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Calpain I inhibition prevents atrial structural remodeling in a canine model with atrial fibrillation

XUE, Hong-jie; LI, Wei-min; LI, Yue; GONG, Yong-tai; YANG, Bao-feng; JIN, Cheng-luo; SHENG, Li; CHU, Shan; ZHANG, Li; SHAN, Hong-bo; LIU, Jie

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Background 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.

Methods 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·kg−1·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.

Results 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 (rs=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.

Conclusions 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.

Chin Med J 2008;121(1):32–37

Department of Cardiology, First Clinical Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China (Xue HJ, Li WM, Li Y, Gong YT, Sheng L, Chu S, Zhang L, Shan HB and Liu J)

Department of Pharmacology and Bio-pharmaceutical Key Laboratory of Heilongjiang Province and State, Harbin Medical University, Harbin, Heilongjiang 150086, China (Yang BF) Department of Uronology, Second Clinical Hospital of Harbin Medical University, Harbin, Heilongjiang 150086, China (Jin CL)

Correspondence to: Dr. LI Wei-min and Dr. LI Yue, Department of Cardiology, First Clinical Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China (Tel: 86-451-53643849. Fax: 86-451-53675733. Email: wml@public.hr.hl.cn; lv991v@163.com)

This work was supported by grants from the National Natural Science Foundation of China (No. 30470686), the Science and Technology Specialized Research Fund for Youth of Heilongjiang Province (No. QC06C076), key Scientific and Technological Project in Heilongjiang Province (No. GB07C32401) and the Innovation Grant of Harbin Medical University for Graduate Student (No. 200401059).

(Received March 15, 2007)

Edited by WANG Mou-yue and LIU Huan

Atrial fibrillation (AF) is characterized by its intrinsically progressive nature, that is, it has the tendency to become more persistent over time. Experimental and clinical studies point to two major mechanisms involved in typical AF: electrical remodeling and structural remodeling.1,2 However, the electrical remodelling is reversible and a return to normal electrophysiological properties is complete within 3 days after restoration of the sinus rhythm.1 This suggests that the electrophysiological abnormalities may not be the dominant factor in the maintenance of AF.3 Recent experimental research has demonstrated that AF-induced atrial structural changes (myolysis and myocyte degeneration) are primarily important for the propensity of AF.4,5 AF-induced structural remodeling leads to the heterogeneity in the electrical activation pattern and the loss of contractility of atrial tissue, which in turn increase the vulnerability to AF.6 A more conclusive insight into the molecular mechanisms underlying AF-induced structural remodeling will be useful for finding an effective method to prevent the maintenance of AF.

Some investigations have suggested that calpains play an important role in the AF related structural remodeling process.7 Calpains are calcium-activated neutral proteases. In vitro assays a large number of proteins can be cleaved by calpains: such as cytoskeletal proteins, kinases, phosphatases and membrane-associated proteins, even including some ion-channel proteins.8 Recent studies suggest that AF is associated with intracellular calcium overloading, an increase in cytosolic calcium levels is known to cause the activation of calcium-dependent calpains.9 The expression and activity of calpain has been found to be increased in the atrial tissue of patients with paroxysmal and persistent lone AF and these changes are accompanied by structural atrial remodeling.9 Since activation of calpain is likely to contribute to further deterioration of atrial tissue, interference with the calpain pathway may represent an important tool to unravel the sequence of molecular events in AF.

In this study, we investigated the significance of activation of calpain in atrial remodeling by assessing the effects of the calpain inhibitor ALLM in the long-term rapid atrial pacing canine model.

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METHODS

Canine model of AF

Fifteen mongrel dogs (13–18 kg) were randomized into three groups: sham-operated group, control group and calpain inhibitor group; each with 5 dogs. Animals were anesthetized with pentobarbital (30 mg/kg, iv). Active fixation leads were implanted in the right atrial (RA) appendage free wall and connected to a pacemaker (Department of Electronic Engineering, Fudan University, Shanghai, China). After recovery, atrial pacing was instituted at a rate of 600 beats per minute for 3 weeks. The sham-operated group underwent sham operations with placement of a nonfunctioning RA lead. After pacing, the dogs in the calpain inhibitor group were given ALLM 1.0 mg·kg−1·d−1 by intravenous injections for 3 weeks, the dogs in the control and in the sham operated groups were administered dimethyl sulphoxide (DMSO) at 1.5 ml/d, iv.

