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Cardiovascular Research 2003 59(2):419-427; doi:10.1016/S0008-6363(03)00385-7
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Decrease in sarcoglycans and dystrophin in failing heart following acute myocardial infarction

Hiroyuki Yoshidaa, Masaya Takahashia, Miki Koshimizua, Kouichi Tanonakaa, Ryo Oikawaa, Teruhiko Toyo-okab and Satoshi Takeoa,*

aDepartment of Pharmacology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
bDepartment of Organ Pathophysiology and Internal Medicine, Tokyo University Hospital, Tokyo 113-8655, Japan

* Corresponding author. Tel.: +81-426-76-4583; fax: +81-426-76-5560. takeos{at}ps.toyaku.ac.jp

Received 28 January 2003; revised 8 March 2003; accepted 9 April 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Genetic defects in several sarcoglycans (SGs) and dystrophin (Dys) play a critical role in cardiomyopathy. The present study was designed to determine whether changes in SGs and Dys might occur in animals with chronic heart failure (CHF) induced by acute myocardial infarction (AMI), which have no genetic defects. Methods: AMI was induced by the left coronary artery ligation (CAL) in rats. The hemodynamic parameters of the 2- and 8-week CAL (2w- and 8w-CAL) rats were measured and the myocardial SGs, Dys, calpain, and calpastatin levels were determined by the Western blot method. Myocardial calpain-like protease activity was evaluated as caseinolysis activity. Results: Increases in left ventricular end-diastolic pressure (LVEDP) and right ventricular systolic pressure, and a decrease in ±dP/dt were observed at the 2nd week, whereas cardiac output index (COI) was preserved. In contrast, the 8w-CAL rats showed a further increment in LVEDP with low COI. {alpha}-SG of the viable left ventricle (LV), and septum (Sep) of the 8w-CAL rat decreased (60–70% of the control). The {alpha}- and β-SGs of the right ventricle (RV) of the 2w- and 8w-CAL rats were reduced, while {gamma}- and {delta}-SGs in the three regions did not change significantly. Dys in the viable LV and RV of the 8w-CAL rat decreased (75% of the control). The amount of m-calpain in the three regions of the 2w- and 8w-CAL rats increased (140–200% of the control), whereas the endogenous calpain inhibitor, calpastatin, did not change significantly. The in vitro degradation studies using purified m-calpain or cytosolic fractions of the 8w-CAL rat heart suggested a reduction in SGs and Dys by calpain. Conclusion: The results suggest that a decrease in SGs and Dys may play an important role in the pathophysiology of CHF following AMI.

KEYWORDS Calpain; Dystrophin; Heart failure; Hemodynamics; Sarcoglycan


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Dystrophin-related protein (DRP) complex consists of dystrophin, sarcoglycans, and dystroglycans and stabilizes sarcolemmal integrity [1]. Inherent mutation of dystrophin gene causes Duchenne- or Becker-type muscular dystrophy and dilated cardiomyopathy (DCM) [2]. Limb-girdle muscular dystrophy (LGMD) is caused by mutation of sarcoglycan (SG) subunits or by the deficiency of SGs [3]. Thus, some phenotypes of LGMD may induce the sarcoglycanopathy [3]. Recent studies have shown that cardiomyopathy in Bio14.6 [4] and TO-2 strain hamsters, which mimic dilated cardiomyopathy of humans [5], is caused by the gene deletion of {delta}-SG [4,6]. These two strains share a common gene mutation with distinct clinical features [7]. Kawada et al. reported that {delta}-SG gene transfection with recombinant adeno-associated virus improves cardiac function and survival of TO-2 hamster [8,9]. These findings suggest that changes in DRP complex may play an important role in the maintenance of muscle contractility.

Our previous studies revealed that the coronary ligation of rats at the proximal artery resulted in chronic heart failure with low cardiac output [10–12]. However, no information is available concerning changes in DRP complex in this heart failure. To verify the hypothesis that cardiac failure following acute myocardial infarction is associated with alteration in DRP complex, we examined alterations in DRP complex in the failing heart and possible involvement of endogenous calpain or calcium-activated neutral protease [13] in the process of DRP disruption.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
Male Wistar rats (SLC, Hamamatsu, Japan), weighing 210–240 g, were used in the present study. The animals were conditioned according to The Guide for the Care and Use of Laboratory Animals as promulgated by the National Research Council. The protocol of this study was approved by The Committee of Animal Use and Welfare of Tokyo University of Pharmacy and Life Science.

