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Am J Physiol Heart Circ Physiol 288: H1674-H1682, 2005. First published December 2, 2004; doi:10.1152/ajpheart.00945.2004
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Sarcoplasmic reticulum Ca2+ transport and gene expression in congestive heart failure are modified by imidapril treatment

Qiming Shao,1 Bin Ren,1 Harjot K. Saini,1 Thomas Netticadan,1 Nobuakira Takeda,2 and Naranjan S. Dhalla1

1Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; and 2Department of Internal Medicine, Jikei University, Tokyo, Japan

Submitted 10 September 2004 ; accepted in final form 10 November 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was designed to test the hypothesis that blockade of the renin-angiotensin system improves cardiac function in congestive heart failure by preventing changes in gene expression of sarcoplasmic reticulum (SR) proteins. We employed rats with myocardial infarction (MI) to examine effects of an angiotensin-converting enzyme inhibitor, imidapril, on SR Ca2+ transport, protein content, and gene expression. Imidapril (1 mg·kg–1·day–1) was given for 4 wk starting 3 wk after coronary artery occlusion. Infarcted rats exhibited a fourfold increase in left ventricular end-diastolic pressure, whereas rates of pressure development and decay were decreased by 60 and 55%, respectively. SR Ca2+ uptake and Ca2+ pump ATPase, as well as Ca2+ release and ryanodine receptor binding activities, were depressed in the failing hearts; protein content and mRNA levels for Ca2+ pump ATPase, phospholamban, and ryanodine receptor were also decreased by ~55–65%. Imidapril treatment of infarcted animals improved cardiac performance and attenuated alterations in SR Ca2+ pump and Ca2+ release activities. Changes in protein content and mRNA levels for SR Ca2+ pump ATPase, phospholamban, and ryanodine receptor were also prevented by imidapril treatment. Beneficial effects of imidapril on cardiac function and SR Ca2+ transport were not only seen at different intervals of MI but were also simulated by another angiotensin-converting enzyme inhibitor, enalapril, and an ANG II receptor antagonist, losartan. These results suggest that blockade of the renin-angiotensin system may increase the abundance of mRNA for SR proteins and, thus, may prevent the depression in SR Ca2+ transport and improve cardiac function in congestive heart failure due to MI.

myocardial infarction; cardiac gene expression; renin-angiotensin system

BY VIRTUE OF THE PRESENCE of a Ca2+ release channel or ryanodine receptor and Ca2+-stimulated ATPase or Ca2+ pump ATPase or sarco/endoplasmic reticulum Ca2+-ATPase type 2 (SERCA2a) proteins, the sarcoplasmic reticulum (SR) is known to release and accumulate Ca2+ in cardiomyocytes and is thus considered to play a critical role in contraction and relaxation in the myocardium (8). Another protein, phospholamban, in the SR membrane has been shown to regulate the function of Ca2+ pump ATPase, whereas calsequestrin binds Ca2+ within the lumen of the SR. Although a wide variety of defects at the level of the ryanodine receptor, Ca2+ pump ATPase, and phospholamban in the SR membrane have been identified, calsequestrin appears to be resistant to different pathophysiological stimuli (8, 10, 11). Ca2+-handling abnormalities in cardiomyocytes from different types of failing hearts have been attributed primarily to changes in the SR membrane (23, 28). Although several investigators have reported depression in the activities of SR Ca2+ transport (Ca2+ uptake and Ca2+ release) in hypertrophied and failing hearts, the results regarding changes in SR gene and protein expression are conflicting (4, 5, 13, 18, 26, 27, 32, 33). Such conflicting findings can be seen to be due to differences in the type and stage of heart failure, drug treatments in patients before use of their tissue for analysis, and methods employed for determination of protein and gene expression. However, most of the previous studies have examined changes in SR function, protein expression, or gene expression in the failing heart and have used tissue at one stage of heart failure. Thus it is critical to examine changes in SR function at different stages of heart failure in each experimental model to reach a meaningful conclusion. Furthermore, extensive studies on changes in gene expression, protein contents, and SR function at any given stage of heart failure are needed to support the concept that remodeling (alterations in the molecular structure) of the SR membrane occurs during the development of heart failure.

