calpain and Atrial-Fibrillation

Calpain is an intracellular Ca(2+)-activated protease and an important mediator of the actions of calcium. Cleavage by calpain is critical in a variety of calcium-regulated cellular processes such as muscle contraction, neuronal excitability, secretion, signal transduction, cell proliferation, differentiation, cell cycle progression, and apoptosis. Deregulation of calpain caused by a disruption of calcium homeostasis during cardiac pathologies such as atrial fibrillation, heart failure, hypertrophy, or ischemia reperfusion, is critically involved in the myocardial damage. This review will summarize the physiologic and pathophysiologic basis of calpain. Atrial fibrillation is chosen as one example to explain the specific consequences of an increased calpain activity in cardiac muscle.

Topics: Animals; Atrial Fibrillation; Calpain; Cardiovascular Agents; Cysteine Proteinase Inhibitors; Enzyme Activation; Fibrinolytic Agents; Heart Atria; Humans; Myocardium; Risk Assessment; Risk Factors

In the heart, changes in velocity and in patterns of conduction of myocardial electrical activity can affect cardiac rhythm and the coordination of contraction. Abnormal electrical coupling between cardiomyocytes through gap junctions is, therefore, considered an important factor in various pathophysiologic conditions. In the present report we summarize the literature on gap junctions and their structural proteins, the connexins, in the normal and fibrillating atrium. Putative implications of the recently reported remodelling of atrial gap junctions for stability of the arrhythmia will be discussed. Also the reversibility of the remodelling process will be addressed in the light of a potentially new therapeutic target for controlling the progression of atrial fibrillation (AF).

Topics: Animals; Atrial Fibrillation; Calpain; Chronic Disease; Connexins; Electrophysiology; Gap Junctions; Heart Atria; Humans; Myocardium

An important acknowledgement of the last several years is that atrial fibrillation (AF) modifies the electrical properties of the atrium in a way that promotes its occurrence and maintenance. This arrhythmogenic electrophysiological remodeling is well established, but can not explain by itself that 'AF begets AF'. This review describes molecular changes involving rapid functional alterations and slower changes in protein expression that cause electrical remodeling and contractile dysfunction in AF. An important molecular feature of AF is the reduction in L-type Ca(2+) channel function and protein expression. This reduction may serve to protect the cell against a potentially lethal Ca(2+) overload resulting from the increased activation rate in AF. Further, the review discusses the possible role of proteolytic systems, notably the calpains, as a mechanism linking Ca(2+) overload to reduced protein expression. Thus, it appears that the elaborate molecular changes in AF are directed primarily at protecting the myocyte from cellular stress. However, such early protection occurs at the expense of electrophysiological changes that promote the long-term maintenance of AF.

Topics: Animals; Atrial Fibrillation; Atrial Function; Calcium; Calcium Channels, L-Type; Calpain; Cardiac Pacing, Artificial; Chronic Disease; Connexins; Gene Expression; Heart Atria; Humans

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

Calpain, calcineurin (CaN), and nuclear factor of activated T cell (NFAT) play a key role in the development of atrial fibrillation. Patients with valvular heart disease (VHD) are prone to develop atrial fibrillation (AF). Thus, our current study was aimed at investigating whether activation of calpain-CaN-NFAT pathway is associated with the incidence of AF in the patients with VHD and diabetes. The expressions of calpain 2 and alpha- and beta-isoforms of CaN catalytic subunit (CnA) as well as NFAT-c3 and NFAT-c4 were quantified by quantitative reverse transcription-polymerase chain reaction in atrial tissues from 77 hospitalized patients with VHD and diabetes. The relevant protein content was measured by Western blot and calpain 2 in human atrium was localized by immunohistochemistry. We found that the expressions of calpain 2, CnA alpha and CnA beta, and NFAT-c3 but not NFAT-c4 were significantly elevated in the samples from patients with AF compared to those with sinus rhythm (SR). Elevated protein levels of calpain 2 and CnA were observed in patients with AF, and so was the enhanced localization of calpain 2. We thereby concluded that CaN together with its upstream molecule, calpain 2, and its downstream effector, NFAT-c3, might contribute to the development of AF in patients with VHD and diabetes.

Topics: Adult; Atrial Appendage; Atrial Fibrillation; Blotting, Western; Calcineurin; Calpain; Diabetes Complications; Female; Heart Valve Diseases; Humans; Immunohistochemistry; Male; Middle Aged; NFATC Transcription Factors; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

To investigate the relationship between the protein expression of calpain-2 and calcineurin (CaN) and atrial fibrillation (AF) in patient with valvular heart disease (VHD).. A total of 40 patients who underwent valve replacement surgery in our hospital from March 2013 to March 2014, right atrial appendages were excised during operation and patients were divided into sinus rhythm (SR) group (n = 17) and AF group (n = 23). The protein expression of calpain-2 and the α-isoform of CaN catalytic subunit (CnA) in the right atrial appendages were determined by Western blot.. The protein levels of the full-length CnAa (60,000), the 45,000 fragment of CnAa without autoinhibitory domain, and calpain-2 were significantly upregulated in the AF group compared to the SR group (1.25 ± 0.51 vs. 0.76 ± 0.37, 1.08 ± 0.37 vs. 0.76 ± 0.25, and 0.82 ± 0.44 vs. 0.51 ± 0.19, respectively, all P < 0.05).. Activated calpain-2-CaN signal pathway might be involved in the pathogenesis of AF.