2.2 Operation
Myocardial infarction was produced in 49 rats by ligation of the left ventricular coronary artery (CAL rats) according to the method described previously [10]. Rats that revealed an abnormal Q wave (more than 0.3 mV) 1 day after the operation were used for the following experiment [11]. Among the coronary artery-ligated rats, 33 rats survived by the 1st week after surgery (approx. 65% of the operated animals). Three rats died between the 2nd and 8th week after surgery. Thirty sham-operated rats without coronary artery ligation (Sham rats) were treated in a similar manner.

2.3 Measurement of hemodymanic parameters
Measurements of in vivo hemodynamic parameters were performed by a method described previously [10]. Briefly, prior to the operation, and 2 and 8 weeks after the operation, the rats were anesthetized with a gas mixture of nitrous oxide/oxygen (3:1) and 0.5–2.5% enflurane. A microtip pressure transducer (SPC 320, Miller Instrument, Houston, TX) was introduced into the left ventricle through the right carotid artery to measure left ventricular systolic and end-diastolic pressures (LVSP and LVEDP, respectively). The arterial blood pressure was measured by means of a pressure transducer attached to a cannula placed into the right femoral artery. Heart rate (HR) measurements were triggered from changes in arterial blood pressure. After 30 min equilibration of the setting, the above parameters were recorded.

After determination of the left ventricular function, the microtip pressure transducer was introduced into the right ventricle through the right jugular vein to measure the right ventricular systolic pressure (RVSP) and the right ventricular end-diastolic pressure (RVEDP). The PO2, PCO2, and pH of the blood samples of the animals under the present experimental conditions ranged from 95 to 104 mmHg, from 37 to 42 mmHg, and from 7.41 to 7.45 (n = 5), respectively.

In another set of experiments, we determined the aortic flow by the method described previously [10]. The rats were inhaled with a gas mixture of nitrous oxide/oxygen and enflurane as described above, intubated, and artificially respirated with air. After dissection of the right thorax, an electro-magnetic flow meter with a diameter of 2.0–2.5 mm (MFV-3100, Nihon Kohden, Tokyo) was placed around the thoracic aorta, and then the aortic blood flow was measured. The systemic blood pressure was monitored through a cannula inserted into the femoral artery, and the heart rate was measured through the pulse of the systemic blood pressure. Cardiac output index was calculated by dividing aortic flow by body weight. The PO2, PCO2, and pH of the blood samples of the animals under the present experimental conditions ranged from 93 to 99 mmHg, 35 to 39 mmHg, and 7.38 to 7.42 (n = 5), respectively.

2.4 Infarct size
After measurement of aortic flow, the left ventricle of rats with CAL or sham-operated rats was isolated and sectioned into seven slices with 1-mm thickness (n = 5) from the base of the heart to the apex in a plane parallel to the atrioventricular groove. The slices were stained at 37°C for 10 min with 1% triphenyltetrazolium chloride (TTC) in saline. After staining, TTC-unstained areas were determined according to a planimetric method [10].

2.5 Western blotting
Myocardial DRP complex proteins were determined by a modified method described previously [12]. Briefly, after determination of hemodynamic parameters for the left/right ventricular function, hearts were quickly isolated and then residual blood in the tissue was washed out with phosphate-buffered saline. The heart was divided into the left ventricular free wall without infarct area (viable LV), septum (Sep), and right ventricular free wall (RV). The tissues were homogenized in buffer containing 320 mM sucrose, 100 µM disodium EDTA, 100 µM phenylmethanesulfonyl fluoride (PMSF), and 10 mM Tris–HCl (pH 7.40). The homogenates were sampled for Western blot analysis. The samples were boiled in the Laemmli buffer containing 250 mM Tris–HCl, 4% SDS, 10% glycerol, 0.006% bromophenol blue, and 2% β-mercaptoethanol (pH 6.8), and fractionated by SDS electrophoresis on a 10% polyacrylamide gel (SDS–PAGE, 10x10 cm) for SGs, calpain, and calpastatin and on a 4% SDS–PAGE for dystrophin, respectively, according to the method of Laemmli [14].

