cardiomegaly

Despite advances in its treatment, heart failure remains a major cause of morbidity and mortality, evidencing an urgent need for novel mechanism-based targets and strategies. Myocardial hypertrophy, caused by a wide variety of chronic stress stimuli, represents an independent risk factor for the development of heart failure, and its prevention constitutes a clinical objective. Recent studies performed in preclinical animal models support the contribution of the Ca

Topics: Animals; Calcium-Binding Proteins; Calpain; Cardiomegaly; Heart Failure

Despite many advances in percutaneous and surgical interventions in the treatment of coronary artery disease (CAD), up to one-third of patients are still either not candidates or receive suboptimal revascularization. Calpains are a class of calcium-activated non-lysosomal cysteine proteases that serve as a proteolytic unit for cellular homeostasis. Uncontrolled activation of calpain has been found to be involved in the pathogenesis of myocardial reperfusion injury, cardiac hypertrophy, myocardial stunning and cardiac ischemia. Inhibition of calpains has been shown to significantly attenuate myocardial stunning and reduced infarct size after ischemia-reperfusion. Calpain inhibition therefore serves as a potential medical therapy for patients suffering from a number of diseases, including CAD.

Topics: Animals; Calpain; Cardiomegaly; Coronary Artery Disease; Enzyme Activation; Humans; Myocardial Reperfusion Injury

Calpains are cytosolic calcium-activated cysteine proteases. Recently, they have been proposed to influence signal transduction processes leading to myocardial remodelling and heart failure. In this review, we will first describe some of these molecular mechanisms. Calpains may contribute to myocardial hypertrophy and inflammation, mainly through the activation of transcription factors such as NF-κB. They play an important role in the fibrosis process partly by activating transforming growth factor β. They are also implicated in cell death as they cause the breakdown of sarcolemma and sarcomeres. Nevertheless, a key to understanding the molecular basis of calpain-mediated myocardial remodelling likely lies in the identification of mechanisms involved in calpain secretion, since cytosolic and extracellular proteases would have different functions. Finally, we will provide an overview of the available evidence that calpains are indeed actively involved in the common causes of heart failure, including hypertension, diabetes, atherosclerosis, ischaemia-reperfusion, atrial fibrillation, congestive failure, and mechanical unloading.

Topics: Animals; Apoptosis; Calpain; Cardiomegaly; Fibrosis; Heart Failure; Humans; Inflammation; Necrosis; Transcription Factors; Ventricular Remodeling

Cardiac hypertrophy develops most commonly in response to hypertension and is an independent risk factor for the development of heart failure. The mechanisms by which cardiac hypertrophy may be reversed to reduce this risk have not been fully determined to the point where mechanism-specific therapies have been developed. Recently, proteases in the calpain family have been implicated in the regulation of the development of cardiac hypertrophy in preclinical animal models. In this review, we summarize the molecular mechanisms by which calpain inhibition has been shown to modulate the development of cardiac (specifically ventricular) hypertrophy. The context within which calpain inhibition might be developed for therapeutic intervention of cardiac hypertrophy is then discussed.

Topics: Animals; Calpain; Cardiomegaly; Cardiovascular Agents; Cysteine Proteinase Inhibitors; Disease Models, Animal; Drug Design; Heart Ventricles; Humans; Signal Transduction; Ventricular Remodeling

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.

Topics: Angiotensin II; Animals; Calpain; Cardiomegaly; Mice; Myocytes, Cardiac; Proto-Oncogene Proteins c-akt; Signal Transduction; Somatomedins