Topics: Atrial Appendage; Atrial Fibrillation; Blotting, Western; Calcineurin; Calpain; Heart Valve Diseases; Humans; Up-Regulation

Atrial fibrillation (AF) is characterized by structural remodeling, contractile dysfunction, and AF progression. Histone deacetylases (HDACs) influence acetylation of both histones and cytosolic proteins, thereby mediating epigenetic regulation and influencing cell proteostasis. Because the exact function of HDACs in AF is unknown, we investigated their role in experimental and clinical AF models.. Tachypacing of HL-1 atrial cardiomyocytes and Drosophila pupae hearts significantly impaired contractile function (amplitude of Ca(2+) transients and heart wall contractions). This dysfunction was prevented by inhibition of HDAC6 (tubacin) and sirtuins (nicotinamide). Tachypacing induced specific activation of HDAC6, resulting in α-tubulin deacetylation, depolymerization, and degradation by calpain. Tachypacing-induced contractile dysfunction was completely rescued by dominant-negative HDAC6 mutants with loss of deacetylase activity in the second catalytic domain, which bears α-tubulin deacetylase activity. Furthermore, in vivo treatment with the HDAC6 inhibitor tubastatin A protected atrial tachypaced dogs from electric remodeling (action potential duration shortening, L-type Ca(2+) current reduction, AF promotion) and cellular Ca(2+)-handling/contractile dysfunction (loss of Ca(2+) transient amplitude, sarcomere contractility). Finally, atrial tissue from patients with AF also showed a significant increase in HDAC6 activity and reduction in the expression of both acetylated and total α-tubulin.. AF induces remodeling and loss of contractile function, at least in part through HDAC6 activation and subsequent derailment of α-tubulin proteostasis and disruption of the cardiomyocyte microtubule structure. In vivo inhibition of HDAC6 protects against AF-related atrial remodeling, disclosing the potential of HDAC6 as a therapeutic target in clinical AF.

Topics: Acetylation; Animals; Atrial Fibrillation; Atrial Remodeling; Calpain; Cardiac Pacing, Artificial; Dogs; Drosophila; Drosophila Proteins; HeLa Cells; Histone Deacetylase 6; Histone Deacetylases; Humans; Hydroxamic Acids; Indoles; Mice; Microtubules; Myocardial Contraction; Myocytes, Cardiac; Tubulin

The aim of this study was to investigate the correlation between the change in the expression of atrial calpains and electrical, molecular and structural remodeling during aging and atrial fibrillation (AF). Adult and aged canines in sinus rhythm (SR) and with persistent AF (induced by rapid atrial pacing) were investigated. A whole-cell patch clamp was used to measure the L-type Ca2+ current (ICa-L) in cells in the left atrium. The mRNA and protein expression of the L-type calcium channel alc subunit (LVDCCa1c) and calpains were measured by quantitative (q)PCR and western blot analysis. Histopathological and ultrastructural changes were analyzed via light and electron microscopy. The quantity of apoptotic myocytes was determined by a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay. In SR groups, atrial cells of the aged canines exhibited a longer action potential (AP) duration to 90% repolarization (APD90), lower AP plateau potential and peak ICa-L current densities (P<0.05). In the adult and aged groups, AF led to a higher maximum diastolic potential, an increase in AP amplitude and decreases in APD90, AP plateau potential and peak ICa-L densities (P<0.05). Compared with the control group, the mRNA and protein expression levels of LVDCCa1c were decreased in the aged groups; however, the mRNA and protein expression of calpain 1 was increased in the adult and the aged groups with AF (P<0.05). Samples of atrial tissue exhibited abnormal histopathological and ultrastructural changes, such as accelerated fibrosis and apoptosis with aging and in AF. Age-related alterations in atrial tissues were attributed to the increased expression of calpain 1. The general pathophysiological alterations in normal aged atria may therefore produce a substrate that is conducive to AF.