By employing a rat model of congestive heart failure (CHF) due to myocardial infarction (MI), we have reported that SR Ca2+ uptake, Ca2+ release, Ca2+ pump ATPase, SR protein content, and SR gene expression were depressed in the failing heart (1, 2, 16, 29, 34, 44). Although some investigators (3, 32) were unable to detect changes in mRNA levels for SERCA2 and phospholamban, others (7, 14, 24, 40) showed decreases in SR Ca2+ uptake, Ca2+ release, Ca2+ pump ATPase, and mRNA levels for SR proteins in hearts failing due to MI. Furthermore, alterations in MI-induced SR Ca2+ transport and heart function were prevented by some angiotensin-converting enzyme (ACE) inhibitors, such as captopril and trandolapril (14, 34, 41). Other ACE inhibitors, enalapril and cilazapril, and an angiotensin II receptor type 1 (AT1R) antagonist, losartan, were shown to attenuate MI-induced changes in SR gene expression (16, 42); however, no report concerning these agents on MI-induced alterations in SR Ca2+ transport is available. In fact, an AT1R antagonist, valsartan, has been reported to prevent changes in SR function without improvements in cardiac performance in pacing-induced heart failure in dogs (30). A close examination of the literature has revealed that the information regarding the effects of ACE inhibitors or AT1R antagonists on SR Ca2+ transport as well as SR protein and gene expression is scattered and insufficient in heart failure due to MI. Thus a comprehensive investigation is warranted to support the hypothesis that blockade of the renin-angiotensin system (RAS) prevents MI-induced heart function and cardiac remodeling by improving alterations in SR function and gene expression. This study therefore was undertaken to provide detailed information regarding the effects of a long-acting ACE inhibitor, imidapril (22), on cardiac function, SR Ca2+ uptake and Ca2+ release activities, SR protein contents, and SR gene expression in heart failure due to MI. Although imidapril has been reported to prevent the MI-induced changes in cardiac performance, myosin ATPase activity and gene expression, PKC activities and isoforms, and phospholipase C and D activities (36–38, 43), no information regarding the effect of this agent on SR function or SR protein as well as gene expression is available in the literature. In this study, some experiments were also carried out with enalapril and losartan to test whether the effects of imidapril on MI-induced changes in cardiac performance and SR Ca2+ transport are simulated by these agents.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental model. All protocols were approved by the University of Manitoba Animal Care Committee in accordance with the standards of the Canadian Council on Animal Care. MI was induced in male Sprague-Dawley rats (175–200 g) by occlusion of the left coronary artery as described by Afzal and Dhalla (1). The animals were anesthetized with 1–5% isoflurane in O2 at a flow rate of 2 l/min, and the heart was exposed through the left thoracotomy. The left anterior descending coronary artery was ligated ~2–3 mm from the origin of the aorta with 6-0 silk suture. The heart was repositioned in the chest, and the incision was closed with a purse-string suture. Sham-operated animals were treated in the same way, except the artery was not ligated. Rats were then divided randomly into four groups: sham untreated (sham), sham + imidapril (sham + IMP), MI, and MI + IMP. Animals were fed rat chow and water ad libitum. Imidapril (1 mg·kg–1·day–1) was administered by gastric gavage starting at 3 wk after the surgery, and the treatment was continued for 4 wk. In some experiments, treatment with imidapril was continued for 8 or 12 wk; in others, the doses of imidapril were 0.2, 2, and 5 mg·kg–1·day–1. The infarcted rats were also treated with and without enalapril (10 mg·kg–1·day–1) or losartan (20 mg·kg–1·day–1) for 4 wk. Tap water was given by gastric gavage to sham and untreated infarcted animals in all experimental groups to avoid the physiological alterations associated with gavage-induced stress. After hemodynamic measurement, the hearts were surgically removed. The viable left ventricle (LV), including the septum, was dissected, weighed, and frozen in liquid nitrogen; the scar tissue was separated from the LV and weighed. Data from animals with low scar weight were excluded according to criteria reported earlier (16, 37). In some experiments, plasma levels of ANG II (45) as well as plasma and LV ACE activities (20) were determined. Lipid peroxidation was assayed by measuring the formation of LV malondialdehyde (MDA) by using the thiobarbituric acid method (9). Other parameters of oxidative stress, such as conjugated diene formation (9) as well as LV reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, were also measured (25).