The proteins separated on the gel were transferred on the PVDF membrane and then detected with their respective antibodies [11]. The following antibodies were used: anti-{alpha}-SG (1:1500 dilution; NCL-a-SARC, Novocastra Lab, Newcastle upon Tyne, UK), anti-β-SG (1:2000 dilution; NCL-b-SARC, Novocastra Lab), anti-{gamma}-SG (1:3000 dilution; NCL-g-SARC, Novocastra Lab), anti-{delta}-SG (1:4000 dilution) [8], anti-dystrophin (1:3000 dilution; NCL-DYS1, Novocastra Lab), anti-m-calpain (1:3000 dilution; SA-255, Biomol, Plymouth Meeting, PA), and anti-calpastatine (1:1000 dilution; MAB3084, Chemicon, Temecula, CA). Thereafter, the proteins on the membrane were visualized by use of an ECLTM (Amersham Pharmacia Biotech, Buckinghamshire, UK) and their bands, developed on the X-ray films, were semi-quantified by a Densitograph® (Atto, Tokyo).

2.6 Preparation of sarcolemmal fraction
Crude sarcolemmal fraction was prepared from the left ventricular muscle of control rats including the septum as described previously [11]. The heart was homogenized with five volumes of cold buffer (300 µM PMSF, 320 mM sucrose, 1 mM EGTA, 20 mM Tris–HCl; pH 7.40). The homogenate was centrifuged at 1000xg at 4°C for 10 min. The supernatant fluid was centrifuged at 8000xg for 20 min at 4°C, and then the resultant supernatant fluid was recentrifuged at 100,000xg at 4°C for 20 min. The resultant pellet was resuspended in the buffer without PMSF. The protein concentration was determined by the method of Lowry et al. [15].

2.7 Incubation of sarcolemma with m-calpain
To examine proteolysis of SGs and dystrophin by m-calpain, the sarcolemma-enriched fraction was incubated in the presence and absence of 10 mM CaCl2 at 30°C for 10–60 min in the reaction buffer of the following composition; 100 mM KCl, 10 mM β-mercaptoethanol, 5 U calpain II [rat recombinant calpain II (Calbiochem, San Diego, CA)], 5 mM CaCl2, 20 mM Tris–HCl, pH 7.40. The reaction was terminated by the addition of 100 µM leupeptin, an exogenous cystein-protease inhibitor [13]. SG and dystrophin contents in the buffer were determined by Western blotting as described above.

2.8 Proteolytic activity ex vivo in cytosolic fraction
Caseinolytic activity of the cytosolic fraction, where calpain is present, isolated from the viable LV, Sep, and RV of the failing heart was estimated ex vivo using the degradation rate of casein. The tissue was homogenized with five volumes of 100 mM imidazole buffer (pH 7.50). The homogenate was centrifuged at 1000xg for 10 min at 4°C. The supernatant was centrifuged at 8000xg for 20 min at 4°C, and then the resultant supernatant fluid was recentrifuged at 100,000xg for 20 min at 4°C. The supernatant fluid was used as a cytosolic fraction. Protein in each cytosolic fraction was incubated in the buffer of the following composition; 0.4% (w/v) casein, 5 mM cysteine, 5 mM CaCl2, 100 mM imidazole/HCl (pH 7.5). After a 30 min-incubation, the reaction was terminated by the addition of ice-cold 5% (w/v) trichloroacetic acid and then the reaction mixture was centrifuged at 10,000xg for 15 min at 4°C. The absorbance of the resultant supernatant at 280 nm, which represents small peptide fragments with aromatic amino acids produced by calpain proteolysis, was measured by a spectrophotometer (U-Best 30, JASCO, Hachioji, Japan). To determine the specificity for caseinolytic activity of calpain, leupeptin was added by 100 µM for the control activity.