Topics: Action Potentials; Aging; Animals; Apoptosis; Atrial Fibrillation; Blotting, Western; Calpain; Cells, Cultured; Dogs; Electrophysiology; Heart Atria; Myocytes, Cardiac; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Atrial fibrillation (AF) in mitral regurgitation (MR) is a complex disease where multiple factors may induce left-atrial structural remodeling (SR). We explored the differential SR of the left-atrial posterior wall (LAPW) of patients affected by MR with or without persistent AF, and the expression of key proteins involved in its pathogenesis.. Light microscopy of LAPW samples from 27 patients with MR and persistent AF (group 1), 33 with MR in sinus rhythm (group 2), and 15 autopsy controls (group 3) was used to measure myocyte diameter, percentage of myocytolytic myocytes, interstitial fibrosis, and capillary density; RT-PCR and Western blotting were used to assess the mRNA and protein levels of SOD-1, SOD-2, HO-1, calpain, MMP-2, MMP-9, TIMP-1, TIMP-2, and VEGF; immunofluorescence was used to locate these proteins. Myocyte diameter was similar in groups 1 and 2, but larger than controls. Compared to group 2, group 1 had more myocytolytic myocytes (20.8 ± 5.6% vs 14.7 ± 4.5%; P < 0.0001), increased interstitial fibrosis (10.4 ± 5.1% vs 7.5 ± 4.2%; P < 0.05), and decreased capillary density (923 ± 107 No/mm(2) vs 1,040 ± 100 No/mm(2); P < 0.0001). All of the proteins were more expressed in groups 1 and 2 than in controls. The protein and mRNA levels of SOD-1, SOD-2, MMP-2, and MMP-9 were higher in group 1 than in group 2.. The LAPW of MR patients with or without AF shows considerable SR. The former has more severe histopathological changes and higher levels of proteins involved in SR, thereby reaching a threshold beyond which the sinus impulse cannot normally activate atrial myocardium.

Topics: Adult; Aged; Aged, 80 and over; Arrhythmia, Sinus; Atrial Fibrillation; Autopsy; Blotting, Western; Calpain; DNA, Complementary; Female; Fluorescent Antibody Technique; Heart Atria; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 2; Microscopy, Confocal; Middle Aged; Mitral Valve Insufficiency; Myocytes, Cardiac; Platelet Endothelial Cell Adhesion Molecule-1; Real-Time Polymerase Chain Reaction; RNA; Superoxide Dismutase; Superoxide Dismutase-1; Vascular Endothelial Growth Factor A

The most common clinical tachycardia, Atrial Fibrillation (AF), is a progressive disease, caused by cardiomyocyte remodeling, which finally results in contractile dysfunction and AF persistence. Recently, we identified a protective role of heat shock proteins (HSPs), especially the small HSPB1 member, against tachycardia remodeling in experimental AF models. Our understanding of tachycardia remodeling and anti-remodeling drugs is currently hampered by the lack of suitable (genetic) manipulatable in vivo models for rapid screening of key targets in remodeling. We hypothesized that Drosophila melanogaster can be exploited to study tachycardia remodeling and protective effects of HSPs by drug treatments or by utilizing genetically manipulated small HSP-overexpressing strains. Tachypacing of Drosophila pupae resulted in gradual and significant cardiomyocyte remodeling, demonstrated by reduced contraction rate, increase in arrhythmic episodes and reduction in heart wall shortening, compared to normal paced pupae. Heat shock, or pre-treatment with HSP-inducers GGA and BGP-15, resulted in endogenous HSP overexpression and protection against tachycardia remodeling. DmHSP23 overexpressing Drosophilas were protected against tachycardia remodeling, in contrast to overexpression of other small HSPs (DmHSP27, DmHSP67Bc, DmCG4461, DmCG7409, and DmCG14207). (Ultra)structural evaluation of the tachypaced heart wall revealed loss of sarcomeres and mitochondrial damage which were absent in tachypaced DmHSP23 overexpressing Drosophila. In addition, tachypacing induced a significant increase in calpain activity, which was prevented in tachypaced Drosophila overexpressing DmHSP23. Tachypacing of Drosophila resulted in cardiomyocyte remodeling, which was prevented by general HSP-inducing treatments and overexpression of a single small HSP, DmHSP23. Thus, tachypaced D. melanogaster can be used as an in vivo model system for rapid identification of novel targets to combat AF associated cardiomyocyte remodeling.