All animals survived the 3-week experimental period. After the stop of the rapid atrial pacing, spontaneous AF occurred in 3 dogs in the control group and in 1 dog in the calpain inhibitor group. AF lasting more than 30 minutes was terminated by direct current electrical cardioversion. The left atrial and right atrial appendage tissues and the interatrial septum were removed after euthanasia.

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Proteolytic assay

Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin (Suc-LLVY-AMC, Biomol, USA) was used as a calpain substrate. Twenty-five μg of protein extract was added to 20 mmol/L Suc-LLVY-AMC in 300 ml of Tris-buffered saline. AMC release was measured by fluorometry, 360-nm excitation and 430-nm emission (Spectrometer LS55, Perkin Elmer, USA) after incubation for 30 minutes at 25°C. Standard curves were generated using known concentrations of 7-amino-4 methyl-coumarin (Biomol) and 25 μg of heat denatured protein. Maximal calpain activation was assessed after reconstitution of calcium at 1 mmol/L.

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Immunohistochemistry

Five μm thick paraffin left atrial sections were made. After three washes, phosphate buffer solution (PBS) for 5 minutes and 30 minutes blocking with 1% bovine serum albumin (BSA) in PBS, sections were incubated with polyclonal rabbit anti-calpain I antibodies (1:100) (BOSTER Biotechnology Co.,Wuhan, China) overnight at 4°C. After three washes with PBS for 5 minutes the sections were incubated with peroxidase conjugated goat anti-rabbit IgG (Santa Cruz, CA., USA) for 20 minutes followed by three washes with PBS. Peroxidase activity was detected using diaminobenzidine (DAB). After staining for 5 minutes sections were rinsed with water, counterstained with haematoxylin and mounted with glycerol gelatin.

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Ultramicrostructure evaluation

For morphological evaluation by electron microscopy, heart tissues were fixed for at least 2 hours at 4°C in 2% glutaraldehyde, in 0.1 mol/L cacodylate buffer, pH 7.4. Then the tissue was prepared for 2 hours in 1% osmium tetroxide, supplemented with 1.5% K4Fe (CN)6 in cacodylate buffer, pH 7.4 at 4°C. After dehydration in ethanol, prepared tissue was cut in ultrathin sections (60 nm) and stained with uranylacetate. The sections were examined in a JEM —1220 (Hitatchi, Japan) electron microscope.

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Quantification of myolysis

Two μm thick tissue sections derived from various sites of the atria were examined by light microscope. To quantify the degree of myolysis, sections were stained with HE. At least six sections per atrial site were examined and at least 400 cells per section were analyzed (Motic Images Advanced 3.0). The extent of cell change was evaluated only in cells in which the nucleus was present in the plane of the section. Cells were scored by morphometry as mildly myolytic, if myolysis involved 10% to 25% of the cytosol, and as severely myolytic if >25% of the sarcomeres were absent.

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Western blotting for TnT and myosin

For electrophoresis, 50 μg of total protein from homogenized total tissue was separated in a SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidenedifluoride (PVDF) (Roth, Germany). Membranes were incubated in a blocking buffer (PBS, 0.05% Tween-20, 1% BSA ) for 1 hour at 37°C and then blots were incubated overnight at 4°C with monoclonal mouse anti-TnT (troponin T) antibody (1:200, Santa Cruz), monoclonal mouse anti-skeletal myosin (slow) antibody (1:200, Boster Biotechnology Co., China) and monoclonal mouse anti-GAPDH antibody (1:500, Santa Cruz). Goat anti mouse IgG (1: 2000, Santa Cruz) conjugated with horseradish peroxidase were used as secondary antibodies and sections subsequently developed with Uptilight HRP Blotting Reagent.

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Echocardiography

Left atrial (LA), left atrial appendage (LAA) structure and function were assessed by transthoracic and transoesophageal echocardiographic examinations (GE VIVID5, USA). LA maximal volume (LAVmax), LAA maximal volume (LAAVmax), LA minimal volume (LAVmin) and LAA minimal volume (LAAVmin) were measured. LA ejection fraction (LAEF) was calculated as (LAVmax - LAVmin) / LAVmax. LAA ejection fraction (LAAEF) was calculated as (LAAVmax - LAAVmin)/LAAVmax. LAA maximal forward flow velocity (V-LAA+) and LAA maximal backward flow velocity (V-LAA”) were also obtained.

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Statistical analysis

Results are expressed as mean ± standard deviation (SD) or medians. Statistical significance of the differences between means was assessed with Kruskal-Wallis test, Spearman correlation analysis and analysis of variance (ANOVA) for the post-hoc comparison (SPSS 11.0). Statistical significance was set as P<0.05.