Furthermore, the cytosolic fraction isolated from the viable LV, Sep, or RV of the 8w-CAL rat was incubated with the sarcolemmal fraction isolated from normal rats, and then sarcoglycan and dystrophin proteins after the incubation were determined by the method described above.

2.9 Experimental groups
In the present study, 30 CAL rats were used. Hemodynamic parameters of the 2w- and 8w-CAL rats (n = 6 each) were measured and followed by determination of DRP complex proteins, calpain, and calpastatin. In another set of experiments, measurement of cardiac output index and subsequent determination of infarct size were carried out in the 2w- and 8w-CAL rats (n = 5 each). The effect of leupeptin on caseinolytic activity of the cytosolic fraction of the 8w-CAL rats was determined (n = 4). Proteolysis of DRP complex in the presence of the cytosolic fraction of the 8w-CAL rat was also determined (n = 4).

2.10 Statistics
The results were expressed as the means±S.E.M. Statistical significance was estimated by analysis of variance (ANOVA) followed by Fisher's multiple comparison. Differences with a probability of less than 5% were considered to be statistically significant (P<0.05).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Body, heart, and lung weight
Body weight of the CAL rat slightly but significantly decreased compared with that of the Sham rat (Table 1). Heart weight, heart weight-to-body weight ratio, RV weight, RV weight-to-body weight ratio of the CAL rat were larger than those of the Sham rat. Lung weight and lung weight-to-body weight ratio were also larger than those of the Sham rat.


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  		Table 1 Cardiac and lung weight parameters of the Sham and CAL rats at the 2nd and 8th weeks after the operation

 
3.2 Hemodynamics
The mean arterial pressure (MAP) and LVSP of the 2w- and 8w-CAL rats was decreased, whereas HR did not change significantly (Table 2). In contrast, the LVEDP of the CAL rat was increased at the 2nd week and elevated further at the 8th week. The RVSPs of the CAL rats were increased at the 2nd and 8th weeks compared with those of the Sham rat, whereas the RVEDPs of these CAL rats did not differ from the corresponding Sham rat. There were no significant differences in these parameters between the 2w- and 8w-Sham rats.


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  		Table 2 Hemodynamic parameters of the Sham and CAL rats at the 2nd and 8th weeks after the operation

 
In another set of experiments, cardiac output index was determined. Cardiac output index of the CAL rat decreased at the 8th week, whereas it did not alter at the 2nd week (Table 2). There were no changes in the cardiac output index of Sham rats throughout the experiment. The infarct area of the CAL rat occupied approximately 40% of the left ventricle (Table 1), but no infarction in the myocardium of Sham rats was seen.

3.3 Expression of SGs in myocardium
After the in vivo measurement of hemodynamics, the contents of myocardial SG and dystrophin proteins in the viable LV, Sep, and RV were quantified by the Western blot method (Fig. 1). {alpha}-SG content in the RV of the 2w-CAL rat decreased, whereas that of the viable LV or Sep did not alter. Myocardial {alpha}-SG content in all three portions of the 8w-CAL rat decreased to approximately 60, 70, and 55% of the control value, respectively. β-SG content in the RV of the 2w- and 8w-CAL rats decreased, whereas those in the viable LV and Sep of either CAL rat did not change significantly. {gamma}- and {delta}-SGs in the three portions of the CAL rat remained constant throughout the experiment. These four SGs in the heart of the Sham rat were similar to those of the control.


Figure 1
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  		Fig. 1 Representatives of Western blot analysis (A) and semi-quantified data (B) for {alpha}-, β-, {gamma}-, and {delta}-sarcoglycan, and dystrophin proteins in the viable left ventricular free wall (LV), intermediate septum (Sep), and right ventricular free wall (RV) of the Sham (open columns) and CAL rats (hatched columns) at the 2nd (left panels) and 8th weeks (right panels) after the operation. Each value represents the mean±S.E.M. of six experiments. *Significantly different from the corresponding Sham group (P<0.05).

 
There were no changes in dystrophin content in the three portions of the 2w-CAL rat. Dystrophin contents in the viable LV and RV of the 8w-CAL rat decreased to approximately 85% and 75% of the control. Dystrophin content in the Sep of the 8w-CAL rat tended to be decreased. Myocardial dystrophin content in the three portions of the Sham rat was similar to that of the control throughout the experiment.

