calpain and Heart-Failure

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

Mitochondrial dysfunction underlines a multitude of pathologies; however, studies are scarce that rescue the mitochondria for cellular resuscitation. Exploration into the protective role of mitochondrial transcription factor A (TFAM) and its mitochondrial functions respective to cardiomyocyte death are in need of further investigation. TFAM is a gene regulator that acts to mitigate calcium mishandling and ROS production by wrapping around mitochondrial DNA (mtDNA) complexes. TFAM's regulatory functions over serca2a, NFAT, and Lon protease contribute to cardiomyocyte stability. Calcium- and ROS-dependent proteases, calpains, and matrix metalloproteinases (MMPs) are abundantly found upregulated in the failing heart. TFAM's regulatory role over ROS production and calcium mishandling leads to further investigation into the cardioprotective role of exogenous TFAM. In an effort to restabilize physiological and contractile activity of cardiomyocytes in HF models, we propose that TFAM-packed exosomes (TFAM-PE) will act therapeutically by mitigating mitochondrial dysfunction. Notably, this is the first mention of exosomal delivery of transcription factors in the literature. Here we elucidate the role of TFAM in mitochondrial rescue and focus on its therapeutic potential.

Topics: Animals; Calcium; Calpain; DNA-Binding Proteins; DNA, Mitochondrial; Exosomes; Gene Expression Regulation; Heart Failure; Humans; Matrix Metalloproteinases; Mice; Mice, Knockout; Mitochondria, Heart; Mitochondrial Proteins; Myocytes, Cardiac; Reactive Oxygen Species; Transcription Factors; Transcriptional Activation

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

Cardiomyocytes are terminally differentiated cells and thus do not have the ability to dilute damaged proteins and organelles by cell division. Thus, proteolytic and recycling systems within the cardiomyocyte are essential to maintain cardiac function. The major proteolytic systems in the cell are: the ubiquitin-proteasome system, autophagy, and calpain. The ubiquitin-proteasome system degrades specific proteins by labelling them with ubiquitin. Autophagy degrades cytosolic proteins and organelles; this is generally believed to be a non-specific type of degradation. Calpain is a Ca(2+)-sensitive cysteine protease that degrades intracellular substrates including cytoskeletal proteins, and participates in Ca(2+)-mediated intracellular processes. All three systems exist in the cardiomyocyte and play pivotal roles in maintaining cardiac function. However, there is still controversy regarding the role of each protein-degradation system in the heart. Our recent reports using cardiac-specific knockout mice have revealed the cardioprotective roles of autophagy and calpain in the development of heart failure. While these proteolytic systems exhibit distinct molecular mechanisms, they work cooperatively (one process can regulate another).

Topics: Animals; Autophagy; Calpain; Dystrophin; Heart Failure; Humans; Myocardium; Myocytes, Cardiac; Proteasome Endopeptidase Complex; Proteolysis; Ubiquitin

The precise mechanism(s) of the progression of advanced heart failure (HF) should be determined to establish strategies for its treatment or prevention. Based on pathological, molecular, and physiological findings in 3 animal models and human cases, we propose a novel scheme that a vicious cycle formed by increased sarcolemma (SL) permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin (Dys) commonly lead to advanced HF. The aim of this article was to assess our recent paradigm that disruption of myocardial Dys is a final common pathway to advanced HF, irrespective of its hereditary or acquired origin, but not intended to provide a comprehensive overview of the various factors that may be involved in the course of HF in different clinical settings. In addition, each component of Dys-associated proteins (DAP) was heterogeneously degraded in vivo and in vitro, i.e. Dys and alpha-sarcoglycan (SG) were markedly destroyed using isolated calpain 2, while delta-SG was not degraded at all. The up-regulation of calpain 2 was confirmed through previously published data that remain insufficient for precise evaluation, supporting our new scheme that the activation of calpain(s) is involved in the steady process of Dys cleavage. In addition, somatic gene therapy is discussed as a potential option to ameliorate the physiological/metabolic indices and to improve the prognosis.

Topics: Animals; Calpain; Cardiomyopathy, Dilated; Disease Models, Animal; Dystrophin; Genetic Therapy; Heart Failure; Humans; Myocardial Infarction; Sarcoglycans; Transduction, Genetic

The precise mechanism of the progression of advanced heart failure is unknown. We assessed a new scheme in two heart failure models: (I) congenital dilated cardiomyopathy (DCM) in TO-2 strain hamsters lacking delta-sarcoglycan (SG) gene and (II) administration of a high-dose of isoproterenol, as an acute heart failure in normal rats. In TO-2 hamsters, we followed the time course of the histological, physiological and metabolic the progressions of heart failure to the end stage. Dystrophin localization detected by immunostaining age-dependently to the myoplasm and the in situ sarcolemma fragility evaluated by Evans blue entry was increased in the same cardiomyocytes. Western blotting revealed a limited cleavage of the dystrophin protein at the rod domain, strongly suggesting a contribution of endogenous protease(s). We found a remarkable up-regulation of the amount of calpain-1 and -2, and no change of their counterpart, calpastatin. After supplementing TO-2 hearts with the normal delta-SG gene in vivo, these pathological alterations and the animals' survival improved. Furthermore, dystrophin but not delta-SG was disrupted by a high dose of isoproterenol, translocated from the sarcolemma to the myoplasm and fragmented. These results of heart failure, irrespective of the hereditary or acquired origin, indicate a vicious cycle formed by the increased sarcolemma permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin would commonly lead to advanced heart failure.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cardiomyopathy, Dilated; Cell Membrane Permeability; Cricetinae; Dependovirus; Disease Models, Animal; Dystrophin; Enzyme Activation; Genetic Therapy; Heart Failure; Isoproterenol; Mesocricetus; Models, Biological; Rats; Sarcoglycans; Sarcolemma

Calpains are calcium-activated neutral proteases involved in the regulation of key signaling pathways. Junctophilin-2 (JP2) is a Calpain-specific proteolytic target and essential structural protein inside Ca

Topics: Animals; Calcium; Calpain; DNA; Heart Failure; Membrane Proteins; Mice; Mice, Knockout; Myocytes, Cardiac