The protein levels and activities of calpain-1 and calpain-2 are increased in cardiac mitochondria under pathological conditions including ischemia, diabetes, and sepsis, and transgenic overexpression of mitochondrial-targeted calpain-1 induces dilated heart failure, which underscores an important role of increased calpain in mitochondria in mediating myocardial injury. However, it remains to be determined whether selective inhibition of calpain in mitochondria protects the heart under pathological conditions. In this study, we generated transgenic mice overexpressing mitochondrial-targeted calpastatin in cardiomyocytes. Their hearts were isolated and subjected to global ischemia/reperfusion. Hyperglycemia was induced in the transgenic mice by injections of STZ. We showed that transgenic calpastatin was expressed exclusively in mitochondria isolated from their hearts but not from other organs including skeletal muscle and lung tissues. Transgenic overexpression of mitochondrial-targeted calpastatin significantly attenuated mitochondrial oxidative stress and cell death induced by global ischemia/reperfusion in isolated hearts, and ameliorated mitochondrial oxidative stress, cell death, myocardial remodeling and dysfunction in STZ-treated transgenic mice. The protective effects of mitochondrial-targeted calpastatin were correlated with increased ATP5A1 protein expression and ATP synthase activity in isolated hearts subjected to global ischemia/reperfusion and hearts of STZ-treated transgenic mice. In cultured rat myoblast H9c2 cells, overexpression of mitochondrial-targeted calpastatin maintained the protein levels of ATP5A1 and ATP synthase activity, prevented mitochondrial ROS production and decreased cell death following hypoxia/reoxygenation, whereas upregulation of ATP5A1 or scavenging of mitochondrial ROS by mito-TEMPO abrogated mitochondrial ROS production and decreased cell death. These results confirm the role of calpain in myocardial injury, suggesting that selective inhibition of calpain in myocardial mitochondria by mitochondrial-targeted calpastatin is an effective strategy for alleviating myocardial injury and dysfunction in cardiac pathologies.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cardiomegaly; Diabetes Mellitus, Experimental; Fibrosis; Male; Mice, Transgenic; Mitochondria, Heart; Mitochondrial Proton-Translocating ATPases; Myocytes, Cardiac; Oxidative Stress; Reactive Oxygen Species

[Figure: see text].

Topics: Adrenergic beta-Agonists; Animals; Calcineurin; Calcium; Calcium Signaling; Calpain; Cardiomegaly; Homeostasis; Ion Channels; Isoproterenol; Mechanotransduction, Cellular; Mice; Mice, Knockout; Myocytes, Cardiac; Pyrazines; Rats; Thiadiazoles

Increased activity of calpain-1 and matrix metalloproteinase (MMP)-2 was observed in different models of arterial hypertension and contribute to thicken the left ventricle (LV) walls and to hypertrophy cardiac myocytes. MMP-2 activity may be regulated by calpain-1 via bioactive molecules activation such as transforming growth factor (TGF)-β in cardiovascular diseases. This study analyzed whether calpain-1 causes cardiac hypertrophy and dysfunction by modulating the expression and activity of MMP-2 in renovascular hypertension.. Male Wistar rats were submitted to two kidneys, one clip (2K1C) model of hypertension or sham surgery and were treated with verapamil (VRP, 8 mg/kg/bid) by gavage from the second to tenth week post-surgery. Systolic blood pressure (SBP) was weekly assessed by tail-cuff plethysmography and morphological and functional parameters of LV were analyzed by echocardiography. MMP-2 activity was analyzed by in situ and gelatin zymography, while calpain-1 activity by caseinolytic assay. MMP-2, calpain-1, TGF-β and MMP-14/TIMP-2 levels were identified in the LV by western blots. Fluorescence assays were performed to evaluate oxidative stress, MMP-2 and calpain-1 levels.. SBP increased in 2K1C rats and was unaltered by VRP. However, VRP notably ameliorated hypertension-induced increase in LV thickness. VRP decreased hypertension-induced enhances in calpain-1 and MMP-2 activities, oxidative stress and mature TGF-β levels. Treatment with VRP also decreased the accentuated MMP-14/TIMP-2 levels in 2K1C.. Treatment with VRP decreases calpain-1 and MMP-2 activities and also reduces TGF-β and MMP-14/TIMP-2 levels in the LV of hypertensive rats, thus contributing to ameliorate cardiac hypertrophy.

Topics: Animals; Calpain; Cardiomegaly; Gene Expression Regulation; Hypertension; Male; Matrix Metalloproteinase 2; Rats; Rats, Wistar; Vasodilator Agents; Ventricular Remodeling; Verapamil

Pathological cardiac hypertrophy is ultimately accompanied by cardiomyocyte apoptosis. Apoptosis mainly related to calpain-1-mediated apoptotic pathways. Studies had proved that taurine can maintain heart health through antioxidation and antiapoptotic functions, but the effect of taurine on cardiac hypertrophy is still unclear. This study aimed to determine whether taurine could inhibit calpain-1-mediated mitochondria-dependent apoptotic pathways in isoproterenol (ISO)-induced hypertrophic cardiomyocytes. We found that taurine could inhibit the increase in cell surface area and reduce the protein expression levels of the hypertrophic markers atrial natriuretic peptide, brain natriuretic polypeptide, and β-myosin heavy chain. Taurine also reduced ROS, intracellular Ca

Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Atrial Natriuretic Factor; bcl-2-Associated X Protein; Calcium; Calcium-Binding Proteins; Calpain; Cardiomegaly; Caspase 3; Caspase 9; Cell Line; Cytochromes c; Isoproterenol; Membrane Potential, Mitochondrial; Mitochondria; Myocytes, Cardiac; Natriuretic Peptide, Brain; Natriuretic Peptides; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Rats; Taurine; Ventricular Myosins

Heart failure (HF) is a functional lack of myocardial performance due to a loss of molecular control over increases in calcium and ROS, resulting in proteolytic degradative advances and cardiac remodeling. Mitochondria are the molecular powerhouse of cells, shifting the sphere of cardiomyocyte stability and performance. Functional mitochondria rely on the molecular abilities of safety factors such as TFAM to maintain physiological parameters. Mitochondrial transcription factor A (TFAM) creates a mitochondrial nucleoid structure around mtDNA, protecting it from mutation, inhibiting NFAT (ROS activator/hypertrophic stimulator), and transcriptionally activates Serca2a to decrease calcium mishandling. Calpain1 and MMP9 are proteolytic degratory factors that play a major role in cardiomyocyte decline in HF. Current literature depicts major decreases in TFAM as HF progresses. We aim to assess TFAM function against Calpain1 and MMP9 proteolytic activity and its role in cardiac remodeling. To this date, no publication has surfaced describing the effects of aortic banding (AB) as a surgical HF model in TFAM-TG mice. HF models were created via AB in TFAM transgenic (TFAM-TG) and C57BLJ-6 (WT) mice. Eight weeks post AB, functional analysis revealed a successful banding procedure, resulting in cardiac hypertrophy as observed via echocardiography. Pulse wave and color doppler show increased aortic flow rates as well as turbulent flow at the banding site. Preliminary results of cardiac tissue immuno-histochemistry of HF-control mice show decreased TFAM and compensatory increases in Serca2a fluorescent expression, along with increased Calpain1 and MMP9 expression. Protein, RNA, and IHC analysis will further assess TFAM-TG results post-banding. Echocardiography shows more cardiac stability and functionality in HF-induced TFAM-TG mice than the control counterpart. These findings complement our published in vitro results. Overall, this suggests that TFAM has molecular therapeutic potential to reduce protease expression.

Topics: Animals; Calpain; Cardiomegaly; Disease Models, Animal; DNA-Binding Proteins; Gene Expression Regulation; Heart Failure; High Mobility Group Proteins; Matrix Metalloproteinase 9; Mice; Mice, Transgenic; Myocardium; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Up-Regulation

BACKGROUND Simvastatin, an HMG-CoA reductase inhibitor, has been reported to exert multiple protective effects on the cardiovascular system. However, the molecular mechanism remains to be examined. The present study was designed to study the effects of simvastatin on cardiac hypertrophy in diabetic rats and to explore its potential mechanism. MATERIAL AND METHODS Sprague-Dawley rats were assigned into a control (Con) group, a streptozotocin (STZ) group, and a STZ+simvastatin (STZ+SIM) group. The level of reactive oxygen species (ROS) was measured by using dihydroethidium (DHE) staining. The protein expressions of p65, IκBα, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), calpain-1, and endothelial nitric oxide synthase (eNOS) were examined by Western blot analysis. qPCR was used to detect the levels of brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP). RESULTS Simvastatin improved the cardiac hypertrophy of diabetic rats, as demonstrated by decreases in the ratios of left ventricular weight/body weight (LVW/BW) and heart weight/body weight (HW/BW) and by the downregulation of mRNA expression of BNP and ANP in the heart tissue. Simvastatin decreased the protein expressions of VCAM-1, ICAM-1, IL-6, and TNF-α, increased eNOS protein expression, and limited an increase in ROS levels in the heart tissue. Simvastatin increased IkBa protein expression in cytoplasm and inhibited the translocation of p65, the subunit of nuclear factor-κB (NF-κB) to the nucleus from the cytoplasm of the heart tissue. Furthermore, simvastatin attenuated the activity of calpain and calpain-1 protein expression in heart tissue. CONCLUSIONS Simvastatin attenuates cardiac hypertrophy in diabetic rats, which might be due to the attenuation of oxidative stress and inflammation induced by calpain-1-mediated activation of NF-κB.