Hemodynamic studies. The animals were anesthetized with an injection of a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg). The right carotid artery was exposed and cannulated with a microtipped pressure transducer (model SPR-249, Millar Instruments, Houston, TX) introduced through a proximal arteriotomy (1). The catheter was advanced carefully into the LV through the lumen of the carotid artery and secured with a silk ligature around the artery. Different hemodynamic parameters, such as heart rate, LV systolic pressure, LV end-diastolic pressure, rate of pressure development (+dP/dt), and rate of pressure decay (–dP/dt), were recorded by a computer program (AcqKnowledge 3.0.3, MP100, BIOPAC Systems, Goleta, CA).

Isolation of SR membranes. Membrane fraction enriched with SR was isolated according to the method described previously (1, 34). Briefly, viable nonischemic ventricular tissue was homogenized in a Waring blender in a medium containing (in mM) 10 NaHCO3, 5 NaN3, and 15 Tris·HCl (pH 6.8) at medium speed for 45 s. The homogenate was centrifuged at 10,000 g for 20 min, the pellet was discarded, and the supernatant was centrifuged again at 40,000 g for 30 min. The pellet was suspended in 0.6 M KCl and 20 mM Tris·HCl (pH 6.8) to solubilize the contractile proteins and centrifuged again at 40,000 g for 5 min. The final pellet was washed and suspended in 0.25 M sucrose and 20 mM Tris·HCl (pH 6.8). The protein concentration in each sample of SR was determined using Lowry's method, and the purity of these SR fractions was checked by monitoring marker enzyme activities (1, 34). SR preparations in all groups were found to be minimally (2–4%) but equally contaminated by other subcellular organelles.

Determination of SR Ca2+ uptake and Ca2+ pump ATPase activities. SR Ca2+ uptake activity was determined using the Millipore filtration technique (1). The SR membrane (0.05 mg/ml) was incubated in the presence of (in mM) 100 KCl, 20 Tris·HCl (pH 6.8), 5 MgCl2, 5 NaN3, and 5 potassium oxalate. The desired concentration of free 45Ca2+ in solution was obtained by addition of EGTA as calculated with a program developed by Fabiato (12). The reaction was started with 5 mM ATP, and a 0.2-ml sample was filtered at the desired time through a Millipore filter (45 µm) and immediately washed twice with 3 ml of ice-cold buffer. The filters were dried, and the radioactivity was counted using a liquid scintillation counter (Beckman Instruments, Mississauga, ON, Canada). Total and basal ATPase activities were determined in an incubation medium similar to that used for the Ca2+ uptake assay (2). When total ATPase was measured, nonradioactive CaCl2 (final 10 µM free Ca2+, except where indicated) was used, and when basal ATPase was measured, Ca2+ was omitted and 0.2 mM EGTA was added. The reaction was started by the addition of 5 mM Tris-ATP after 3 min of preincubation with 50 µg of membrane. The reaction was terminated by addition of 12% ice-cold trichloroacetic acid. Inorganic phosphate liberated during the reaction was estimated in the protein-free filtrate by a spectrophotometric method (2). The Ca2+-stimulated Mg2+-dependent ATPase (Ca2+ pump ATPase) activity was calculated as the difference between the total and basal ATPase activities.

SR 3[H]ryanodine receptor binding and Ca2+ release activities. High-affinity ryanodine binding was determined (31) by incubation of the cardiac SR membranes at 37°C for 1 h in a buffered medium containing 25 mM 3[H]ryanodine (6 Ci/mM); free Ca2+ concentration was 20 µM. The binding reaction was terminated by filtration through cellulose nitrate filters with a pore size of 0.45 µm. The filters were washed with cold buffer (twice with 5 ml each time). Each sample was processed in duplicate, and the radioactivity for each vial was measured with a liquid scintillation counter (Beckman Instruments) for 5 min. Nonspecific binding was measured in the presence of 10 µM ryanodine. The Ca2+ release activity of SR vesicles was determined by a method described earlier (31). Briefly, the SR fraction was incubated with 10 µM 45CaCl2 (20 mCi/ml) and 5 mM ATP for 45 min in a medium containing (in mM) 100 KCl, 5 MgCl2, 5 NaN3, 20 Tris·HCl (pH 6.8), and 5 potassium oxalate. The Ca2+ release was induced by addition of 1 mM EGTA and 1 mM Ca2+ to the reaction mixture and terminated at 15 s by Millipore filtration. Radioactivity in the filter was counted in 10 ml of scintillation fluid by using a liquid scintillation counter (Beckman Instruments). Treatment of the SR preparation with 20 µM ryanodine prevented 95–100% of this Ca2+-induced Ca2+ release.