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RESULTS

Proteolytic activity and expression of calpain I

Proteolytic activity was significantly increased in atrial tissue from dogs in the control group compared to the sham-operated group (2.35-fold, P<0.01). The proteolytic activity was significantly reduced in the calpain inhibitor group compared to the control group (0.51-fold, P<0.01). Furthermore, tissue calpain activity in all groups was significantly correlated with the degree of myolysis (rs=0.90 961, P<0.0001).

Calpain I expression and localization was studied by immunohistochemistry (Figure 1). Calpain I was predominantly localized in the nucleus, intercalated discs and in the cytoplasm of atrial myocytes. To quantify the expression level of calpain I protein in myocytes, the staining intensity was scored semiquantitatively (Motic Images Advanced 3.0). Staining intensity was significantly increased in the control group compared to the sham-operated group (P<0.01) and the calpain inhibitor group (P<0.01).

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Structural changes under light microscopy

Atrial myocytes from dogs in the sham-operated group showed compact sarcomeres occupying the entire cytoplasm (Figure 2A). In contrast, atrial myocytes in the control group were depleted of contractile material. In these cells, myolysis started in the perinuclear area and often extended toward the plasma membrane (Figure 2B). The myolysis was seen at all different atrial sites in the control group, but it was found in fewer areas in the calpain inhibitor group (Figure 2C). The percentage of myolytic myocytes (medians) was significantly higher in the control group than in the sham-operated group (Table 1), but lower in the calpain inhibitor group than in the control group.

Figure 1.

Figure 1.

Table 1

Table 1

Cells were considered to be mildly affected when 10%-25 % of the sarcomeres had disappeared. Cells with >25% myolysis were considered to be severely affected. The results of the morphometric analysis are displayed in Table 1.

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Ultramicrostructure under electron microscopy

At the ultrastructural level, atrial myocytes from dogs in the sham-operated group showed a highly organized sarcomeric structure with rows of uniformly sized mitochondria (Figure 3A). The atrial myocytes from dogs in the control group had undergone typical alterations: nuclear redistribution of heterochromatin and typical changes in size and shape of mitochondria (Figure 3B), depleted contractile material (myolysis) (Figure 3C), and giant mitochondria were also seen in dogs in the control group. In the calpain inhibitor group, the giant mitochondria were absent and the disappearance of sarcomeres was more moderate than that in the control group (Figure 3D).

Figure 2.

Figure 2.

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Specific expression of TnT and myosin

TnT expression was significantly different in the three groups (Figure 4). The expression of TnT decreased 0.51±0.12 fold in the control group compared to the sham-operated group (P<0.01), however it was increased 1.36±0.28 fold in the calpain inhibitor group compared to the control group (P<0.01). Myosin expression was decreased 0.43±0.11 fold in the control group compared to the sham-operated group (P<0.01). In the calpain inhibitor group, myosin expression was 1.49±0.23 fold higher than in the control group (P<0.01), but it was still 0.72±0.16 fold lower than in the sham-operated group (P<0.01).

Figure 3.

Figure 3.

Figure 4.

Figure 4.

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Echocardiography

After 3 weeks of continuous rapid atrial pacing, haemodynamic parameters of the left atrium were measured by transthoracic echocardiography after sinus rhythm restoration (Table 2). Left atrial functional parameters in the control group were significantly impaired after pacing compared with before pacing. In dogs treated with ALLM, values of these parameters were significantly improved compared with the control group (Table 2).

Table 2

Table 2

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DISCUSSION

The present study shows that rapidly paced canine atria display the same key characteristics of ultrastructural remodeling observed in atrial tissue from patients with chronic AF. We found rapid pacing in canine atrium to cause myolysis, glycogen accumulation, typical chromation changes and to induce calpain activity. Furthermore, the level of myocyte remodeling correlated well with the activity of calpain and the ratio of atrial myolysis, one of the main parameters of quantification of atrial structural changes, and positively correlated with the calpain activity. We also found that the pacing induced myolysis and degradation of TnT and myosin were partially prevented by treatment with the calpain inhibitor ALLM, which may contribute to the survival of atrial function. These results suggest that activation of calpain represents a key feature in the AF related myocyte remodeling processes.