Calpain proteolysis contributes to the pathogenesis of heart failure but the calpain isoforms responsible and their substrate specificities have not been rigorously defined. One substrate, Junctophilin-2 (JP2), is essential for maintaining junctional cardiac dyads and excitation-contraction coupling. We previously demonstrated that mouse JP2 is cleaved by calpain-1 (CAPN1) between Arginine 565 (R565) and Threonine 566 (T566). Recently, calpain-2 (CAPN2) was reported to cleave JP2 at a novel site between Glycine 482 (G482) and Threonine 483 (T483). We aimed to directly compare the contributions of each calpain isoform, their Ca2+ sensitivity, and their cleavage site selection for JP2. We find CAPN1, CAPN2 and their requisite CAPNS1 regulatory subunit are induced by pressure overload stress that is concurrent with JP2 cleavage. Using in vitro calpain cleavage assays, we demonstrate that CAPN1 and CAPN2 cleave JP2 into similar 75 kD N-terminal (JP2NT) and 25 kD C-terminal fragments (JP2CT) with CAPNS1 co-expression enhancing proteolysis. Deletion mutagenesis shows both CAPN1 and CAPN2 require R565/T566 but not G482/T483. When heterologously expressed, the JP2CT peptide corresponding to R565/T566 cleavage approximates the 25 kD species found during cardiac stress while the C-terminal peptide from potential cleavage at G482/T483 produces a 35 kD product. Similar results were obtained for human JP2. Finally, we show that CAPN1 has higher Ca2+ sensitivity and cleavage efficacy than CAPN2 on JP2 and other cardiac substrates including cTnT, cTnI and β2-spectrin. We conclude that CAPN2 cleaves JP2 at the same functionally conserved R565/T566 site as CAPN1 but with less efficacy and suggest heart failure may be targeted through specific inhibition of CAPN1.

Topics: Animals; Arginine; Calpain; Disease Models, Animal; Glycine; Heart Failure; HEK293 Cells; Humans; Male; Membrane Proteins; Mice; Muscle Proteins; Mutagenesis, Site-Directed; Myocytes, Cardiac; Proteolysis; Signal Transduction; Threonine; Transfection

Xin-Ji-Er-Kang (XJEK) as a herbal formula of traditional Chinese medicine (TCM) has shown the protective effects on myocardial function as well as renal function in mouse models of myocardial infarction.. We investigated the effects of XJEK on cardiovascular- and renal-function in a heart failure mouse model induced by high salt (HS) and the associated mechanisms.. For the purpose of assessing the effects of XJEK on a hypertensive heart failure model, mice were fed with 8% high salt diet. XJEK was administered by oral gavage for 8 weeks. Cardiovascular function parameters, renal function associated biomarkers and XJEK's impact on renin-angiotensin-aldosterone system (RAAS) activation were assessed. To determine the underlying mechanism, the calpain1/junctophilin-2 (JP2)/sarcoplasmic reticulum Ca. Mice on HS-diet exhibited hypertensive heart failure along with progressive kidney injury. Similar to fosinopril, XJEK ameliorated hypertension, cardiovascular-and renal- dysfunction in mice of HS-diet group. XJEK inhibited HS-induced activation of RAAS and reversed the abnormal expression pattern of calpain1and JP2 protein in heart tissues. XJEK significantly improved cell viability of angiotensin II-challenged AC16 cells. Moreover, XJEK's impact on calpain1/JP2 pathway was partly diminished in AC16 cells transfected with calpastatin siRNA.. XJEK was found to exert cardiovascular- and renal protection in HS-diet induced heart failure mouse model. XJEK inhibited HS-diet induced RAAS activation by inhibiting the activity and expression of calpain1 and protected the junctional membrane complex (JMC) in cardiomyocytes.

Topics: Animals; Blood Pressure; Calpain; Drugs, Chinese Herbal; Heart Failure; Hypertension; Kidney; Membrane Proteins; Mice; Muscle Proteins; Oxidative Stress; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction

Mechanoelectric feedback (MEF) was related to malignant arrhythmias in heart failure (HF). Desmin is a cytoskeleton protein and could be involved in MEF as a mechanoelectrical transducer. In this study, we will discuss the role of desmin alterations in mechanical electrical feedback in heart failure and its mechanisms.. We used both an in vivo rat model and an in vitro cardiomyocyte model to address this issue. For the in vivo experiments, we establish a sham group, an HF group, streptomycin (SM) group, and an MDL-28170 group. The occurrence of ventricular arrhythmias (VA) was recorded in each group. For the in vitro cardiomyocyte model, we established an NC group, a si-desmin group, and a si-desmin + NBD IKK group. The expression of desmin, IKKβ, p-IKKβ, IKBα, p-NF-κB, and SERCA2 were detected in both in vivo and in vitro experiments. The content of Ca. An increased number of VAs were found in the HF group. SM and MDL-28170 can reduce desmin breakdown and the number of VAs in heart failure. The knockdown of desmin in the cardiomyocyte can activate the NF-κB pathway, decrease the level of SERCA2, and result in abnormal distribution of Ca. Overall, desmin may participate in MEF through the NF-κB pathway. This study provides a potential therapeutic target for VA in HF.

Topics: Animals; Calcium; Calpain; Cells, Cultured; Desmin; Dipeptides; Disease Models, Animal; Electrocardiography; Feedback, Physiological; Gene Knockdown Techniques; Heart Failure; Male; Myocytes, Cardiac; NF-kappa B; Rats, Sprague-Dawley; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Tachycardia, Ventricular

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. Patients with HF exhibit a loss of junctophilin-2 (JPH2), a structural protein critical in forming junctional membrane complexes in which excitation-contraction takes place. Several mechanisms have been proposed to mediate the loss of JPH2, one being cleavage by the calcium-dependent protease calpain. The downstream mechanisms underlying HF progression after JPH2 cleavage are presently poorly understood. In this study, we used Labcas to bioinformatically predict putative calpain cleavage sites on JPH2. We identified a cleavage site that produces a novel C-terminal JPH2 peptide (JPH2-CTP) using several domain-specific antibodies. Western blotting revealed elevated JPH2-CTP levels in hearts of patients and mice with HF, corresponding to increased levels of calpain-2. Moreover, immunocytochemistry demonstrated nuclear localization of JPH2-CTP within ventricular myocytes isolated from a murine model of pressure overload-induced HF as well as rat ventricular myocytes treated with isoproterenol. Nuclear localization of JPH2-CTP and cellular remodeling were abrogated by a genetic mutation of the nuclear localization sequence within JPH2-CTP. Taken together, our studies identified a novel C-terminal fragment of JPH2 (JPH2-CTP) generated by calpain-2 mediated cleavage which localizes within the cardiomyocyte nucleus during HF. Blocking nuclear localization of JPH2-CTP protects cardiomyocytes from isoproterenol-induced hypertrophy in vitro. Future in vivo studies of the nuclear role of JPH2-CTP may reveal a causal association with adverse remodeling during HF and establish CTP as a therapeutic target.