Isolation of total RNA and Northern blot analysis. Total RNA was isolated from the viable LV (including the septum) of sham control and experimental rats with or without imidapril treatment by the acid guanidinium thiocyanate-phenol-chloroform method (TRIzol Reagent, GIBCO-BRL Life Technologies, Burlington, ON, Canada), as described previously (16, 34). Briefly, frozen samples of viable LV were ground with a pestle and mortar and homogenized with a Polytron (model PT3000) at 12,000 rpm twice for 15 s each, with 20 s between homogenizations, in the presence of 1.5 ml of TRIzol Reagent. The mixture was cooled on ice for an additional 15 min and centrifuged at 12,000 g (model J2-HS, Beckman Instruments) for 10 min at 4°C. The supernatant was incubated with chloroform (0.3 ml/sample) for 5 min at room temperature and then centrifuged at 12,000 g for 15 min at 4°C. The RNA containing the upper aqueous phase was kept at –20°C for 4 h after addition of 0.75 ml of isopropyl alcohol. After centrifugation at 12,000 g for 10 min at 4°C, RNA pellets were suspended in 75% molecular biology-grade ethanol diluted with diethyl pyrocarbonate (DEPC)-treated water. After sedimentation at 12,000 g for 10 min at 4°C, RNA pellets were washed again with 75% ethanol, centrifuged at 12,000 g for 10 min at 4°C, and vacuum dried by Speed Vac (model SC110, Sarvant Instruments, Farmingdale, NY). Samples were dissolved in DEPC-treated water, and the RNA concentration was calculated from the absorbance at 260 and 280 nm with SPECTRAmax PLUS (Molecular Devices, Sunnyvale, CA). The final RNA pellet was resuspended in sterile distilled water containing 0.1% DEPC and stored at –70°C. Total RNA (20 µg) was denatured at 65°C for 10 min and size fractionated on a 1.2% agarose gel containing 1.1 M formaldehyde. The blotted samples were transferred onto nylon membranes (Schleicher & Schuell, Keene, NH), UV cross-linked, and hybridized to randomly primed cDNA probes. The membranes were washed with standard saline citrate containing 0.1% SDS at 42°C for 20 min and exposed to Kodak X-Omat-AR film using an intensifying screen. After autoradiography, the mRNA bands were quantitated using a densitometer (model GS-670, Bio-Rad, Mississauga, ON, Canada). The optical density of Ca2+ pump ATPase, phospholamban, ryanodine receptor, and calsequestrin bands was divided by the GAPDH or 18S rRNA band optical density. The relative level of these messages, corrected against the GAPDH or 18S rRNA value in each sample, was calculated as a percentage of the mean value of the corresponding message level in the sham control group.

Western blot analysis. The relative contents of SR Ca2+ pump ATPase, phospholamban, and ryanodine receptor protein in SR membranes of sham and experimental animals with or without imidapril treatment were separated on 6% (for ryanodine receptor), 10% (for Ca2+ pump ATPase), and 15% (for phospholamban) gels by SDS-PAGE according to the method described elsewhere (16). The same volume (10 µl in each well, 20 µl in the ryanodine experiment) of samples was loaded for each group. Protein samples (20–25 µg of SR) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for all except the ryanodine receptor, which was transferred onto nitrocellulose membranes. Membranes were probed with monoclonal mouse anti-SERCA2a (1:2,000; Affinity Bioreagents, Golden, CO), monoclonal anti-ryanodine receptor antibodies (1:1,000; Research Diagnostic, Flanders, NJ), and monoclonal antiphospholamban (1:5,000; Upstate Biotechnology, Lake Placid, NY). The membranes were subsequently incubated for 40 min with secondary antibody (IgG antibody 1:3,000; Amersham, Arlington Heights, IL) and for 30 min with streptavidin-conjugated horseradish peroxidase (1:5,000; Amersham) at room temperature. The antibody-antigen complexes in all membranes were detected by the enhanced chemiluminescence kit (Amersham Life Sciences, Oakville, ON, Canada). An imaging densitometer (model GS-800, Bio-Rad, Hercules, CA) was used to scan the protein bands, and results were quantified using Quantity one 4.4.0 software (Bio-Rad).