Topics: Animals; Atrial Fibrillation; Calpain; Disease Models, Animal; Diterpenes; Drosophila melanogaster; Drosophila Proteins; Gene Expression; Gene Expression Regulation; Heart; Heat-Shock Proteins; Heat-Shock Proteins, Small; Myocardial Contraction; Oximes; Piperidines; Tachycardia

Changes in connexins and calpains of the myocardial sleeve of the pulmonary vein and the left atrium were investigated in chronic atrial fibrillation (AF) animal models.. There are no reports of changes in the calpain system and connexins in the pulmonary vein where AF is initiated.. An AF animal model was prepared by rapid pacing of the right atrium for 8 weeks. Histological changes of pulmonary veins were analyzed by Masson trichrome staining, and mRNA as well as protein expression of connexins and calpains were measured by real-time fluorescence quantitative PCR and Western blotting.. In AF dogs, the fibrous collagen reticulum surrounding individual myocardial cells was reduced or disrupted. In the myocardial sleeve of the AF dogs, Cx40 protein expression was significantly downregulated compared to the control group (60.78 +/- 10.91 vs. 88.31 +/- 14.73, p < 0.05), but calpain 1 was significantly upregulated (94.00 +/- 7.24 vs. 81.77 +/- 5.82, p < 0.05), and they were negatively correlated (r = -0.66, p < 0.05). Cx40 protein expression was significantly lower in the myocardial sleeve tissue than in the left atrium in the AF dogs (60.78 +/- 10.91 vs. 91.38 +/- 17.16, p < 0.05).. Varied gap junctional remodeling around the pulmonary vein may be one of the underlying mechanisms for pulmonary vein-left atrial reentry. During AF, the calpain system of the myocardial sleeve tissue is activated and may hydrolyze Cx40 protein, which is a possible important molecular mechanism for gap junctional remodeling that merits further investigation.

Topics: Animals; Atrial Fibrillation; Calcium-Binding Proteins; Calpain; Connexin 43; Connexins; Disease Models, Animal; Dogs; Female; Gap Junction alpha-5 Protein; Gap Junctions; Heart Atria; Male; Pacemaker, Artificial; Pulmonary Veins; RNA, Messenger

Oxidative stress has recently been implicated in atrial fibrillation (AF); however, the mechanisms remain unclear. Herein, we hypothesize that probucol can attenuate atrial structure remodeling.. Twenty dogs were randomly divided into sham-operated, control, and probucol-treated groups. We identified apoptosis and histopathological changes in the atria. Oxidative stress was measured by lipid peroxidation and echocardiographic examinations were performed.. Atrial apoptosis indexes were dramatically decreased in the probucol-treated group compared to the control group. Relative to the control group, the percentage of myolysis was dramatically decreased in the probucol-treated group (p < 0.01). There was less lipid peroxidation in the probucol-treated group than the control group. Atrial function was dramatically elevated in the probucol-treated group.. The results of this study indicate that the antioxidant probucol suppresses atrial structural remodeling and may act as a new therapy for AF.

Topics: Animals; Antioxidants; Apoptosis; Atrial Fibrillation; bcl-2-Associated X Protein; Calpain; Caspase 3; Dogs; Female; Heart Atria; Lipid Peroxidation; Male; Myocytes, Cardiac; Oxidative Stress; Probucol; Proto-Oncogene Proteins c-bcl-2

Topics: Animals; Apoptosis; Atrial Fibrillation; Calpain; Caspase 7; Dogs; Myocytes, Cardiac; Oligopeptides

This study was designed to evaluate the effects of a calpain inhibitor on cardiac muscle apoptosis in rapid pacing canine atrial fibrillation (AF) models.. Twenty one dogs were divided into three groups: a sham operation group, a control AF group and a calpain inhibitor group. Sustained AF was induced by rapid right atrium pacing at 600 beats per minute. N-Acetyl-Leu-Leu-Met (1.0 mg/kg/day) was administered in the calpain inhibitor group for three weeks. The activity of calpain I and cardiomyocyte apoptosis were measured by fluorometry and TUNEL assay, respectively. Protein expression of caspase-3 was detected by Western blot. The localizations of caspase-3, caspase-8, bcl-2 and ARC were assessed by immunohistochemistry.. In comparison to the sham operation group, the activity of calpain I was significantly increased in the control AF group (2.3 fold, p < 0.001), and decreased in the calpain inhibitor group (1.1 fold, p < 0.005). The calpain activity correlated with the apoptosis index (r = 0.9, p < 0.05). The apoptosis index was 1.0 +/- 0.2%, 11.8 +/- 6.8% and 3.5 +/- 2.1% in the sham operation group, control AF group and calpain inhibitor group, respectively. In the sham operation group, control AF group and calpain inhibitor group, the expressions of caspase-3 (13.0 +/- 1.9%, 52.8 +/- 4.3% and 33.6 +/- 3.7%), caspase-8 (40.1 +/- 5.3%, 92.6 +/- 6.5% and 55.3 +/- 5.9%), bcl-2 (65.8 +/- 6.1%, 52.0 +/- 5.7% and 69.9 +/- 5.3%) and ARC (70.2 +/- 8.6%, 68.8 +/- 7.3% and 81.5 +/- 8.8%) were calculated as immunohistochemical indexes, respectively.. The calpain inhibitor N-Acetyl-Leu-Leu-Met attenuated apoptosis through a complicated network of apoptosis-related proteins, which may result in improvement of structural remodeling in atrial fibrillation.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Atrial Fibrillation; Blotting, Western; Body Weight; Calpain; Caspase 3; Dogs; Immunohistochemistry; In Situ Nick-End Labeling; Myocardial Contraction; Myocytes, Cardiac; Oligopeptides; Organ Size; Protease Inhibitors; RNA, Messenger