Topics: Animals; Calpain; Cell Nucleus; Female; Heart Failure; Humans; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Myocytes, Cardiac

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

We and others have reported that calpain-1 was increased in myocardial mitochondria from various animal models of heart disease. This study investigated whether constitutive up-regulation of calpain-1 restricted to mitochondria induced myocardial injury and heart failure and, if so, whether these phenotypes could be rescued by selective inhibition of mitochondrial superoxide production. Transgenic mice with human CAPN1 up-regulation restricted to mitochondria in cardiomyocytes (Tg-mtCapn1/tTA) were generated and characterized with low and high over-expression of transgenic human CAPN1 restricted to mitochondria, respectively. Transgenic up-regulation of mitochondria-targeted CAPN1 dose-dependently induced cardiac cell death, adverse myocardial remodeling, heart failure, and early death in mice, the changes of which were associated with mitochondrial dysfunction and mitochondrial superoxide generation. Importantly, a daily injection of mitochondria-targeted superoxide dismutase mimetics mito-TEMPO for 1 month starting from age 2 months attenuated cardiac cell death, adverse myocardial remodeling and heart failure, and reduced mortality in Tg-mtCapn1/tTA mice. In contrast, administration of TEMPO did not achieve similar cardiac protection in transgenic mice. Furthermore, transgenic up-regulation of mitochondria-targeted CAPN1 induced a reduction of ATP5A1 protein and ATP synthase activity in hearts. In cultured cardiomyocytes, increased calpain-1 in mitochondria promoted mitochondrial permeability transition pore (mPTP) opening and induced cell death, which were prevented by over-expression of ATP5A1, mito-TEMPO or cyclosporin A, an inhibitor of mPTP opening. In conclusion, this study has provided direct evidence demonstrating that increased mitochondrial calpain-1 is an important mechanism contributing to myocardial injury and heart failure by disrupting ATP synthase, and promoting mitochondrial superoxide generation and mPTP opening.

Topics: Animals; Calpain; Cardiomyopathy, Dilated; Cell Death; Cyclic N-Oxides; Disease Models, Animal; Heart Failure; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Proton-Translocating ATPases; Myocytes, Cardiac; Superoxides

Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder with variable genetic etiologies. Here we focused on understanding the precise molecular pathology of a single clinical variant in DSP, the gene encoding desmoplakin. We initially identified a novel missense desmoplakin variant (p.R451G) in a patient diagnosed with biventricular ACM. An extensive single-family ACM cohort was assembled, revealing a pattern of coinheritance for R451G desmoplakin and the ACM phenotype. An in vitro model system using patient-derived induced pluripotent stem cell lines showed depressed levels of desmoplakin in the absence of abnormal electrical propagation. Molecular dynamics simulations of desmoplakin R451G revealed no overt structural changes, but a significant loss of intramolecular interactions surrounding a putative calpain target site was observed. Protein degradation assays of recombinant desmoplakin R451G confirmed increased calpain vulnerability. In silico screening identified a subset of 3 additional ACM-linked desmoplakin missense mutations with apparent enhanced calpain susceptibility, predictions that were confirmed experimentally. Like R451G, these mutations are found in families with biventricular ACM. We conclude that augmented calpain-mediated degradation of desmoplakin represents a shared pathological mechanism for select ACM-linked missense variants. This approach for identifying variants with shared molecular pathologies may represent a powerful new strategy for understanding and treating inherited cardiomyopathies.

Topics: Adult; Arrhythmias, Cardiac; Calpain; Cardiomyopathies; Desmoplakins; Female; Genetic Predisposition to Disease; Glycine; Heart; Heart Failure; Humans; Male; Middle Aged; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Mutation, Missense; Pedigree; Phenotype; Recombinant Proteins; Stem Cells

Ischemia/reperfusion injury is associated with contractile dysfunction and increased cardiomyocyte death. Overexpression of the hematopoietic lineage substrate-1-associated protein X-1 (HAX-1) has been shown to protect from cellular injury but the function of endogenous HAX-1 remains obscure due to early lethality of the knockout mouse. Herein we generated a cardiac-specific and inducible HAX-1 deficient model, which uncovered an unexpected role of HAX-1 in regulation of sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) in ischemia/reperfusion injury. Although ablation of HAX-1 in the adult heart elicited no morphological alterations under non-stress conditions, it diminished contractile recovery and increased infarct size upon ischemia/reperfusion injury. These detrimental effects were associated with increased loss of SERCA2a. Enhanced SERCA2a degradation was not due to alterations in calpain and calpastatin levels or calpain activity. Conversely, HAX-1 overexpression improved contractile recovery and maintained SERCA2a levels. The regulatory effects of HAX-1 on SERCA2a degradation were observed at multiple levels, including intact hearts, isolated cardiomyocytes and sarcoplasmic reticulum microsomes. Mechanistically, HAX-1 ablation elicited increased production of reactive oxygen species at the sarco/endoplasic reticulum compartment, resulting in SERCA2a oxidation and a predisposition to its proteolysis. This effect may be mediated by NAPDH oxidase 4 (NOX4), a novel binding partner of HAX-1. Accordingly, NOX inhibition with apocynin abrogated the effects of HAX-1 ablation in hearts subjected to ischemia/reperfusion injury. Taken together, our findings reveal a role of HAX-1 in the regulation of oxidative stress and SERCA2a degradation, implicating its importance in calcium homeostasis and cell survival pathways.