Statistical analysis. Values are means ± SE. Differences between the control and experimental groups were calculated by one-way analysis of variance followed by the Newman-Keuls test. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General characteristics and hemodynamics. Sham control and infarcted rats were treated with imidapril for 4 wk starting 3 wk after the operation. The infarct in this experimental model is fully healed at 3 wk, and the clinical signs of CHF, such as ascites and dyspnea, begin to appear 4 wk after coronary artery occlusion. Thus the drug treatment was started 3 wk after the operation to test whether changes associated with CHF are prevented. The results in Table 1 indicate that cardiac hypertrophy, as reflected by increased LV and right ventricular weights in the infarcted animals, was prevented by imidapril treatment. However, imidapril treatment did not affect the scar weight or body weight. Hemodynamic parameters given in Table 1 indicate that LV systolic pressure did not change, but LV end-diastolic pressure was increased about fourfold in the MI group. Both +dP/dt and –dP/dt were decreased by ~50% of their respective control values (Table 1). All these changes in cardiac performance in the infarcted animals were attenuated by imidapril treatment. Neither the heart weight nor the hemodynamic parameters in the sham group were affected by imidapril treatment (Table 1).


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  		Table 1. General and hemodynamic characteristics of sham and MI rats with or without 4 wk of imidapril starting 3 wk after MI

 
SR Ca2+ uptake activity and SR Ca2+-stimulated ATPase activities. Ca2+ uptake by SR from sham control and infarcted animals with or without imidapril treatment for 4 wk was studied at different times of incubation (Fig. 1A) as well as at different concentrations of Ca2+ (Fig. 1B). The results show that SR Ca2+ uptake activities in the infarcted hearts were markedly depressed at all times of incubation and at different concentrations of Ca2+. These changes in cardiac SR Ca2+ uptake activity were prevented by treatment of infarcted animals with imidapril (Fig. 1, A and B). Because SR Ca2+ uptake is an energy-dependent process, we examined the SR Ca2+-stimulated ATPase or Ca2+ pump ATPase activity in sham control and experimental preparations. SR Ca2+ pump ATPase activity was depressed in the infarcted group, whereas imidapril treatment prevented this change in the failing heart (Fig. 1, C and D). Mg2+-ATPase activities in SR preparations were not altered in different groups (Fig. 1D). Imidapril treatment did not affect SR Ca2+ uptake and Ca2+ pump activities in the sham control group (Fig. 1).



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  		Fig. 1. Sarcoplasmic reticulum (SR) Ca2+ transport activities in sham rats and rats subjected to myocardial infarction (MI) with or without imidapril treatment. A: Ca2+ uptake at different times of incubation. B: Ca2+ uptake at different concentrations of Ca2+. C: SR Ca2+-stimulated ATPase activity. D: SR Mg2+-ATPase activity. Imidapril (IMP) was given (1 mg·kg–1·day–1 po) for 4 wk starting 3 wk after coronary occlusion. Values are means ± SE of 6 samples in each group. *P < 0.05 compared with sham (control). #P < 0.05 compared with MI.

 
SR Ca2+ release and [3H]ryanodine receptor binding activities. To examine Ca2+ release activities in SR preparations, Ca2+-induced Ca2+ release and specific [3H]ryanodine binding were measured. Values for maximal binding and dissociation constant (Kd) for ryanodine receptor binding were obtained from the Scatchard plot analysis of the data. SR Ca2+ release activity and maximal binding for ryanodine receptor were depressed in the infarcted heart without any change in the affinity (1/Kd) for ryanodine (Table 2). These alterations were prevented by treatment of the infarcted animals with imidapril. SR Ca2+ release and ryanodine receptor binding activities in the sham group were not affected by imidapril treatment (Table 2).


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  		Table 2. SR Ca2+ release and ryanodine receptor binding in sham and MI rats with or without 4 wk of imidapril starting 3 wks after MI

 
Cardiac performance and SR Ca2+ transport activities. To gain further information regarding the relation between changes in cardiac performance and SR Ca2+ transport activities, Ca2+ uptake and Ca2+ release activities were examined in SR preparations from hemodynamically assessed sham control and experimental animals. Treatment of the infarcted animals with high doses of imidapril (2 and 5 mg·kg–1·day–1), in contrast to treatment with a low dose (0.2 mg·kg–1·day–1), attenuated the depressed cardiac performance, SR Ca2+ uptake, and SR Ca2+ release activities (Table 3). The hypertrophic response of the infarcted heart was also prevented by high doses of imidapril without any changes in the scar weight. Comparison of these data in Table 3 with those in Fig. 1 and Tables 1 and 2 indicates that high doses of imidapril did not produce effects greater than those produced by imidapril at 1 mg·kg–1·day–1.