Topics: Animals; Calpain; Cardiomegaly; Diabetes Complications; Diabetes Mellitus, Experimental; Disease Models, Animal; Inflammation; Intercellular Adhesion Molecule-1; Male; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase Type III; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Simvastatin; Streptozocin; Vascular Cell Adhesion Molecule-1

Junctophilin-2 (JP2) is a structural protein required for normal excitation-contraction (E-C) coupling. After cardiac stress, JP2 is cleaved by the calcium ion-dependent protease calpain, which disrupts the E-C coupling ultrastructural machinery and drives heart failure progression. We found that stress-induced proteolysis of JP2 liberates an N-terminal fragment (JP2NT) that translocates to the nucleus, binds to genomic DNA, and controls expression of a spectrum of genes in cardiomyocytes. Transgenic overexpression of JP2NT in mice modifies the transcriptional profile, resulting in attenuated pathological remodeling in response to cardiac stress. Conversely, loss of nuclear JP2NT function accelerates stress-induced development of hypertrophy and heart failure in mutant mice. These data reveal a self-protective mechanism in failing cardiomyocytes that transduce mechanical information (E-C uncoupling) into salutary transcriptional reprogramming in the stressed heart.

Topics: Active Transport, Cell Nucleus; Adaptation, Physiological; Animals; Calpain; Cardiomegaly; Cell Nucleus; Excitation Contraction Coupling; Gene Expression Regulation; Humans; MEF2 Transcription Factors; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle Proteins; Myocardial Contraction; Myocytes, Cardiac; Proteolysis; Transcription, Genetic; Ventricular Remodeling

Calpain is activated following myocardial infarction and ablation of calpastatin (CAST), an endogenous inhibitor of calpains, promotes left ventricular remodeling after myocardial infarction (MI). The present study aimed to investigate the effect of transgenic over-expression of CAST on the post-infarction myocardial remodeling process.. We established transgenic mice (TG) ubiquitously over-expressing human CAST protein and produced MI in TG mice and C57BL/6J wild-type (WT) littermates.. The CAST protein expression was profoundly upregulated in the myocardial tissue of TG mice compared with WT littermates (P < 0.01). Overexpression of CAST significantly reduced the infarct size (P < 0.01) and blunted MI-induced interventricular hypertrophy, global myocardial fibrosis and collagen I and collagen III deposition, hypotension and hemodynamic disturbances at 21 days after MI. Moreover, the MI-induced up-regulation and activation of calpains were obviously attenuated in CAST TG mice. MI-induced down-regulation of CAST was partially reversed in TG mice. Additionally, the MI-caused imbalance of matrix metalloproteinases and their inhibitors was improved in TG mice.. Transgenic over-expression of CAST inhibits calpain activation and attenuates post-infarction myocardial remodeling.

Topics: Animals; Calpain; Cardiomegaly; Collagen Type I; Collagen Type III; Cytoskeletal Proteins; Disease Models, Animal; Enzyme Activation; Female; Fibrosis; Gene Expression Regulation; Humans; Isoenzymes; Male; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases, Secreted; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardial Infarction; Survival Analysis; Ventricular Function, Left; Ventricular Remodeling

Apoptosis of cardiomyocytes plays an important role in the transition from cardiac hypertrophy to heart failure. Hypertrophied cardiomyocytes show enhanced susceptibility to apoptosis. Therefore, the aim of this study was to determine the susceptibility to apoptosis and its mechanism in hypertrophied cardiomyocytes using a rat model of transverse abdominal aortic constriction (TAC). Sixteen weeks of TAC showed compensatory and pathological hypertrophy in the left ventricle. TUNEL-positive nuclei were significantly increased in TAC with angiotensin II (Ang II) treatment. Calpain inhibitor, PD150606, effectively inhibited Ang II-induced apoptosis of hypertrophied cardiomyocytes. Ang II increased nuclear translocation of intracellular Ca(2+) activated calpain-2 in hypertrophied cardiomyocytes. Ang II enhanced the interaction between activated calpain-2 and Ca(2+)/calmodulin-dependent protein kinase II δB (CaMKIIδB), and promoted the degradation of CaMKIIδB by calpain-2 in the nuclei of hypertrophied cardiomyocytes. Consequently, the depressed CaMKIIδB downregulated the expression of antiapoptotic Bcl-2 leading to mitochondrial depolarization and release of cytochrome c led to apoptosis of hypertrophied cardiomyocytes. In conclusion, hypertrophied cardiomyocytes show increased susceptibility to apoptosis during Ang II stimulation via nuclear calpain-2 and CaMKIIδB pathway.