Topics: Adaptor Proteins, Signal Transducing; Aged; Animals; Calpain; Endoplasmic Reticulum; Female; Gene Deletion; Heart Failure; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice, Inbred C57BL; Middle Aged; Myocardial Contraction; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; NADPH Oxidase 4; Oxidation-Reduction; Oxidative Stress; Proteins; Proteolysis; Rats, Wistar; Reactive Oxygen Species; Recovery of Function; RNA, Messenger; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases

In human heart failure, Ser199 (equivalent to Ser200 in mouse) of cTnI (cardiac troponin I) is significantly hyperphosphorylated, and in vitro studies suggest that it enhances myofilament calcium sensitivity and alters calpain-mediated cTnI proteolysis. However, how its hyperphosphorylation affects cardiac function in vivo remains unknown.. To address the question, 2 transgenic mouse models were generated: a phospho-mimetic cTnIS200D and a phospho-silenced cTnIS200A, each driven by the cardiomyocyte-specific α-myosin heavy chain promoter. Cardiac structure assessed by echocardiography and histology was normal in both transgenic models compared with littermate controls (n=5). Baseline in vivo hemodynamics and isolated muscle studies showed that cTnIS200D significantly prolonged relaxation and lowered left ventricular peak filling rate, whereas ejection fraction and force development were normal (n=5). However, with increased heart rate or β-adrenergic stimulation, cTnIS200D mice had less enhanced ejection fraction or force development versus controls, whereas relaxation improved similarly to controls (n=5). By contrast, cTnIS200A was functionally normal both at baseline and under the physiological stresses. To test whether either mutation impacted cardiac response to ischemic stress, isolated hearts were subjected to ischemia/reperfusion. cTnIS200D were protected, recovering 88±8% of contractile function versus 35±15% in littermate controls and 28±8% in cTnIS200A (n=5). This was associated with less cTnI proteolysis in cTnIS200D hearts.. Hyperphosphorylation of this serine in cTnI C terminus impacts heart function by depressing diastolic function at baseline and limiting systolic reserve under physiological stresses. However, paradoxically, it preserves heart function after ischemia/reperfusion injury, potentially by decreasing proteolysis of cTnI.

Topics: Adrenergic beta-Agonists; Animals; Calpain; Disease Models, Animal; Genetic Predisposition to Disease; Heart Failure; Hemodynamics; Isolated Heart Preparation; Male; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Myocardial Contraction; Myocardial Reperfusion Injury; Myofibrils; Myosin Heavy Chains; Phenotype; Phosphorylation; Promoter Regions, Genetic; Protein Domains; Protein Stability; Proteolysis; Recovery of Function; Serine; Time Factors; Troponin I; Ventricular Function, Left

β2-Spectrin is critical for integrating membrane and cytoskeletal domains in excitable and nonexcitable cells. The role of β2-spectrin for vertebrate function is illustrated by dysfunction of β2-spectrin-based pathways in disease. Recently, defects in β2-spectrin association with protein partner ankyrin-B were identified in congenital forms of human arrhythmia. However, the role of β2-spectrin in common forms of acquired heart failure and arrhythmia is unknown. We report that β2-spectrin protein levels are significantly altered in human cardiovascular disease as well as in large and small animal cardiovascular disease models. Specifically, β2-spectrin levels were decreased in atrial samples of patients with atrial fibrillation compared with tissue from patients in sinus rhythm. Furthermore, compared with left ventricular samples from nonfailing hearts, β2-spectrin levels were significantly decreased in left ventricle of ischemic- and nonischemic heart failure patients. Left ventricle samples of canine and murine heart failure models confirm reduced β2-spectrin protein levels. Mechanistically, we identify that β2-spectrin levels are tightly regulated by posttranslational mechanisms, namely Ca(2+)- and calpain-dependent proteases. Furthermore, consistent with this data, we observed Ca(2+)- and calpain-dependent loss of β2-spectrin downstream effector proteins, including ankyrin-B in heart. In summary, our findings illustrate that β2-spectrin and downstream molecules are regulated in multiple forms of cardiovascular disease via Ca(2+)- and calpain-dependent proteolysis.

Topics: Adult; Aged; Animals; Ankyrins; Atrial Fibrillation; Calcium; Calpain; Case-Control Studies; Disease Models, Animal; Dogs; Down-Regulation; Female; Heart Failure; Heart Ventricles; Humans; Male; Mice, Inbred C57BL; Middle Aged; Proteolysis; Signal Transduction; Spectrin; Stroke Volume; Ventricular Function, Left

Sumoylation is a posttranslational modification that regulates a wide spectrum of cellular activities. Cardiomyopathy is the leading cause of heart failure. Whether sumoylation, particularly SUMO-2/3 conjugation, is involved in cardiomyopathy has not been investigated. We report here that SUMO-2/3 conjugation was elevated in the human failing hearts, and we investigated the impact of increased SUMO-2 conjugation on heart function by using the gain-of-function approach in mice, in which cardiac specific expression of constitutively active SUMO-2 was governed by alpha myosin heavy chain promoter (MHC-SUMO-2 transgenic, SUMO-2-Tg). Four of five independent SUMO-2-Tg mouse lines exhibited cardiomyopathy with various severities, ranging from acute heart failure leading to early death to the development of chronic cardiomyopathy with aging. We further revealed that SUMO-2 directly regulated apoptotic process by at least partially targeting calpain 2 and its natural inhibitor calpastatin. SUMO conjugation to calpain 2 promoted its enzymatic activity, and SUMO attachment to calpastatin mainly promoted its turnover and altered its subcellular distribution. Thus, enhanced SUMO-2 conjugation led to increased apoptosis and played a pathogenic role in the development of cardiomyopathy and heart failure.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cardiomyopathies; Heart Failure; HeLa Cells; Humans; Mice; Protein Binding; Protein Transport; Small Ubiquitin-Related Modifier Proteins; Ubiquitins

A highly organized transverse tubule (T-tubule) network is necessary for efficient Ca(2+)-induced Ca(2+) release and synchronized contraction of ventricular myocytes. Increasing evidence suggests that T-tubule remodeling due to junctophilin-2 (JP-2) downregulation plays a critical role in the progression of heart failure. However, the mechanisms underlying JP-2 dysregulation remain incompletely understood.. A mouse model of reversible heart failure that is driven by conditional activation of the heterotrimeric G protein Gαq in cardiac myocytes was used in this study. Mice with activated Gαq exhibited disruption of the T-tubule network and defects in Ca(2+) handling that culminated in heart failure compared with wild-type mice. Activation of Gαq/phospholipase Cβ signaling increased the activity of the Ca(2+)-dependent protease calpain, leading to the proteolytic cleavage of JP-2. A novel calpain cleavage fragment of JP-2 is detected only in hearts with constitutive Gαq signaling to phospholipase Cβ. Termination of the Gαq signal was followed by normalization of the JP-2 protein level, repair of the T-tubule network, improvements in Ca(2+) handling, and reversal of heart failure. Treatment of mice with a calpain inhibitor prevented Gαq-dependent JP-2 cleavage, T-tubule disruption, and the development of heart failure.. Disruption of the T-tubule network in heart failure is a reversible process. Gαq-dependent activation of calpain and subsequent proteolysis of JP-2 appear to be the molecular mechanism that leads to T-tubule remodeling, Ca(2+) handling dysfunction, and progression to heart failure in this mouse model.