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  		Table 3. Cardiac performance and SR Ca2+ uptake and release in sham and MI rats with or without imidapril for 4 wk starting 3 wk after MI

 
Treatment of the infarcted animals with imidapril (1 mg·kg–1·day–1) for 8 or 12 wk also prevented the MI-induced hypertrophic response and attenuated the MI-induced changes in cardiac performance as well as SR Ca2+ uptake and Ca2+ release activities (Table 4). To test whether the beneficial effects of imidapril on heart function and SR Ca2+ transport are simulated by blockade of the RAS, the infarcted animals were treated with another ACE inhibitor, enalapril, and an AT1R antagonist, losartan, for 4 wk. Treatment of the infarcted animals with enalapril or losartan prevented the MI-induced hypertrophic response and attenuated the depressed cardiac performance as well as SR Ca2+ uptake and Ca2+ release activities (Table 5). Neither enalapril nor losartan treatment affected cardiac performance and SR Ca2+ transport activities in the sham control animals. The mean arterial pressure in sham control and infarcted animals (102 ± 2.6 and 99 ± 2.8 mmHg, respectively) was not significantly different from that in animals treated for 4 wk with imidapril, enalapril, or losartan.


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  		Table 4. Cardiac performance and SR Ca2+ uptake and release in sham and MI rats with or without 8 or 12 wk of imidapril starting 3 wk after MI

 

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  		Table 5. Cardiac performance and SR Ca2+ uptake and release in sham and MI rats with or without enalapril or losartan for 4 wk starting 3 wk after MI

 
SR protein content and gene expression. To study the molecular mechanisms of the beneficial effects of imidapril on SR Ca2+ transport activities in the infarcted heart, alterations in SR protein content and gene expression were measured in the sham and infarcted animals with or without imidapril treatment. Protein contents of the LV SR ryanodine receptor, Ca2+ pump ATPase, and phospholamban were identified by enhanced chemiluminescence Western blotting (Fig. 2). The SR Ca2+ pump ATPase, phospholamban, and ryanodine receptor protein levels were reduced by 55%, 46%, and 30%, respectively, in failing LV (Fig. 2). The reduction in protein contents of cardiac SR ryanodine receptor, Ca2+ pump ATPase, and phospholamban in the infarcted animals was prevented by imidapril treatment significantly. Because the protein content of calsequestrin in SR did not change in the infarcted hearts (16), protein content of calsequestrin was not determined in the present study.



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  		Fig. 2. Typical Western blot (A) and summaries of protein contents for SR ryanodine receptor (B), Ca2+ pump ATPase (C), and phospholamban (D) in sham and MI rats with or without imidapril treatment. Imidapril was given (1 mg·kg–1·day–1 po) for 4 wk starting 3 wk after coronary occlusion. Values are means ± SE of 6 experiments. *P < 0.05 compared with sham. #P < 0.05 compared with MI.

 
Levels of mRNA encoding SR Ca2+-cycling proteins were examined using Northern blot from the LV of sham, MI, sham + IMP, and MI + IMP groups (Fig. 3). mRNA levels for SERCA2, phospholamban, and ryanodine receptor proteins were decreased by ~55–65% with no changes in calsequestrin mRNA level in the MI group (Fig. 4). This decrease in LV mRNA levels for the ryanodine receptor, SERCA2, and phospholamban was prevented by imidapril treatment.



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  		Fig. 3. Typical Northern blot for SR ryanodine receptor (RyR), Ca2+ pump ATPase [sarco/endoplasmic reticulum Ca2+-ATPase type 2 (SERCA2)], phospholamban (PLB), and calsequestrin (CQS) mRNA in sham and MI rats with or without imidapril treatment. GAPDH and 18S mRNA levels were used as internal standards for correction of loading variation in each RNA sample. Imidapril was given (1 mg·kg–1·day–1 po) for 4 wk starting 3 wk after coronary occlusion. Quality of mRNA preparation is evident from ethidium bromide staining of 28S and 18S rRNA.