Topics: Angiotensin II; Animals; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calpain; Cardiomegaly; Constriction, Pathologic; Gene Expression Regulation; Humans; Myocytes, Cardiac; Rats; Signal Transduction

Calpain-1 activation and oxidative stress are two critical factors contributing to apoptosis of hypertrophic cardiomyocyte. Astragaloside IV (ASIV) exhibits protective effect against various heart diseases. The present study was designed to investigate whether the inhibitory effect of ASIV on isoproterenol (ISO)-induced apoptosis of hypertrophic cardiomyocyte was associated with the anti-oxidation and calpain-1 inhibition. Hypertrophy, apoptosis, mitochondrial oxidative stress and calpain-1 expression were measured in the heart tissue of Sprague-Dawley (SD) rats and H9C2 cells treated with ISO alone or combination with ASIV. The results showed that ASIV attenuated apoptotic rate, increased Bcl-2 expression, decreased Bax expression, ameliorated the integrity of mitochondrial structure and improved mitochondrial membrane potential (MMP). Moreover, ASIV combination reduced both calpain-1 protein expression and calpain activity, down-regulated mitochondrial NOX4 (mito-NOX4) expression, increased activity of mitochondrial superoxide dismutase (mito-SOD) and mitochondrial catalase (mito-CAT) compared to ISO treated alone. The results suggested that ASIV exerted anti-apoptosis effect on ISO-induced hypertrophic cardiomyocyte by attenuating oxidative stress and calpain-1 activation.

Topics: 2-Propanol; Animals; Antioxidants; Apoptosis; Calpain; Cardiomegaly; Cardiotonic Agents; Cell Line; Gene Expression Regulation; Membrane Potential, Mitochondrial; Mitochondria; Myocytes, Cardiac; Oxidative Stress; Rats; Rats, Sprague-Dawley; Saponins; Triterpenes

The gene ankyrin repeat domain 1 (Ankrd1) is an enigmatic gene and may exert pleiotropic function dependent on its expression level, subcellular localization and even types of pathological stress, but it remains unclear how these factors influence the fate of cardiomyocytes. Here we attempted to investigate the role of CARP on cardiomyocyte hypertrophy. In neonatal rat ventricular cardiomyocytes (NRVCs), angiotensin II (Ang II) increased the expression of both calpain 1 and CARP, and also induced cytosolic translocation of CARP, which was abrogated by a calpain inhibitor. In the presence of Ang-II in NRVCs, infection with a recombinant adenovirus containing rat Ankrd1 cDNA (Ad-Ankrd1) enhanced myocyte hypertrophy, the upregulation of atrial natriuretic peptide and β-myosin heavy chain genes and calcineurin proteins as well as nuclear translocation of nuclear factor of activated T cells. Cyclosporin A attenuated Ad-Ankrd1-enhanced cardiomyocyte hypertrophy. Intra-myocardial injection of Ad-Ankrd1 in mice with transverse aortic constriction (TAC) markedly increased the cytosolic CARP level, the heart weight/body weight ratio, while short hairpin RNA targeting Ankrd1 inhibited TAC-induced hypertrophy. The expression of calcineurin was also significantly increased in Ad-Ankrd1-infected TAC mice. Olmesartan (an Ang II receptor antagonist) prevented the upregulation of CARP in both Ang II-stimulated NRVCs and hearts with pressure overload. These findings indicate that overexpression of Ankrd1 exacerbates pathological cardiac remodeling through the enhancement of cytosolic translocation of CARP and upregulation of calcineurin.