Topics: Animals; Calcium; Calpain; Disease Notification; Down-Regulation; GTP-Binding Protein alpha Subunits, Gq-G11; Heart Failure; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle Cells; Muscle Proteins; Proteolysis; Signal Transduction

Cardiac sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is central to the maintenance of normal Ca(2+) homeostasis and contraction. Studies indicate that the Ca(2+)-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca(2+) binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met(369) antibody identified a novel calpain cleavage site at Met(369). Engineering NCX1-Met(369) into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met(369) inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met(369) cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.

Topics: Aged; Amino Acid Sequence; Animals; Aortic Valve Stenosis; Binding Sites; Calpain; Female; Heart Failure; Heart Ventricles; HEK293 Cells; Humans; Male; Molecular Sequence Data; Myocardium; Myocytes, Cardiac; Primary Cell Culture; Protein Binding; Proteolysis; Rats; Rats, Wistar; Sodium-Calcium Exchanger

Calpain is an intracellular Ca²⁺-activated protease that is involved in numerous Ca²⁺ dependent regulation of protein function in many cell types. This paper tests a hypothesis that calpains are involved in Ca²⁺-dependent increase of the late sodium current (INaL) in failing heart. Chronic heart failure (HF) was induced in 2 dogs by multiple coronary artery embolization. Using a conventional patch-clamp technique, the whole-cell INaL was recorded in enzymatically isolated ventricular cardiomyocytes (VCMs) in which INaL was activated by the presence of a higher (1 μM) intracellular [Ca²⁺] in the patch pipette. Cell suspensions were exposed to a cell- permeant calpain inhibitor MDL-28170 for 1-2 h before INaL recordings. The numerical excitation-contraction coupling (ECC) model was used to evaluate electrophysiological effects of calpain inhibition in silico. MDL caused acceleration of INaL decay evaluated by the two-exponential fit (τ₁ = 42±3.0 ms τ₂ = 435±27 ms, n = 6, in MDL vs. τ₁ = 52±2.1 ms τ₂ = 605±26 control no vehicle, n = 11, and vs. τ₁ = 52±2.8 ms τ₂ = 583±37 ms n = 7, control with vehicle, P<0.05 ANOVA). MDL significantly reduced INaL density recorded at -30 mV (0.488±0.03, n = 12, in control no vehicle, 0.4502±0.0210, n = 9 in vehicle vs. 0.166±0.05pA/pF, n = 5, in MDL). Our measurements of current-voltage relationships demonstrated that the INaL density was decreased by MDL in a wide range of potentials, including that for the action potential plateau. At the same time the membrane potential dependency of the steady-state activation and inactivation remained unchanged in the MDL-treated VCMs. Our ECC model predicted that calpain inhibition greatly improves myocyte function by reducing the action potential duration and intracellular diastolic Ca²⁺ accumulation in the pulse train.. Calpain inhibition reverses INaL changes in failing dog ventricular cardiomyocytes in the presence of high intracellular Ca²⁺. Specifically it decreases INaL density and accelerates INaL kinetics resulting in improvement of myocyte electrical response and Ca²⁺ handling as predicted by our in silico simulations.

Topics: Animals; Calpain; Computer Simulation; Dipeptides; Disease Models, Animal; Dogs; Heart Failure; Heart Ventricles; Membrane Potentials; Models, Biological; Myocytes, Cardiac; Sodium

Right heart failure from right ventricular (RV) pressure overload is a major cause of morbidity and mortality, but its mechanism is incompletely understood. We tested the hypothesis that right heart failure during 4 hours of RV pressure overload is associated with alterations of the focal adhesion protein talin, and that the inhibition of calpain attenuates RV dysfunction and preserves RV talin. Anesthetized open-chest pigs treated with the calpain inhibitor MDL-28170 (n = 20) or inactive vehicle (n = 23) underwent 4 hours of RV pressure overload by pulmonary artery constriction (initial RV systolic pressure, 64 ± 1 and 66 ± 1 mm Hg in MDL-28170 and vehicle-treated pigs, respectively). Progressive RV contractile dysfunction was attenuated by MDL-28170: after 4 hours of RV pressure overload, RV systolic pressure was 44 ± 4 mm Hg versus 49 ± 6 mm Hg (P = 0.011), and RV stroke work was 72 ± 5% of baseline versus 90 ± 5% of baseline, (P = 0.027), in vehicle-treated versus MDL-28170-treated pigs, respectively. MDL-28170 reduced the incidence of hemodynamic instability (death or systolic blood pressure of < 85 mm Hg) by 46% (P = 0.013). RV pressure overload disrupted talin organization. MDL-28170 preserved talin abundance in the RV free wall (P = 0.039), and talin abundance correlated with the maintenance of RV free wall stroke work (r = 0.58, P = 0.0039). α-actinin and vinculin showed similar changes according to immunohistology. Right heart failure from acute RV pressure overload is associated with reduced talin abundance and disrupted talin organization. Calpain inhibition preserves the abundance and organization of talin and RV function. Calpain inhibition may offer clinical utility in treating acute cor pulmonale.

Topics: Actinin; Acute Disease; Animals; Calpain; Dipeptides; Electrophoresis, Polyacrylamide Gel; Heart Failure; Heart Ventricles; Hypertension, Pulmonary; Swine; Talin

Calpains make up a family of Ca(2+)-dependent intracellular cysteine proteases that include ubiquitously expressed μ- and m-calpains. Both are heterodimers consisting of a distinct large catalytic subunit (calpain 1 for μ-calpain and calpain 2 for m-calpain) and a common regulatory subunit (calpain 4). The physiological roles of calpain remain unclear in the organs, including the heart, but it has been suggested that calpain is activated by Ca(2+) overload in diseased hearts, resulting in cardiac dysfunction. In this study, cardiac-specific calpain 4-deficient mice were generated to elucidate the role of calpain in the heart in response to hemodynamic stress. Cardiac-specific deletion of calpain 4 resulted in decreased protein levels of calpains 1 and 2 and showed no cardiac phenotypes under base-line conditions but caused left ventricle dilatation, contractile dysfunction, and heart failure with interstitial fibrosis 1 week after pressure overload. Pressure-overloaded calpain 4-deficient hearts took up a membrane-impermeant dye, Evans blue, indicating plasma membrane disruption. Membrane repair assays using a two-photon laser-scanning microscope revealed that calpain 4-deficient cardiomyocytes failed to reseal a plasma membrane that had been disrupted by laser irradiation. Thus, the data indicate that calpain protects the heart from hemodynamic stresses, such as pressure overload.