3.4 Amount of myocardial calpain and calpastatin
Changes in myocardial calpain and calpastatin of the operated rats are shown in Fig. 2. Calpain content of the viable LV, Sep, and RV of the 2w-CAL rat increased to approximately 165, 140, or 205% of the control, respectively. Calpain content of these three regions in the 8w-CAL rat also increased to a level similar to those of the 2w-CAL rat. In contrast, calpastatin content of the heart remained unchanged in the 2w- and the 8w-CAL rats. There were no significant changes in myocardial calpain and calpastatin contents in the Sham rat.


Figure 2
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  		Fig. 2 Representatives of Western blot analysis (upper panels) and semi-quantified data (lower panels) for m-calpain and calpastatin proteins in the viable left ventricular free wall (LV), intermediate septum (Sep), and right ventricular free wall (RV) of the Sham (open columns) and CAL rats (hatched columns) at the 2nd (left panel) and 8th weeks (right panel) after the operation. Each value represents the mean±S.E.M. of six experiments. *Significantly different from the corresponding Sham group (P<0.05).

 
3.5 Incubation of sarcolemma with calpain
Changes in SGs and dystrophin proteins of the sarcolemmal fraction in the presence of m-calpain are shown in Fig. 3. When the sarcolemmal fraction from normal hearts was incubated with rat recombinant m-calpain, {alpha}-SG decreased to 50, 20, and 15% of the control (in the absence of calpain) at 5, 10, and 15 min after starting the incubation, respectively. Although β-SG also decreased in the presence of calpain, the protein decreased more gradually than {alpha}-SG. In contrast, {gamma}- and {delta}-SGs did not decrease within 30 min incubation in the presence of calpain. Dystrophin decreased 20 min after the onset of incubation, whereas this protein did not significantly decrease until 15 min after the incubation with calpain.


Figure 3
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  		Fig. 3 Time course of changes in {alpha}- ({alpha}-SG; upper left), β- (β-SG; upper right), {gamma}- ({gamma}-SG; middle left), and {delta}-sarcoglycan proteins ({delta}-SG; lower right), and dystrophin (lower left) of the isolated sarcolemmal fraction in the presence of rat recombinant m-calpain. Each value represents the mean±S.E.M. of four experiments. *Significantly different from pre-incubation value (0 min; P<0.05).

 
3.6 Calpain-like proteolytic activity
Differences in proteolytic activity in the cytosolic fraction between the Sham and CAL rat hearts were examined. The left panel in Fig. 4 shows changes in absorbance at 280 nm during incubation of the cytosolic fraction prepared from the viable LV of the 8w-CAL rat and the LV of the 8w-Sham rat. Incubation of casein with the cytosolic fraction prepared from the LV of the 8w-Sham rat resulted in a gradual increase in the absorbance at 280 nm in a time-dependent manner. When casein was incubated with Sep or RV of the 8w-Sham rat, the degree of increase in the absorbance was similar to that of LV (data not shown).


Figure 4
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  		Fig. 4 Changes in casein proteolysis (caseinolysis) in the myocardial cytosolic fraction prepared from the Sham (square and open columns) or the 8w-CAL rat (circle and hatched columns) (left panel) and the effect of leupeptin (Leu) on the caseinolysis of the cytosolic fraction (right panel). Each value represents the mean±S.E.M. of four experiments. *Significantly different from the Sham rat (Sham; P<0.05). #Significantly different from leupeptin-untreated group (Un; P<0.05).

 
The increases in the absorbance at 280 nm during incubation of casein with the cytosolic fraction of the viable LV, Sep, or RV of the 8w-CAL rat were all greater than that of the 8w-Sham rat (the left panel in Fig. 4). The degree of caseinolytic activity in the cytosolic fraction of the Sep or RV of the 8w-CAL rat was similar to that of the viable LV (data not shown).

The right panel in Fig. 4 shows the effect of leupeptin, an exogenous calpain inhibitor, on the casein proteolysis in the cytosolic fraction prepared from the 8w-CAL rat heart. Increased caseinolytic activity of the 8w-CAL rat was attenuated by the presence of 100 µM leupeptin (to approx. 45% of the leupeptin-untreated value).