Previous studies have demonstrated that AF induced reduction in the expression of multiple proteins is likely mechanisms which promote the susceptibility of the human atrium to arrhythmia. Interestingly, the reduction in protein expression occurs in the absence of changes in mRNA.10,11 The activation of protein degradation seems the likely mechanism, and is consistent with increased proteolytic activity due to the activation of calpains that were recently found in human and experimental AF.9 Studies also found that increased proteolytic activity was predominantly due to increased expression of calpain I rather than calpain II.9 Consistent with these results, our study found that proteolytic activity was significantly increased in atrial tissue from dogs in the control group compared to the sham-operated group, and the proteolytic activity was consistent with the level of calpain I protein staining intensity. Furthermore, we found that calpain I was predominantly localized in the nucleus, intercalated discs and in the cytoplasm of atrial myocytes; which may be vital for the action potential conduction and structural integrity of the atrial myocytes. At the intercalated discs, the activated calpain I may degrade important ion-channels, like the Na-channel and Kv1.5,12,13 and even proteins involved in excitation-contraction coupling and conduction. The increased expression of calpain I at the nucleus might be suggestive of a role in promoting cell death14 while Calpain I may also cleave cytoskeletal proteins in the cytoplasm.2 The proteolytic activity was significantly suppressed by the calpain I inhibitor ALLM.

A significant increase in calpain activity has been observed to be positively correlated with the extent of structural changes. In this study we found atrial tissue calpain activity significantly and positively correlated with myolysis, one of the main parameters of atrial structural changes in all groups. ALLM dramatically attenuated the AF induced atrial myolysis, which indicates that myolysis was mediated by activation of calpain. Calpains are calcium-activated neutral proteases. In in vitro assays a large number of proteins can be cleaved by the calpains; such as cytoskeletal proteins, kinases, phosphatases, membrane-associated proteins, even including some ion-channel proteins.8 Calpain is known to activate the downstream protease caspase-3,14 whose activity has been reported to be increased in patients with chronic AF.15 The activation of cysteine proteases is widely known to initiate and execute programmed cell death, however, particularly in cardiac myocytes, when programmed cell death is not completed it will result in myolysis.16 AF induced ultrastructural changes of atrial myocytes included dispersed chromatin, an increase in number and size of the mitochondria and an increase in glycogen in the myolytic space, which have also been observed in the present study. Most of these changes are consistent with the results of morphological analysis reported by Ausma et al.4 Furthermore, we found that in the calpain inhibitor group the giant mitochondria were absent and the disappearance of sarcomeres was more moderate than in the control group, which may be due to the inhibition of ALLM on activation of calpain.

In addition to atrial myolysis, a causal relationship between calpain activation and the degradation of TnT and myosin has been shown in our study. We measured protein expression of TnT and myosin and found that expression of TnT and myosin decreased after 3 weeks of rapid pacing, and the degradation was prevented by treatment with ALLM. These results suggested that TnT and myosin were possibly cleaved by calpain during AF. Studies proved calpains can rapidly cleave troponins T and I while they cleave myosin and actin, the two major proteins in striated muscle, very slowly. Because of the limited specificity of the calpains, these proteins are only cleaved into ‘limited fragments', the further degradation of fragments of actin and myosin to amino acids requires participation of other proteases.17 Further studies are needed to clarify the proteases involved in this process.

Previous study suggested that atrial structural remodeling in AF may be the dominant factor in the formation of atrial contractile dysfunction and should be responsible for the delayed recovery of atrial function after conversion to sinus rhythm.18 Atrial structural alterations, induced by proteolysis due to activation of calpain, such as the loss of sarcoplasmic reticulum and contractile proteins, dramatically damage atrial contractile force and lead to atrial myocyte stunting.19 Contractile dysfunction might also be caused by myolysis. Schotten et al20 reported that the contractility of atrial muscle bundles isolated from AF patients was markedly reduced and correlated positively with the extent of atrial myolysis. As described in the previous section, atrial myolysis is mediated by calpains, so activation of calpains may be the potential mechanism implicated in the impaired atrial mechanic dysfunction. This hypothesis has been confirmed in our study when we found that in the calpain inhibitor group the parameters of atrial function, such as LAEF and LAAEF, were significantly improved compared with those of the control group. These results demonstrated that calpain activity indeed contributes to the atrial contractile dysfunction; calpain inhibitors may be effective at preventing atrial contractile dysfunction and promoting recovery of atrial function after conversion to sinus rhythm.

In conclusion, we found in rapidly paced canine atria the same characteristics of structural remodeling and contractile dysfunction found in atria from patients with chronic AF. Furthermore, our data underline the importance of calpain activation as the molecular mechanism of the remodeling processes. Inhibition of calpain protease activity is effective in preventing atrial structural remodeling and dysfunction induced by AF, which may be a new option for therapeutic intervention in AF.

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Keywords:

atrial fibrillation; structural remodeling; calpain; inhibitor; N-acetyl-leu-leu-met

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