To evaluate the effects of oxidative stress on the protein expression of atrial calpain I and pathohistological and ultrastructural changes of atrial myocardium in atrial fibrillation (AF).. Twenty dogs were all implanted with pacemaker in a subcutaneous pocket and attached to a screw-in epicardial lead in right atrial appendage. They were randomly divided into 3 groups: sham-operation group (n = 6 without pacing), control group (n = 7 per minutes for 6 weeks), and probucol group (n = 7, pacing 1 week after recovery for 6 weeks, and administration of probucol 100 mg x kg(-1) x d(-1) 1 week before pacing till the end of pacing). One thin silicon plaque containing 4 pairs of electrodes were sutured to the right atrium. The dogs in control group, probucol group were paced at 400 beats per minutes for 6 weeks. Then the dogs were killed with their hearts taken out. The expression of atrial calpain I was measured by Western-blotting and immunohistochemistry. The pathohistological and ultrastructural changes in atrial tissue were tested by light and electron microscopy. The inducibility and duration of AF were measured in the control group and probucol group. The indexes of oxidative stress total anti-oxidation capability (T-AOC), malonyldiadehyde (MDA), and scavenging activities of superoxide anion (O2-) radical were measured by colorimetric method.. The percentage of myolysis in the left and right atria of the control group were (53.6 +/- 11.8)% and. (58.5 +/- 9.2)% respectively, significantly higher than those of the sham operation group [(4.4 +/- 3.1)% and (4.1 +/- 2.9)% respectively, both P < 0.01]. The percentage of myolysis in the left and right atria of the probucol group were (12.3 +/- 3.2)% and (12.0 +/- 2.6)% respectively, both significantly lower than those of the control group (both P < 0.01). The protein expression of calpain I of the control group was significantly higher than that of the sham-operation group, and the protein expression of calpain I of the probucol group was significantly lower than that of the control group. The AF inducibility rate after pacing of the probucol group was 60%, significantly lower than that of the control group (92.9%, P < 0.01). The average AF duration time after pacing of the probucol group was (601 +/- 328) s, significantly shorter than that of the control group (1458 +/- 498) s. The indexes of oxidative stress in probucol group were lower than the level in control group. The MDA levels of the probucol group was (3.08 +/- 0.20) mmol/mg protein, significantly lower than that of the control group (4.15 +/- 0.23) mmol/mg protein). The anti-O2- and T-AOC level of the probucol group were 279 +/- 20 U/g protein and 30.5 +/- 1.3 nmol/mg protein, both significantly higher than those of the control group (215 +/- 16 U/g protein and 25.6 +/- 1.5 nmol/mg protein respectively, both P < 0.01). There were more sarcomere vacuolization and dissolution in atrial myocytes in the control group than in the sham operation group. And the pathohistological and ultrastructural changes of the probucol were lighter than those of the control group.. Probucol prevents the pathohistological and ultrastructural changes in atrial myocardium by inhibiting calpain I expression, thus suppressing atrial structural remodeling, and preventing the induction and promotion of AF.

Topics: Animals; Atrial Fibrillation; Calpain; Disease Models, Animal; Dogs; Female; Heart Atria; Male; Microscopy, Electron, Transmission; Myocardium; Oxidative Stress

The self-perpetuation of atrial fibrillation (AF) is associated with atrial remodeling, including the degradation of the myofibril structure (myolysis). Myolysis is related to AF-induced activation of cysteine proteases and underlies loss of contractile function. In this study, we investigated which proteases are involved in the degradation of myofibrillar proteins during AF and whether their inhibition leads to preservation of contractile function after AF. In tachypaced HL-1 cardiomyocytes and atrial tissue from AF and control patients, degradation of myofibrillar proteins troponin (cTn) T, I, C, human cTnT and actin was investigated by Western blotting, and contractile function was analyzed by cell-shortening measurements. The role of major proteases was determined by applying specific inhibitors. Tachypacing of HL-1 cardiomyocytes induced a gradual and significant degradation of cTns but not actin, and caused contractile dysfunction. Both were prevented by inhibition of calpain but not by inhibition of caspases or the proteasome. In patients with persistent AF, a significant degradation of cTnT, cTnI and cTnC was found compared to sinus rhythm or paroxysmal AF, which correlated significantly with both calpain activity and the amount of myolysis. Additionally, by utilizing tachypaced human cTnT-transfected HL-1 cardiomyocytes, we directly showed that the degradation of human cTnT was mediated by calpain and not by caspases or proteasome. Our results suggest that calpain inhibition may therefore represent a key target in combating AF-related structural and functional remodeling.