Topics: Adenoviridae; Angiotensin II; Animals; Animals, Newborn; Aorta; Atrial Natriuretic Factor; Calcineurin; Calpain; Cardiomegaly; Constriction, Pathologic; Cyclosporine; Gene Expression Regulation; Genetic Vectors; Glycoproteins; Imidazoles; Mice; Muscle Proteins; Myocytes, Cardiac; Myosin Heavy Chains; Nuclear Proteins; Primary Cell Culture; Protein Transport; Rats; Repressor Proteins; RNA, Small Interfering; Signal Transduction; Tetrazoles

Topics: Animals; Calpain; Cardiomegaly; Heart Ventricles; Humans; Ventricular Remodeling

Although cardiac hypertrophy initially ensues as a compensatory mechanism, it often culminates in congestive heart failure. Based on our earlier studies that calpain and beta3 integrin play cell death and survival roles, respectively, during pressure-overload (PO) hypertrophy, we investigated if the loss of beta3 integrin signaling is a potential mechanism for calpain-mediated cardiomyocyte death during PO. beta3 Integrin knockout (beta3) and wild-type mice were used to induce either moderate or severe PO in vivo for short-term (72-hour) and long-term (4-week) transverse aortic constriction. Whereas wild-type mice showed no changes during moderate PO at both time points, beta3 mice exhibited both enrichment of the mu-calpain isoform and programmed cell death of cardiomyocytes after 4-week PO. However, with severe PO that caused increased mortality in both mice groups, cell death was observed in wild-type mice also. To study calpain's role, calpeptin, a specific inhibitor of calpain, was administered through an osmotic mini-pump at 2.5 mg/kg per day beginning 3 days before moderate transverse aortic constriction or sham surgery. Calpeptin administration blocked both calpain enrichment and myocardial cell death in the 4-week PO beta3 mice. Because beta3 integrin contributes to cardioprotective signaling, these studies indicate that the loss of specific integrin function could be a key mechanism for calpain-mediated programmed cell death of cardiomyocytes in PO myocardium.

Topics: Animals; Apoptosis; Calpain; Cardiomegaly; Dipeptides; Heart Failure; Hypertrophy; Integrin beta3; Integrins; Mice; Mice, Knockout; Myocardium; Myocytes, Cardiac; Pressure; Signal Transduction

Topics: Angiotensin II; Animals; Calcium; Calpain; Cardiomegaly; Humans; Inositol 1,4,5-Trisphosphate Receptors; Myocardium; NF-kappa B; Signal Transduction

Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular (RV) PO model, we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24- to 48-h-PO myocardium, and these changes returned to basal level by 1 wk of PO. Histochemical studies in 24-h-PO myocardium revealed the presence of TdT-mediated dUTP nick-end label (TUNEL)-positive cardiomyocytes, which exhibited enrichment of calpain and gelsolin. Biochemical studies showed an increase in histone H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, which indicate programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin (0.6 mg/kg iv) by bolus injections twice, 15 min before and 6 h after induction of 24-h PO. Calpeptin blocked the following PO-induced changes: calpain enrichment and activation, decreased calpastatin level, caspase-3 activation, enrichment and cleavage of gelsolin, TUNEL staining, and histone H2B phosphorylation. Although similar administration of a caspase inhibitor, N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VD-fmk), blocked caspase-3 activation, it did not alleviate other aforementioned changes. These results indicate that biochemical markers of cardiomyocyte cell death, such as sarcomeric disarray, gelsolin cleavage, and TUNEL-positive nuclei, are mediated, at least in part, by calpain and that calpeptin may serve as a potential therapeutic agent to prevent cardiomyocyte loss and preserve myocardial structure and function during cardiac hypertrophy.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calpain; Cardiomegaly; Caspase 3; Caspase Inhibitors; Cats; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Enzyme Activation; Gelsolin; Heart Failure; Histones; In Situ Nick-End Labeling; Injections, Intravenous; Ligation; Male; Myocytes, Cardiac; Phosphorylation; Pulmonary Artery; Time Factors

Cardiomyocytes stop dividing after birth and postnatal heart growth is only achieved by increase in cell volume. In some species, cardiomyocytes undergo an additional incomplete mitosis in the first postnatal week, where karyokinesis takes place in the absence of cytokinesis, leading to binucleation. Proteins that regulate the formation of the actomyosin ring are known to be important for cytokinesis. Here we demonstrate for the first time that small GTPases like RhoA along with their downstream effectors like ROCK I, ROCK II and Citron Kinase show a developmental stage specific expression in heart, with high levels being expressed in cardiomyocytes only at stages when cytokinesis still occurs (i.e. embryonic and perinatal). This suggests that downregulation of many regulatory and cytoskeletal components involved in the formation of the actomyosin ring may be responsible for the uncoupling of cytokinesis from karyokinesis in rodent cardiomyocytes after birth. Interestingly, when the myocardium tries to adapt to the increased workload during pathological hypertrophy a re-expression of proteins involved in DNA synthesis and cytokinesis can be detected. Nevertheless, the adult cardiomyocytes do not appear to divide despite this upregulation of the cytokinetic machinery. The inability to undergo complete division could be due to the presence of stable, highly ordered and functional sarcomeres in the adult myocardium or could be because of the inefficiency of degradation pathways, which facilitate the division of differentiated embryonic cardiomyocytes by disintegrating myofibrils.