 


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  		Fig. 4. Quantitation of Northern blot analysis results for SR ryanodine receptor (A), calsequestrin (B), Ca2+ pump ATPase (C), and phospholamban (D) mRNA levels in sham and MI rats with or without imidapril treatment. Imidapril was given (1 mg·kg–1·day–1 po) for 4 wk starting 3 wk after coronary occlusion. Values are means ± SE of 6 experiments. *P < 0.05 compared with sham. #P < 0.05 compared with MI.

 
To gain some information regarding the mechanisms of the beneficial effects of drug treatments on SR function, some parameters reflecting the status of the RAS and oxidative stress were measured in the infarcted animals with or without imidapril, enalapril, and losartan. The results in Table 6 indicate an increase in plasma ANG II levels, plasma ACE activity, and LV ACE activity in the infarcted animals. Such an increase in these parameters, reflecting activation of the RAS due to MI, was attenuated by treatments with imidapril and enalapril, in contrast to losartan. Furthermore, MDA content, conjugated diene formation, and GSSG content were increased, whereas GSH content was decreased, in the LV of infarcted animals. These changes, indicating an increased degree of oxidative stress in the infarcted hearts, were attenuated by treatments of animals with imidapril, enalapril, and losartan.


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  		Table 6. Status of RAS and oxidative stress in sham and MI rats with or without drug treatments for 4 wk starting 3 wk after MI

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results in this study show cardiac hypertrophy, heart dysfunction, depressed SR Ca2+ release and Ca2+ uptake activities, and decreased SR Ca2+ pump ATPase activities in the infarcted myocardium. These findings are in agreement with previous reports on the MI model of CHF (1, 2, 34, 40, 41). The depression in SR Ca2+ uptake appears to be related to the SR Ca2+ pump mechanism, because SR Ca2+-stimulated ATPase activity was also decreased in MI-induced heart failure. Defective SR Ca2+ transport activities have also been reported in a rabbit model of heart failure due to MI (32) and other experimental models of heart failure, as well as in humans (1, 8, 10, 11, 17, 23, 45). The observed change in SR Ca2+-ATPase activity is specific in nature, because Mg2+-ATPase activity in the SR preparation from the failing heart was unaltered. Similarly, alterations in SR Ca2+ transport activities in the infarcted heart may not be due to any artifact, because marker enzyme activities showed minimal cross-contamination in control and experimental SR preparations. Furthermore, the depression in SR Ca2+ uptake activity in the failing heart was evident at different times of incubation as well as at different concentrations of Ca2+. A similar magnitude of depression (~50%) in the SR Ca2+ release channel in the failing heart was apparent when it was monitored by two different techniques: Ca2+-induced Ca2+ release and ryanodine receptor binding assay. Our results for Ca2+ release and Ca2+ uptake activities obtained by employing isolated SR preparations are similar to those of other investigators (40, 41) who assessed the SR Ca2+ transport activities in the infarcted hearts by employing skinned fiber preparations. Because SR Ca2+ release and Ca2+ uptake are intimately involved in the processes of cardiac contraction and relaxation, respectively, the observed depression in SR Ca2+ release and Ca2+ uptake activities may explain the reduction in cardiac +dP/dt and –dP/dt in the failing heart. These results indicate that the function of the SR with respect to regulation of intracellular Ca2+ is abnormal in the failing heart and support the view that a defect in the SR membrane may be closely linked to contractile abnormalities in CHF.