Topics: Actins; Aged; Atrial Fibrillation; Biomarkers; Calpain; Caspases; Gene Expression Regulation; Heart Atria; Humans; Middle Aged; Models, Biological; Muscle Contraction; Myocytes, Cardiac; Proteasome Endopeptidase Complex; Troponin

Atrial fibrillation (AF) is accompanied by atrial structural remodeling. Calpain activity is induced during AF. To test a causal relationship between calpain activation and atrial structural changes, N-acetyl-Leu-Leu-Met (ALLM), a calpain inhibitor, was utilized in a canine AF model.. Fifteen dogs were randomly divided into 3 groups: sham-operated group, control group and calpain inhibitor group; each with 5 dogs. Sustained AF was induced by rapid right atrium pacing at 600 beats per minute for 3 weeks. ALLM was administered at a dosage of 1.0 mg x kg(-1) x d(-1) in the calpain inhibitor group. Three weeks later, the proteolysis, protein expression of TnT and myosin, calpain I localization and expression and structural changes were examined in left atrial free walls, right atrial free walls and the interatrial septum respectively. Atrial size and contractile function were also measured by echocardiography.. Long-term rapid atrial pacing induced marked structural changes such as enlarged atrial volume, myolysis, degradation of TnT and myosin, accumulation of glycogen and changes in mitochondrial shape and size, which were paralleled by an increase in calpain activity. The positive correlation between calpain activity and the degree of myolysis (r(s) = 0.90 961, P < 0.0001) was demonstrated. In addition to structural abnormalities, pacing-induced atrial contractile dysfunction was observed in this study. The pacing-induced atrial structural alterations and loss of contractility were partially prevented by the calpain inhibitor ALLM.. Activation of calpain represents key features in the progression towards overt structural remodeling. Calpain inhibitor, ALLM, suppressed the increased calpain activity and reversed structural remodeling caused by sustained atrial fibrillation in the present model. Calpain inhibition may therefore provide a possibility for therapeutic intervention in AF.

Topics: Animals; Atrial Fibrillation; Calpain; Cysteine Proteinase Inhibitors; Disease Models, Animal; Dogs; Heart Atria; Myosins; Troponin T

Owing to relative inefficacy and side effects of currently available antiarrhythmic drugs, current interest has shifted to treatments that target atrial fibrillation (AF) substrate. It has been suggested that calpain-induced atrial structural remodelling is under the control of renin-angiotensin system during AF. The purpose of this study is to investigate the effects of cilazapril and valsartan on the mRNA and protein expression of atrial calpains and atrial structural remodelling in AF dogs induced by chronic rapid atrial pacing. Twenty-seven dogs were randomly divided into sham-operated group (n = 6), control group (n = 7), cilazapril group (n = 7) and valsartan group (n = 7). One thin silicon plaque containing 4 pairs of electrodes was sutured to each atrium. A pacemaker was implanted in a subcutaneous pocket and attached to a screw-in epicardial lead in the right atrial appendage. The dogs in control group, cilazapril group and valsartan group were paced at 400 beats per minutes for 6 weeks. The dogs in cilazapril and valsartan groups received cilazapril (1mg x kg(-1)x d(-1)) or valsartan (30mg x kg(-1) x d(-1)) 1 week before rapid atrial pacing until pacing stop respectively. Transthoracic and transoesophageal echocardiographic examinations were performed in order to detect the changes of left atrium volume and contractile function. The inducibility and duration of AF were measured in all the groups. The expressions of atrial calpain I and calpain II mRNA were semi-quantified by reverse transcription-polymerase chain reaction. The protein levels of calpain I and calpain II in atrial myocardium were measured by Western-blot method. Pathohistological and ultrastructural changes in atrial tissue were tested by light and electron microscopy. Compared with the sham-operated control group, dramatic smaller left atrium and left atrial appendage volumes and significant higher atrial contractile function were observed in the cilazapril and valsartan groups. After 6-week atrial tachy-pacing, the mRNA and protein expressions of calpain I increased dramatically in the control group than that in the sham group, tissue calpain protein expression in all groups significantly correlated with the myolysis (r = 0.89, P < 0.01). Cilazapril and valsartan could significantly inhibit the gene and protein expressions of calpain I. No differences were found in the expression of calpain II mRNA and protein between the groups. Compared with atrial myocytes obtained from sham

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Atrial Fibrillation; Blotting, Western; Calpain; Cilazapril; Disease Models, Animal; Dogs; Female; Gene Expression; Gene Expression Regulation; Heart Atria; Male; Myocytes, Cardiac; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazoles; Ultrasonography; Valine; Valsartan