Topics: Animals; Blood Pressure; Calcium; Calpain; Cell Membrane; Heart Failure; Mice; Mice, Knockout; Muscle Proteins; Myocardial Contraction; Myocardium; Stress, Physiological

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

The angiotensin (Ang) II/Ang II receptor (ATR)-associated calcium signaling pathway is the major cause of ventricular remodelling in patients with congestive heart failure (CHF). However, the calcium-regulated proteinases responsible for Ang II-induced remodelling are not well understood. We investigated the profiles of the Ang II/ATR/calpain/calcineurin (CaN) pathway in human failing heart. We measured both the plasma and cardiac levels of Ang II and cardiac mRNA expression of ATR in 39 patients with CHF and 38 healthy controls. Importantly, protein expression of calpains, cleavage of cain/cabin1 and activity of CaN were tested. Both plasma and cardiac levels of Ang II were significantly increased in patients with CHF (both p<0.01), and the plasma Ang II concentration was closely correlated with the parameters of ventricular remodelling (r=+/-0.29-0.65, p<0.05 or <0.01). In addition, the cardiac level of AT1R but not AT2R was significantly upregulated in mild failing hearts (p<0.05) but dramatically downregulated in severe failing ones (p<0.01). CHF was associated with a marked upregulation of calpains, an increased cleavage of cain/cabin1, and the activation of CaN in the failing ventricular tissue. In patients with CHF, calpain upregulation was associated with an increase in cleavage of cain/cabin1 and the activation of CaN, indicating that these changes in calcium-regulated proteinases contribute to Ang II-induced cardiac remodelling.

Topics: Adult; Angiotensin II; Blotting, Western; Calcineurin; Calpain; Female; Gene Expression Profiling; Heart Failure; Humans; Male; Middle Aged; Myocardium; Radioimmunoassay; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Reverse Transcriptase Polymerase Chain Reaction

Many physiological processes and pharmacological agents modulate the ryanodine receptor (RyR), the primary sarcoplasmic reticulum (SR) Ca(2+) release channel in the heart. However, how such modulations translate into functional effects during cardiac excitation-contraction coupling (ECC) is much less clear. Using a low dose (250 microM) of caffeine we sensitized the RyR and examined SR Ca(2+) release using dynamic measurements of cytosolic Ca(2+) ([Ca(2+)](i)) and free Ca(2+) within the SR ([Ca(2+)](SR)). In field stimulated (1 Hz) rabbit ventricular myocytes, application of 250 microM caffeine caused an initial 33% increase in SR Ca(2+) release, which was followed by a decrease in SR Ca(2+) load (28%) and steady-state SR Ca(2+) release (23%). To investigate the effects of caffeine on local SR Ca(2+) release, we measured [Ca(2+)](SR) from individual release junctions during ECC as well as during spontaneous Ca(2+) sparks. In intact myocytes during ECC, caffeine increased global fractional SR Ca(2+) release by decreasing the [Ca(2+)](SR) level at which local release terminated by 21%. Similarly, in permeabilized myocytes during spontaneous Ca(2+) sparks, caffeine decreased the [Ca(2+)](SR) level for release termination by 12%. Finally, we examined if Ca(2+) release termination was changed in myocytes from failing hearts, where remodelling processes lead to altered RyR function. In myocytes from failing rabbit hearts, the [Ca(2+)](SR) termination level for Ca(2+) sparks was 13% lower than that of non-failing myocytes. Collectively, these data suggest that altering the termination level for local Ca(2+) release may represent a novel mechanism to increase SR Ca(2+) release and contractility during ECC.

Topics: Animals; Caffeine; Calcium; Calcium Signaling; Calpain; Calsequestrin; Cells, Cultured; Central Nervous System Stimulants; Heart Failure; Myocytes, Cardiac; Rabbits; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

Genetic depletion of the dystrophin-related glycoprotein (DRGP) complex causes cardiomyopathy in animals and humans. The present study was undertaken to explore the possible involvement of alterations in DRGP in the development of the right ventricular failure in monocrotaline-administered rats (MCT rats). At the 6th and 8th weeks after subcutaneous administration of 60 mg/kg monocrotaline, echocardiographic examination showed that cardiac output indices were decreased and that the right ventricular Tei indices were increased, suggesting that right ventricular failure occurs, at the latest, by 6 weeks after monocrotaline-administration. The levels of alpha- and beta-sarcoglycan and beta-dystroglycan in the right ventricle of the MCT rats at the 6th and 8th weeks were markedly decreased, and these decreases were inversely related to the increase in the right ventricular Tei index of the MCT-administered animals. The content and activity of the Ca(2+)-activated neutral protease m-calpain in the right ventricle of the MCT rats were increased at the 4th to 8th weeks and those of matrix metalloproteinase-2, at the 6th and 8th weeks. These results suggest that m-calpain- and/or matrix metalloproteinase-2-mediated alterations in the contents of alpha-sarcoglycan, beta-sarcoglycan, and beta-dystroglycan may be involved in the development of right ventricular failure in MCT rats.

Topics: Animals; Calpain; Dystroglycans; Dystrophin-Associated Protein Complex; Heart Failure; Heart Ventricles; Hemodynamics; Hypertrophy, Right Ventricular; Male; Matrix Metalloproteinase 2; Monocrotaline; Myocardium; Rats; Rats, Wistar; Sarcoglycans; Time Factors; Ventricular Dysfunction, Right