3.7 Incubation of the sarcolemma isolated from normal rat heart with cytosolic fraction from 8w-CAL rat heart
Fig. 5 shows myocardial sarcoglycan and dystrophin proteins released from the sarcolemmal fraction of normal rats upon incubation with the cytosolic fraction of the viable LV of the 8w-CAL rat. After 60-min incubation, {alpha}- and β-SGs, and dystrophin proteins reduced to approximately 40, 60, and 80% of the pre-incubation levels, respectively. In contrast, {gamma}- and {delta}-SGs after the incubation did not change. The sarcoglycans and dystrophin upon incubation with the cytosolic fraction from the 8w-Sham rat did not release. The degree of proteolytic activity in the cytosolic fraction of the Sep or RV of the 8w-CAL rat was similar to that of the viable LV (data not shown).


Figure 5
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  		Fig. 5 Sarcoglycan and dystrophin proteins released from the isolated sarcolemmal fraction upon 60-min incubation with the cytosolic fraction of the viable left ventricle of the 8w-CAL (open columns) and 8w-Sham rats (dotted columns). Each value represents the mean±S.E.M. of four experiments. *Significantly different from pre-incubation value (P<0.05).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Genetic defects of SGs and/or dystrophin may result in the development of cardiomyopathy and/or muscular dystrophies [2,3,6,16]. The roles of the changes in DRP complex of the heart without genetic mutation, however, have not been clarified. In the present study, we have shown for the first time that alterations in myocardial DRP complex are detected in failing hearts following acute myocardial infarction of the rat that has no genetic mutation of DRP complex.

We observed an increase in LVEDP and decreases in LVSP and ±dP/dt of the CAL rat at the 2nd week without a decrease in COI, suggesting that cardiac function of the 2w-CAL rat was compensated. In contrast, we found the further rise in LVEDP and a decline in COI of the CAL rat at the 8th week, suggesting signs for chronic heart failure (CHF) with low cardiac output as were the cases in previous studies [10–12]. We also found a marked increase in RVSP of both 2w- and 8w-CAL rats without any change in RVEDP. In addition, the lung weight of the CAL rats for both periods increased. These changes may be more or less linked to hypertrophy of the right ventricular free wall of the CAL rat.

We found diverse changes in SG complexes in the failing heart, i.e. {alpha}-SG content of the viable LV and Sep of the 8w-CAL rat decreased, whereas {alpha}- and β-SG contents decreased only in the RV of the 2w- and 8w-CAL rats. Dystrophin content was decreased in the LV and RV of the 8w-CAL rat, but not of the 2w-CAL rat. In contrast, {gamma}- and {delta}-SGs in the three portions remained constant throughout the experiment. The findings showed that {alpha}-SG content was decreased most prominently among the SG proteins and that alterations in {alpha}-SG and dystrophin were associated with the genesis of chronic heart failure with low cardiac output. These results suggest that decreases in {alpha}-SG and dystrophin may play an important role in the pathophysiology of cardiac failure after CAL in rats. This is partially comparable to the previous findings that LGMD 2D, a muscular dystrophy, which reduces tolerance in exercise capacity [17], is caused by mutations in the {alpha}-SG gene [18].

β-SG in the RV of the 2w- and 8w-CAL rats decreased, whereas β-SG in the viable LV and Sep did not change significantly. Zimmer et al. suggest that in this rat model, the left ventricular muscle is capable of adapting to volume overload and the right ventricular muscle, to pressure overload [19]. In accordance with this, we observed that functional failure in either ventricle of the CAL rat was associated with the alterations in β-SG. Thus, the changes in β-SG may contribute to the genesis of contractile dysfunction of the CHF animals. This may account for an increase in RVSP, an indication of right ventricular dysfunction of the 8w-CAL animals.