To study the relation of the structural remodeling processes and activation of calpain I.. Fifteen dogs were randomly divided into three groups. The dogs in pacing group (n=5) and inhibitor group (n=5) were subjected to 3 weeks of rapid atrial pacing at 600 beats/min, control dogs (n=5) were in sham-operated group. The dogs in inhibitor group were administered intravenous N-Acetyl-Leu-Leu-Met (ALLM), a calpain inhibitor, and in pacing group and sham-operated group were administered intravenous DMSO. The activity of calpain I was measured by hydrolyzing Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin. The ultrastructure of atrium was examined by light and electron microscopy. TnT expression was assessed by Western blot. Echocardiography examination was performed in all the three groups.. Calpain I activity was significantly increased in pacing group (2.3-fold, P<0.01), and decreased in inhibitor group (1.1-fold, P>0.05), compared to sham-operated group respectively. The percentages of myolysis were (76.7 +/- 5.9)% and (20.8 +/- 8.1)% in pacing group and inhibitor group respectively (P<0.01). TnT expression decreased in the rapid pacing-induced persistent atrial fibrillation, and these effects were inhibited by calpain I inhibitor ALLM. The area and volume of left atrium tended to increase after 3 weeks ALLM treatment in inhibitor group, but the change was not as prominent as in pacing group (P<0.05).. ALLM can decrease calpain I activity, and prevent canine atrial cardiomyocyte structural remodeling during atrial fibrillation. This study provided a capacity of atrial cardiomyocyte protection.

Topics: Animals; Atrial Fibrillation; Atrial Function, Left; Calpain; Cardiac Pacing, Artificial; Disease Models, Animal; Dogs; Heart Atria; Myocardium; Troponin T

To test the causal relationship between calpain activation and atrial structural changes during atrial fibrillation (AF).. The tip of a spiral mono-polar pacing lead was fixed to the right atrial appendages of 15 dogs randomly divided into 3 equal groups: calpain inhibitor group, undergoing continuous pacing with the impulse of 600 beats/min for 3 weeks and intravenous injection of N-acetyl-Leu-Leu-Met (ALLM), a calpain inhibitor for 3 weeks; control group, undergoing continuous pacing and intravenous injection of dimethyl sulfoxide (DMSO; and sham operation group, given DMSO injection without pacing. Ultrasonography was used to observe the changes of the structures of left atrium and left atrial appendage and the heart function as well. Specimens of atrial muscles were obtained. Calpain 1 activity was detected by Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin method. HE staining was conducted to observe the myolysis. Western blotting was used to detect the protein expression of troponin I (TnI) and myosin.. The left atrial ejection fraction (LAEF) of the ALLM group was (41 +/- 6)%, significantly higher than that of the control group [(34 +/- 9)%, P < 0.05]. The left atrial appendage ejection fraction (LAAEF) of the ALLM group was (41 +/- 6)%, significantly higher than that of the control group [(35 +/- 6)%, P < 0.05]. Myolysis was extensive in the control group [(71.5 +/- 10.2)%], relatively rare in the ALLM group [(12.3 +/- 16.5)%], and was not seen in the sham operation group, with significantly differences among the 3 groups (all P < 0.01). The calpain 1 activity was positively correlated with the degree of myolysis (r(s) = 0.90 961, P < 0.01). The TnI level of the control group was (43 +/- 12)% that of the sham operation group (P = 0.001), the TnI level of the ALLM group was (51 +/- 11)% that of the sham operation group (P = 0.002) and was significant higher than that of the control group (P = 0.01). The level of myosin of the control group was (51 +/- 11)% that of the sham operation group (P = 0.002), and that of the ALLM group was (149 +/- 33)% that of the control group (P = 0.005).. Activation of and upregulation of expression of calpain participate in the structural remodeling of left atrial cardiac muscle and contractile dysfunction. Calpain inhibitor suppresses the increased calpain activity and reverses the structural remodeling of sustained atrial fibrillation. Calpain inhibition may therefore provide a possibility for therapeutic intervention in AF.

Topics: Animals; Atrial Fibrillation; Blotting, Western; Calpain; Disease Models, Animal; Dogs; Heart; Injections, Intravenous; Myocardial Contraction; Myocardium; Myosins; Oligopeptides; Random Allocation