We have observed that SR protein contents for the ryanodine receptor, SERCA2, and phospholamban, as well as mRNA levels for SR proteins, are decreased in the 7-wk failing heart due to MI. These results are in agreement with earlier reports in this experimental model (16, 19, 24, 34, 44), except mRNA levels for the ryanodine receptor were increased and mRNA levels for SERCA2 were unaltered in the viable LV of 16-wk-infarcted animals (39). Such differences in gene expression for SR proteins may be dependent on the stage of heart failure 7 and 16 wk after MI (1). Similar findings in SR protein and/or gene expressions have also been reported in other types of failing hearts in animals and humans (5, 10, 23). Although the observed depression in SR Ca2+ release and Ca2+ uptake activities in MI-induced heart failure is likely to be due to decreased levels of protein contents for the ryanodine receptor and SERCA2, the reduced content for phospholamban in the failing heart would tend to augment SR Ca2+ pump ATPase activity. However, from the data presented here, it is obvious that the ratio of phospholamban to Ca2+ pump ATPase is increased, and this may contribute to inhibition of the SR Ca2+-ATPase pump. Thus it appears that alterations in SR protein contents of the ryanodine receptor and SERCA2 may be responsible for SR dysfunction and contractile abnormalities. Nonetheless, decreased levels of SR proteins may be due to changes in gene expression for SR proteins, because mRNA levels for the ryanodine receptor, SERCA2, and phospholamban were depressed in MI-induced heart failure. Because the mRNA level for calsequestrin did not change in hearts failing as a result of MI, it appears that alterations in gene expression for other SR proteins are specific. From these results, it is likely that alterations in gene and protein expressions may change the molecular structure of the SR membrane with respect to its protein contents, and such a remodeling of the SR membrane may be involved in development of CHF. The results presented here do not rule out other mechanisms, such as proteolysis and phospholipid hydrolysis, which may modify protein and phospholipid composition of the SR membrane, respectively; thus these changes may produce SR remodeling and alter its function (10).

In this study, we have shown that MI-induced hypertrophic response, heart dysfunction, alterations in SR Ca2+ release and Ca2+ uptake activities, and changes in gene and protein expressions are attenuated by treatment of infarcted animals with imidapril, an ACE inhibitor. Imidapril has also been shown to prevent changes in myofibrillar ATPase, myosin protein content, and gene expression for myosin isozymes (37). Because other ACE inhibitors such as captopril, enalapril, trandolapril, and cilazapril have also been reported to attenuate SR function or SR gene expression in hearts failing as a result of MI (14, 16, 34, 41, 42), it appears that ACE inhibitors may improve cardiac function by preventing alterations in SR gene expression. In view of the observations that imidapril prevented changes in PKC isozymes in the infarcted heart, it is possible that imidapril may produce beneficial effects on subcellular organelles through this mechanism in heart failure due to MI. Takeishi et al. (35) also suggested that ramipril may prevent downregulation of Ca2+-cycling protein expression in pressure-overloaded hearts in guinea pigs by attenuation of PKC translocation. The beneficial effects of imidapril on the MI-induced changes in SR Ca2+ transport activities are simulated by enalapril and losartan, and thus it is likely that the action of imidapril may be mediated through angiotensin blockade. This view is supported by the fact that imidapril and enalapril, in contrast to the AT1R antagonist (losartan), depressed the elevated plasma levels of ANG II and increased ACE activities in the infarcted animals. Furthermore, enalapril and losartan have also been shown to attenuate the MI-induced changes in cardiac SR gene and protein expressions (16). Such findings suggest that blockade of the RAS may prevent SR remodeling and, thus, may improve cardiac function in heart failure due to MI.

Because treatments of infarcted animals with imidapril, enalapril, and losartan did not alter the mean arterial pressure, the observed beneficial effects of these drugs may not be due to afterload reduction. Although it can be argued that improvement in SR function is a consequence of improved cardiac performance by RAS blockade, valsartan treatment has been reported to improve SR function without changes in cardiac performance in pacing-induced heart failure (30). Because imidapril prevented most of the MI-induced changes, other mechanisms, such as activation of the sympathetic nervous system (6) and oxidative stress (25) in subcellular remodeling and cardiac dysfunction in heart failure, cannot be ruled out. The increased degree of oxidative stress, as seen by changes in GSSG, GSH, and MDA contents as well as conjugated diene formation in the infarcted hearts, was prevented by imidapril, enalapril, and losartan treatments. However, it remains to be investigated whether the effects of imidapril on SR function and gene expression are mediated through the elevated levels of bradykinin (15) or nitric oxide release (21). Irrespective of the exact mechanism for the beneficial effects of imidapril, the data in the present study support the view that ACE inhibitors improve cardiac performance in CHF partly by preventing alterations in SR gene expression and SR remodeling in the failing heart.


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 METHODS
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This work was supported by grants from the Canadian Institute of Health Research (CIHR) Group in Experimental Cardiology and the CIHR Institute of Circulatory and Respiratory Health on Gene Environment Interaction in Heart Failure. N. S. Dhalla held the CIHR Research and Development Chair in Cardiovascular Research supported by Merck Frosst Canada.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, MB, Canada R2H 2A6 (E-mail: nsdhalla{at}sbrc.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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