Questions arise regarding the mechanism underlying the decreases in SGs and dystrophin after CAL. In this context, we focused on proteolysis of the components in the DRP complex. We found an increase in m-calpain protein in the failing heart, which is one of the Ca2+-dependent neutral cysteine proteases present in all mammalian cells. Sandmann et al. reported that transcription and translation of calpain in the viable left ventricular muscle were increased after myocardial infarction [20]. We found an increase in m-calpain of the viable LV, Sep, and RV in the failing heart at both the 2nd and 8th weeks after myocardial infarction. Calpain content in the RV of the 2w-CAL rat was greater than those in the viable LV and Sep, and β-SG in the RV, but not in the viable LV and Sep of the 2w-CAL rat was decreased. In contrast, calpastatin, an endogenous inhibitor of calpain, in the failing heart was constant. Since calpastatin has been shown to fully inhibit proteolytic activity of all calpain isoforms at a 1:1 ratio of calpain to calpastatin [21], the predominant increase in calpain with no significant changes in calpstatin in the CAL animal may lead to relative enhancement of proteolytic activity for the intracellular proteins such as DRP complex.

Furthermore, we found higher caseinolytic activity in the myocardial cytosolic fraction of the rat with CHF. The study on the incubation with the sarcolemmal fraction from control animals showed a rapid decrease in {alpha}-SG, a gradual decrease in β-SG, and a more delayed decrease in dystrophin. In contrast, {gamma}- and {delta}-SGs were unchanged in the presence of calpain. These patterns of changes in DRP complex in the incubation with m-calpain are comparable with those of DRP complex content in the failing heart. We found calpain-induced increase in the caseinolytic activity in the cytosolic fraction of the 8w-CAL rat heart, which was partially attenuated by a calpain inhibitor leupeptin. Furthermore, the cytosolic fraction of the 8w-CAL rat heart also showed enhanced degradation of DRP complex proteins. These results suggest that the increase in calpain levels relative to calpastatin in the failing heart may contribute to decreases in {alpha}- and β-SGs and dystrophin during the development of heart failure.

In the present study, we found a greater degree of the decrease in {alpha}-SG in the failing heart than that in β-SG and dystrophin. Yoshida et al. [22,23] showed that β-, {gamma}-, and {delta}-SG were cross-linked by succinimidyl propionate, whereas {alpha}-SG was not linked to others. The three SGs except {alpha}-SG have the N-terminal in the intracellular space and a large extracellular domain including a cystein-rich cluster near the C-terminal regions [1]. In contrast, {alpha}-SG has the C-terminal amino acid residue in the intracellular space and a cystein-rich cluster near the transmembrane domain [1]. Therefore, it is likely that the structure of {alpha}-SG in the sarcolemma may have high affinity to the intracellular proteases such as calpain.

{gamma}- and {delta}-SGs contents in the failing heart were similar to those of the heart in the Sham rats. Incubation with purified calpain did not alter {gamma}- and {delta}-SGs in the isolated sarcolemma. Nigro et al. showed that amino acid sequences of {gamma}-SG revealed approximately 55% amino acid identity and approximately 70% similarity to that of {delta}-SG [24]. Thus, these SGs may have structures and/or location in the sarcolemma with high tolerance to calpain-induced proteolysis.

In summary, we found that the DRP complex in the failing heart following AMI was altered in rats without genetic defects. The acquired reduction was detected in the viable LV, Sep, and RV. The non-proportional decreases in {alpha}- and β-SGs and dystrophin may result in a reduction in the amount of functional DRP complex in the sarcolemma, suggesting that a decrease in DRP complex may play an important role in the genesis of contractile dysfunction in heart failure. The advanced heart failure following AMI may be developed by multiple decreases in DRP proteins, but not by a decrease in single protein. The present ex vivo and in vitro findings also suggest that calpain may contribute to a decrease in DRP complex.

Time for primary review 28 days.


   Acknowledgements
 
This work was partly supported by the Grants of the Promotion and Mutual Aid Corporation for Private Schools of Japan, and of Ministry of Education, Culture, Sports, Science and Technology.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Ozawa E., Noguchi S., Mizuno Y., Hagiwara Y., Yoshida M. From dystrophinopathy to sarcoglycanopathy: evolution of a concept of muscular dystrophy. Muscle Nerve (1998) 21:421–438.[CrossRef][Web of Science][Medline]
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