Topics: Actomyosin; Amides; Angiotensin II; Animals; Antihypertensive Agents; Biomarkers; Calpain; Cardiomegaly; Cell Nucleus Division; Cullin Proteins; Cytokinesis; Heart; Hypertension; Intracellular Signaling Peptides and Proteins; Mice; Myocytes, Cardiac; Myofibrils; Protein Serine-Threonine Kinases; Pyridines; Rats; Rats, Inbred Dahl; rho GTP-Binding Proteins; rho-Associated Kinases; Up-Regulation

Calcineurin (CnA) is important in the regulation of myocardial hypertrophy. We demonstrated that targeted proteolysis of the CnA autoinhibitory domain under pathological myocardial workload leads to increased CnA activity in human myocardium. Here, we investigated the proteolytic mechanism leading to activation of CnA.. In patients with diseased myocardium, we found strong nuclear translocation of CnA. In contrast, in normal human myocardium, there was a cytosolic distribution of CnA. Stimulation of rat cardiomyocytes with angiotensin (Ang) II increased calpain activity significantly (433+/-11%; P<0.01; n=6) and caused proteolysis of the autoinhibitory domain of CnA. Inhibition of calpain by a membrane-permeable calpain inhibitor prevented proteolysis. We identified the cleavage site of calpain in the human CnA sequence at amino acid 424. CnA activity was increased after Ang II stimulation (310+/-29%; P<0.01; n=6) and remained high after removal of Ang II (214+/-17%; P<0.01; n=6). Addition of a calpain inhibitor to the medium decreased CnA activity (110+/-19%; P=NS; n=6) after removal of Ang II. Ang II stimulation of cardiomyocytes also translocated CnA into the nucleus as demonstrated by immunohistochemical staining and transfection assays with GFP-tagged CnA. Calpain inhibition and therefore suppression of calpain-mediated proteolysis of CnA enabled CnA exit from the nucleus.. Ang II stimulation of cardiomyocytes increased calpain activity, leading to proteolysis of the autoinhibitory domain of CnA. This causes an increase in CnA activity and results in nuclear translocation of CnA. Loss of the autoinhibitory domain renders CnA constitutively nuclear and active, even after removal of the hypertrophic stimulus.

Topics: Active Transport, Cell Nucleus; Amino Acid Sequence; Animals; Animals, Newborn; Calcineurin; Calpain; Cardiomegaly; Cell Extracts; Cell Nucleus; Cells, Cultured; Enzyme Activation; Frozen Sections; Humans; Hydrolysis; Molecular Sequence Data; Myocardium; Myocytes, Cardiac; Phosphoric Monoester Hydrolases; Proteins; Rats; Rats, Wistar; Transcription, Genetic

Diseases due to mutations in the lamin A/C gene (LMNA) are highly heterogeneous, including neuromuscular and cardiac dystrophies, lipodystrophies, and premature ageing syndromes. In this study we characterized the neuromuscular and cardiac phenotypes of patients bearing the heterozygous LMNA R482W mutation, which is the most frequent genotype associated with the familial partial lipodystrophy of the Dunnigan type (FPLD). Fourteen patients from two unrelated families, including 10 affected subjects, were studied. The two probands had been referred for lipoatrophy and/or diabetes. Lipodystrophy, exclusively observed in LMNA-mutated patients, was of variable severity and limited to postpubertal subjects. Lipodystrophy and metabolic disturbances were more severe in women, even if an enlarged neck was a constant finding. The severity of hypertriglyceridemia and hirsutism in females was related to that of insulin resistance. Clinical muscular alterations were only present in LMNA-mutated patients. Clinical and histological examination showed an invalidating, progressive limb-girdle muscular dystrophy in a 42-yr-old woman that had been present since childhood, associated with a typical postpubertal FPLD phenotype. Six of eight adults presented the association of calf hypertrophy, perihumeral muscular atrophy, and a rolling gait due to proximal lower limb weakness. Muscular histology was compatible with muscular dystrophy in one of them and/or showed a nonspecific excess of lipid droplets (in three cases). Immunostaining of lamin A/C was normal in the six muscular biopsies. Surprisingly, calpain 3 expression was undetectable in the patient with the severe limb-girdle muscular dystrophy, although the gene did not reveal any molecular alterations. At the cardiac level, cardiac septal hypertrophy and atherosclerosis were frequent in FPLD patients. In addition, a 24-yr-old FPLD patient had a symptomatic second degree atrioventricular block. In conclusion, we showed that most lipodystrophic patients affected by the FPLD-linked LMNA R482W mutation show muscular and cardiac abnormalities. The occurrence and severity of the myopathic and lipoatrophic phenotypes varied and were not related. The muscular phenotype was evocative of limb girdle muscular dystrophy. Cardiac hypertrophy and advanced atherosclerosis were frequent. FPLD patients should receive careful neuromuscular and cardiac examination whatever the underlying LMNA mutation.