The aim of the present study was to detect the expression of calpain-I, calpastatin, caspase-3 and apoptosis in the left atria of patients with rheumatic heart disease (RHD), and to find the association of these factors. Also, it was intended to investigate the effect of the above factors on the mechanism of atrial fibrillation (AF).. 43 patients with RHD undergoing valve-replacement were included, 15 patients with regular sinus rhythm (Group RSR), 8 patients with paroxysmal AF (Group AF1) and 20 patients with permanent AF (Group AF2). Western blot was used to examine the content of calpain-I, caspase-3 and calpastatin. The apoptosis index (AI) was measured by TUNEL.. (1) Expression of calpain-I in group AF2 was increased to (344.0 +/- 101.9)%, and caspase-3 was increased to (394.0 +/- 99.4)% compared to group RSR (P < 0.01, respectively). Amount of calpastatin was reduced to (27.0 +/- 12.8)% (P < 0.01). The expressions of these proteins were unchanged in group AF1. (2) AI in group AF2 was higher than that in groups RSR and AF1 (P < 0.01). (3) In group AF2, the levels of calpain-I, caspase-3 and AI were positively relative to left atrial dimension and AF duration, P < 0.05 - 0.01, respectively, whereas calpastatin was negatively correlated with left atrial dimension and AF duration (P = 0.007 and P = 0.001, respectively). (4) The protein content of calpain-I was positively related with that of caspase-3 and AI (P < 0.01, respectively), and the content of calpastatin was negatively related with that of calpain-I and caspase-3 (P < 0.01, respectively).. Apoptosis of atrial cell increased in left atria and the protein contents of calpain-I, caspase-3 and calpastatin significantly altered during AF in humans with RHD. The observed interactions suggest that these factors compose a system to cause the structural remodeling and dysfunction of atria. The course may play a key role in promoting the onset and maintenance of AF.

Topics: Adult; Apoptosis; Atrial Fibrillation; Atrial Function; Calcium-Binding Proteins; Calpain; Caspase 3; Female; Heart Atria; Humans; Male; Middle Aged; Myocytes, Cardiac; Rheumatic Heart Disease

Atrial fibrillation (AF) is accompanied by electrical, structural and ion-channel protein remodeling. We tested if proteolysis by calpain and proteasome is activated during AF, and studied the relation with the remodeling processes.. Right atrial appendages were obtained from patients with paroxysmal (n=7) or persistent (n=10) lone AF and compared to controls (n=10) in sinus rhythm undergoing coronary artery bypass grafting (CABG). Proteolysis was measured using Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin. Protein expression of calpain I and II was assessed by Western-blot and calpain I localization by immunohistochemistry. Structural changes were quantified by counting atrial myocytes with contraction bands or hibernation.. Calpain activity was significantly increased in paroxysmal AF (2-fold, P<0.001) and persistent AF (3-fold, P<0.001), mainly due to calpain I activation. Increased calpain I protein expression was found in AF with Western blot and immunohistochemistry. Myocytes from all AF groups showed increased contraction bands, whereas hibernation was only found in persistent AF. Calpain activity correlated with L-type Ca(2+) channel and Kv1.5 protein amounts (r=-0.80, P<0.001 and r=-0.72, P<0.001, respectively), degree of structural changes (r=0.90, P<0.001), shortening of atrial effective refractory period (AERP) (basic cycle length 500 ms, r=-0.60, P<0.001) and AERP rate adaptation (r=-0.80, P<0.001).. Calpain activity is induced during AF and correlates with parameters of ion-channel protein, structural and electrical remodeling. The results suggest that calpain activation represents an important mechanism linking calcium overload to cellular adaptation mechanisms in human AF.

Topics: Adult; Analysis of Variance; Atrial Appendage; Atrial Fibrillation; Blotting, Western; Calpain; Case-Control Studies; Cell Count; Humans; Immunohistochemistry; Microscopy, Electron; Middle Aged; Proteins

Atrial fibrillation (AF) is accompanied by intracellular calcium overload. The purpose of this study was to assess the role of calcium-dependent calpains and cytokines during AF. Atrial tissue samples from 32 patients [16 with chronic AF and 16 in sinus rhythm (SR)] undergoing open heart surgery were studied. Atrial expression of calpain I and II, calpastatin, troponin T (TnT), troponin C (TnC), and cytokines [interleukin (IL)-1 beta, IL-2, IL-6, IL-8, IL-10, transforming growth factor (TGF)-beta 1, and tumor necrosis factor-alpha] were determined. Expression of calpain I was increased during AF (461 +/- 201% vs. 100 +/- 34%, P < 0.05). Amounts of calpain II and calpastatin were unchanged. Total calpain enzymatic activity was more than doubled during AF (35.2 +/- 17.7 vs. 12.4 +/- 9.2 units, P < 0.05). In contrast to TnC, TnT levels were reduced in fibrillating atria by 26% (P < 0.05), corresponding to the myofilament disintegration seen by electron microscopy. Small amounts of only IL-2 and TGF-beta 1 mRNA and protein were detected regardless of the underlying cardiac rhythm. In conclusion, atria of patients with permanent AF show evidence of calpain I activation that might contribute to structural remodeling and contractile dysfunction, whereas there is no evidence of activation of tissue cytokines.

Topics: Actin Cytoskeleton; Adult; Aged; Atrial Appendage; Atrial Fibrillation; Blotting, Western; Calcium-Binding Proteins; Calpain; Cytokines; Female; Humans; In Vitro Techniques; Interleukin-2; Male; Middle Aged; Myocardium; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1