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

The mechanism for the decrease in cardiomyocyte size with mechanical unloading is unknown. The calpain system regulates cardiomyocyte atrophy. We obtained samples from failing human hearts at the time of implantation and explantation of a left ventricular assist device. For mechanical unloading, we also heterotopically transplanted rat or mouse hearts for 1 week. The effect of calpain inhibition on cardiac atrophy was assessed in transplanted hearts overexpressing calpastatin. We measured transcript levels of calpain 1 and 2 in the human and the rodent model, as well as calpain activity, a calpain-specific degradation product and cardiomyocyte size in the two rodent models. Mechanical unloading of the failing human heart significantly increased calpain 2 gene expression. Transcript levels of calpain 1 and 2, calpain activity and a calpain-specific degradation product all significantly increased in the unloaded rat heart. Unexpectedly, in hearts of animals overexpressing calpastatin, cardiomyocyte size also decreased. Mechanical unloading of the mammalian heart activates the calpain system, although other proteolytic systems may compensate for decreased calpain activity when calpastatin is overexpressed.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Size; Disease Models, Animal; Gene Expression Regulation; Heart Failure; Heart Transplantation; Heart-Assist Devices; Humans; Male; Mice; Middle Aged; Myocardium; Myocytes, Cardiac; Rats; Time Factors; Transplantation, Heterotopic

Regulating the balance between synthesis and proteasomal degradation of cellular proteins is essential for tissue growth and maintenance, but the critical pathways regulating protein ubiquitination and degradation are incompletely defined. Although participation of calpain calcium-activated proteases in post-necrotic myocardial autolysis is well characterized, their importance in homeostatic turnover of normal cardiac tissue is controversial. Hence, we evaluated the consequences of physiologic calpain (calcium-activated protease) activity in cultured cardiomyocytes and unstressed mouse hearts. Comparison of in vitro proteolytic activities of cardiac-expressed calpains 1 and 2 revealed calpain 1, but not calpain 2, activity at physiological calcium concentrations. Physiological calpain 1 activation was evident in adenoviral transfected cultured cardiomyocytes as proteolysis of specific substrates, generally increased protein ubiquitination, and accelerated protein turnover, that were each inhibited by coexpression of the inhibitor protein calpastatin. Conditional forced expression of calpain 1, but not calpain 2, in mouse hearts demonstrated substrate-specific proteolytic activity under basal conditions, with hyperubiquitination of cardiac proteins and increased 26S proteasome activity. Loss of myocardial calpain activity by forced expression of calpastatin diminished ubiquitination of 1 or more specific myocardial proteins, without affecting overall ubiquitination or proteasome activity, and resulted in a progressive dilated cardiomyopathy characterized by accumulation of intracellular protein aggregates, formation of autophagosomes, and degeneration of sarcomeres. Thus, calpain 1 is upstream of, and necessary for, ubiquitination and proteasomal degradation of a subset of myocardial proteins whose abnormal accumulation produces autophagosomes and degeneration of cardiomyocytes with functional decompensation.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cardiomyopathy, Dilated; Cells, Cultured; Heart Failure; Homeostasis; Mice; Mice, Transgenic; Microscopy, Electron; Myocardium; Myocytes, Cardiac; Osmolar Concentration; Proteasome Endopeptidase Complex; Protein Isoforms; Proteins; Substrate Specificity; Transfection; Ubiquitin

Changes in proteolytic activity of the myocardium during the development of heart failure after left coronary artery ligation (CAL) of rats were examined. Hemodynamics of the rats at the eighth week (8w-CAL rat), but not at the second week (2w-CAL rat), after CAL showed the symptoms of chronic heart failure. Contents of mu-calpin and m-calpain, but not an intrinsic calpain inhibitor calpastatin, in the viable left ventricular muscle (viable LV) and the right ventricular muscle (RV) of the 2w-CAL and 8w-CAL rats were increased, which was associated with an elevation of intrinsic activities of leupeptin-sensitive, Ca(2+)-activated proteolysis in the cytosolic fractions of the viable LV and RV. Oral administration of 3 mg/kg/d trandolapril or 1 mg/kg/d candesartan from the second to eighth week after CAL improved the hemodynamics of 8w-CAL rats. The drug treatment attenuated the increases in mu-calpain and m-calpain contents and the elevation of the proteolytic activity of the viable LV and RV in the 8w-CAL rat. The drug treatment increased calpastatin content of the RV in the 8w-CAL rat. These results suggest that sustained activation of calpain is involved in the development of chronic heart failure and that trandolapril and candesartan prevent the activation of calpains after CAL.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Calcium-Binding Proteins; Calpain; Cytosol; Enzyme Activation; Heart Failure; Male; Myocardial Infarction; Myocardium; Organ Size; Rats; Rats, Wistar

To investigate the relation among myocardial angiotension II receptor (AT1/AT2) expression, calpain and cardiac function in patients with congestive heart failure (CHF).. Message RNA (mRNA) expression of AT1/AT2 receptor in myocardial tissue of 39 patients with CHF due to valvular heart disease and 8 control subjects were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR). Immunoprecipitation was used to assay the protein expression of u-calpain and m-calpain.. Pathological changes of myocardial tissue in CHF due to valvular heart disease showed typical myocardial remodeling. AT1 receptor mRNA expression was slightly increased in the patients with mild CHF than in the control subjects, but decreased in the moderate and severe CHF patients. No difference was observed in AT2 receptor mRNA expression among all the groups, but the ratio of AT1/AT2 decreased. The protein expression of u-calpain and m-calpain were positively correlated to the levels of cardiac function in patient with CHD.. The expression of AT1 receptor is down-regulated in moderate and severe CHF, the dominant receptor subtype is transformed to AT2. Cardiomyocyte apoptosis or death may be induced via AT2 receptor to activate calpain system, leading the deterioration of cardiac function.

Topics: Adult; Calpain; Case-Control Studies; Female; Heart Failure; Humans; Male; Middle Aged; Myocardium; Receptor, Angiotensin, Type 2; Ventricular Remodeling; Young Adult