Topics: Adolescent; Adult; Arteriosclerosis; Calpain; Cardiomegaly; Child; Diabetes Mellitus, Lipoatrophic; Female; Humans; Lamin Type A; Leptin; Male; Middle Aged; Muscles; Muscular Dystrophies, Limb-Girdle; Mutation; Triglycerides

Two inhibitors of the calcium-dependent cysteine protease, calpain, have markedly different effects on the extent of hypertrophy induced by the alpha-adrenergic agonist, phenylephrine, of cultured neonatal rat ventricular myocytes. E64c, an inhibitor of calpain and other cysteine proteases, stimulated the hypertrophy by 59%. PD 150606, a specific calpain inhibitor, reduced the hypertrophy by 38%. Phenylephrine decreased the proteolysis of a calpain substrate by the cells 1-2 h after its addition but not at 24 h. PD 150606 inhibited proteolytic activity at all times, and the combination of phenylephrine and PD 150606 did not give greater inhibition. This suggests that cysteine proteases of the papain sub-family are involved with the hypertrophic response at two points, promoting hypertrophy at the first and limiting it at the second. Calpain appears to be the protease involved at the first point, and there may be another cysteine protease acting at the second site.

Topics: Acrylates; Adrenergic alpha-Agonists; Animals; Animals, Newborn; Calpain; Cardiomegaly; Cells, Cultured; Cysteine Proteinase Inhibitors; Glycoproteins; Heart Ventricles; Leucine; Myocytes, Cardiac; Phenylephrine; Rats; Time Factors; Ventricular Function, Left

The purpose of this study was to test the relationship between biochemical and functional changes accompanying beta-agonist induced cardiac hypertrophy and the activation of a calcium stimulated cysteine protease. Because the ultrastructural and ionic changes accompanying beta-agonist induced cardiac hypertrophy are reminiscent of the actions of the calcium activated neutral protease, calpain, it was hypothesized that lowering calpain activity (by the use of an exogenous inhibitor(s)) would reduce the extent of hypertrophy. Rats (275-300 g) were randomly assigned to either a control, beta-agonist (iso) or cysteine protease inhibitor (E64c) group. Isoproterenol administration (1 mg/kg) resulted in changes for ventricular weight to body weight ratio (increases 19%), ventricular [RNA] (increases 105.6%), rate of pressure development (increases 22% for +dP/dt) and maximum developed left ventricular pressure (increases 19%) (p < 0.05) after 3 days. Calpain-like activity (assessed by microplate method) increased by 45% (p < 0.05), while [cAMP] returned to control levels (following a transient rise at 1 day; 606.03 +/- 124.1 pmol/g/wet/wt to 937.9 +/- 225 (p < 0.05)). E64c (administered 1 h prior to iso) reduced the extent of hypertrophy, from 19 to 12%, and prevented the increases in; total [RNA], left ventricular function, the initial [cAMP] increase and calpain-like activity. It is concluded that a calcium stimulated cysteine protease(s), such as calpain, may be involved in the biochemical and functional changes associated with isoproterenol induced cardiac hypertrophy.

Topics: Adrenergic beta-Agonists; Animals; Calcium; Calpain; Cardiomegaly; Cyclic AMP; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Isoproterenol; Leucine; Male; Rats; Rats, Wistar; RNA; Ventricular Function, Left