Genetic depletion of dystrophin-related protein (DRP) complex causes cardiomyopathy in animals and humans. We found in a previous study that some types of DRP were degraded and that calpain content was increased in rats with non-genetically induced heart failure. The present study was aimed at examining the effects of an angiotensin-I-converting enzyme inhibitor (ACEI) trandolapril (Tra) or an angiotensin II type 1 receptor blocker (ARB) candesartan (Can), both of which are known to improve the pathophysiology of chronic heart failure (CHF) on degradation of DRP in failing hearts.. Coronary artery-ligated (CAL) and sham-operated rats (Sham rats) were treated orally with 3 mg/kg/day trandolapril (Tra) or 1 mg/kg/day candesartan (Can) from the 2nd to 8th week after surgery.. Hemodynamic parameters of CAL rats at the 8th week after CAL (8w-CAL) indicated heart failure. alpha-Sarcoglycan (SG) and dystrophin in the surviving left ventricle (surviving LV) of 8w-CAL rats decreased, whereas beta-, gamma-, and delta-SGs remained unchanged. Calcium-activated neutral proteases mu-calpain and m-calpain increased in the surviving LV at the 8th week of postmyocardial infarction. Proteolytic activity in the presence of 5 mM Ca2+ markedly increased at the 2nd and 8th weeks, whereas 50 microM Ca2+ slightly but significantly increased proteolysis of casein. Tra or Can treatment improved the hemodynamic parameters, attenuated changes in alpha-SG and dystrophin, and reversed both calpain contents and activities of the failing heart back to sham levels.. These results suggest that attenuation in calpain-induced degradation of DRP complex is a possible mechanism for the Tra- or Can-mediated improvement of the pathogenesis of CHF following myocardial infarction.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Benzimidazoles; Biphenyl Compounds; Blotting, Western; Calpain; Cytosol; Heart Failure; Hemodynamics; Indoles; Male; Models, Animal; Myocardium; Protein Isoforms; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Reverse Transcriptase Polymerase Chain Reaction; Sarcoglycans; Tetrazoles

To investigate the regulation of calcium sensitive signal substance calpain in signal transduction of myocardial remodeling in patients with congestive heart failure.. All 39 congestive heart failure (CHF) patients with rheumatic mitral valve stenosis disease were selected and 38 cases of healthy persons were included as controls. Cardiac function parameters were measured by echocardiography. The concentration of angiotension II (AngII) in plasma and myocardial tissues was determined by radio immunoassay (RIA). Western blot was used to assay the protein expression of calpain, cain/cabin 1, cain/cabin 1Delta, and calcineurin (CaN) phosphorylation.. The AngII concentrations in the plasma and myocardial tissues in patients with CHF were higher than those in the control group. Meanwhile the AngII concentrations positively correlated to the parameters of the cardiac dilation respectively but negatively correlated to the parameters of cardiac function. Pathological changes of myocardial tissues in CHF with valvular heart disease showed typical myocardial remodeling. The hypertrophy was dominant at early stage of CHF, while at the end stage the characteristics include disordered alignment of the myocytes, the discontinuity and dissolving of cardiomyofibrills, destroyed subcellular organs, and the hyperplasia of interstitial tissue. Compared to the control group, u-calpain, m-calpain, and cain/cabin 1Delta protein expression, CaN phosphorylation in myocardial tissues in CHF groups were highly expressed and their expressions were positively correlated to the severity of CHF. The expression of cain/cabin1 deceased and its expression was negatively correlated to the severity of CHF.. The degradation of cain/cabin 1 by calpain may play an important role by causing the activation of CaN signal pathway in myocardial remodeling mediated by renin angiotension system in CHF.

Topics: Adaptor Proteins, Signal Transducing; Adult; Angiotensin II; Calcineurin; Calpain; Female; Heart Failure; Humans; Male; Middle Aged; Myocardium; Signal Transduction; Ventricular Remodeling

An analytic method based on simulation and modeling of long-term 45Ca(2+) efflux data was used to estimate steady-state Ca(2+) contents (nmolCa(2+)g(-1)tissuewetwt.) and exchange fluxes (nmolCa(2+)min(-1)g(-1)tissuewetwt.) for extracellular and intracellular compartments in in vitro resting diaphragm from congestive heart failure (CHF, n=12) and sham-operated (SHAM, n=10) rats. Left hemidiaphragms were excised from experimental animals, loaded with 45Ca(2+) for 1h, and washed out with 45Ca(2+)-free perfusate for 8h. Tissue from the right hemidiaphragm was used to assess single-fiber cross-sectional area (CSA) as well as the relative proteolytic activity of Ca(2+)-dependent calpain. Kinetic analysis of 45Ca(2+) efflux data revealed that CHF was associated with increased Ca(2+) contents of extracellular and intracellular compartments as well as increased Ca(2+) exchange fluxes for all compartments. This accounted for the model prediction of a 250% increase in total diaphragm Ca(2+). Furthermore, single-fiber CSA was decreased 12% and proteolytic activity of calpain was increased twofold in CHF diaphragm relative to SHAM.. The kinetic data are consistent with the hypothesis that diaphragm Ca(2+) overload in CHF required all intercompartmental Ca(2+) fluxes to increase. The potential relationships among Ca(2+) overload, increased activity of calpain, and wasting of the diaphragm in CHF are discussed.

Topics: Animals; Calcium; Calcium Signaling; Calpain; Cell Compartmentation; Chronic Disease; Diaphragm; Disease Models, Animal; Extracellular Space; Heart Failure; Homeostasis; Intracellular Fluid; Kinetics; Male; Models, Biological; Muscle Fibers, Skeletal; Muscular Atrophy; Rats; Rats, Wistar; Respiration Disorders; Up-Regulation

A hypothesis is presented that explains one of the mechanisms by which a heart starts to fail. The hypothesis is that myocardial function of an overloaded or otherwise stressed heart may become impaired by cellular troponin degradation in vital cardiomyocytes. The troponins (I, T and C) regulate actin-myosin interaction, thereby controlling contraction and relaxation. Troponins have been shown to be targets of activated calpain I. This enzyme, that is activated by elevated intracellular Ca2+ concentrations, such as occurs during ischemia, degrades troponins, leading to impaired interaction between actin and myosin and, thereby, less contractile force. Several reports about troponin degradation in viable myocardium support this hypothesis. Also, results are discussed that demonstrate the presence of immunoreactive troponin fragments in plasma under conditions in which myocardial necrosis can be excluded or is unlikely. The hypothesis implicates that release of troponin and/or troponin degradation products is not specific for necrotic myocardium but may occur from viable myocardium as well. To test this hypothesis, several lines of research are suggested. If the hypothesis is not rejected in the near future, the concept that a positive troponin test reflects 'even microscopic zones of myocardial necrosis' as used by the Joint ESC/ACC Committee for the Redefinition of Myocardial Infarction [The Joint European Society of Cardiology/American College of Cardiology Committee for the Redefinition of Myocardial Infarction. Myocardial infarction redefined-A consensus document of the Joint European Society of Cardiology/American College of Cardiology Committee for the Redefinition of Myocardial Infarction. Eur Heart J 2000;21:1502-1513], should be withdrawn.

Topics: Animals; Calcium; Calpain; Enzyme Activation; Heart Failure; Humans; Myocardial Stunning; Myocytes, Cardiac; Physical Endurance; Troponin; Ventricular Dysfunction, Left