Influence of long-term treatment of imidapril on mortality, cardiac ...

1118

Influence of long-term treatment of imidapril on mortality, cardiac function, and gene expression in congestive heart failure due to myocardial infarction Bin Ren, Qiming Shao, Pallab K. Ganguly, Paramjit S. Tappia, Nobuakira Takeda, and Naranjan S. Dhalla

Abstract: Although it is generally accepted that the efficacy of imidapril, an angiotensin-converting enzyme inhibitor, in congestive heart failure (CHF) is due to improvement of hemodynamic parameters, the significance of its effect on gene expression for sarcolemma (SL) and sarcoplasmic reticulum (SR) proteins has not been fully understood. In this study, we examined the effects of long-term treatment of imidapril on mortality, cardiac function, and gene expression for SL Na+/K+ ATPase and Na+–Ca2+ exchanger as well as SR Ca2+ pump ATPase, Ca2+ release channel (ryanodine receptor), phospholamban, and calsequestrin in CHF due to myocardial infarction. Heart failure subsequent to myocardial infarction was induced by occluding the left coronary artery in rats, and treatment with imidapril (1 mg·kg–1·day–1) was started orally at the end of 3 weeks after surgery and continued for 37 weeks. The animals were assessed hemodynamically and the heart and lung were examined morphologically. Some hearts were immediately frozen at –70 °C for the isolation of RNA as well as SL and SR membranes. The mortality of imidapril-treated animals due to heart failure was 31% whereas that of the untreated heart failure group was 64%. Imidapril treatment improved cardiac performance, attenuated cardiac remodeling, and reduced morphological changes in the heart and lung. The depressed SL Na+/K+ ATPase and increased SL Na+–Ca2+ exchange activities as well as reduced SR Ca2+ pump and SR Ca2+ release activities in the failing hearts were partially prevented by imidapril. Although changes in gene expression for SL Na+/K+ ATPase isoforms as well as Na+–Ca2+ exchanger and SR phospholamban were attenuated by treatments with imidapril, no alterations in mRNA levels for SR Ca2+ pump proteins and Ca2+ release channels were seen in the untreated or treated rats with heart failure. These results suggest that the beneficial effects of imidapril in CHF may be due to improvements in cardiac performance and changes in SL gene expression. Key words: sarcolemmal Na+/K+ ATPase, Na+–Ca2+ exchange, sarcoplasmic reticulum, heart failure, ACE inhibition. 1127 Résumé : Le lien entre l’efficacité de l’imidapril, un inhibiteur de l’enzyme de conversion de l’angiotensine, et l’amélioration des paramètres hémodynamiques durant l’insuffisance cardiaque congestive (ICC) est généralement reconnu; toutefois, l’importance de son effet sur l’expression génique des protéines du sarcolemme (SL) et du réticulum sarcoplasmique (RS) demeure vague. Dans la présent étude, nous avons examiné les effets d’un traitement prolongé à l’imidapril sur la mortalité, la fonction cardiaque et l’expression des gènes pour la Na+/K+ ATPase et l’échangeur Na+–Ca2+ du SL et ainsi que pour la pompe Ca2+ ATPase du RS, le canal de libération du Ca2+ (récepteur à la ryanodine), le phospholamban et la calséquestrine durant une ICC causée par un infarctus du myocarde. L’insuffisance cardiaque par infarctus du myocarde a été induite par l’occlusion de l’artère coronaire gauche de rats, et le traitement à l’imidapril (1 mg·kg–1·jour–1), administré par voie orale, a commencé 3 semaines après la chirurgie et s’est poursuivi pendant 37 semaines. Les animaux ont été évalués sur le plan hémodynamique, et le cœur et le poumon ont été examinés sur le plan morphologique. Certains cœurs ont été congelés immédiatement à –70 °C pour isoler l’ARN ainsi que le SL et le RS. Le taux de mortalité des animaux traités à l’imidapril en raison de l’insuffisance cardiaque a été de 31 %, alors que celui du groupe en insuffisance cardiaque non traité a été de 64 %. Le traitement à l’imidapril a amélioré les performances du coeur, atténué le remodelage cardiaque et réduit les modifications morphologiques dans le cœur et le poumon. La diminution de l’activité Na+/K+ ATPase et l’augmentation de l’activité de l’échangeur Na+–Ca2+ dans le SL ainsi que l’augmentation de l’activité de la pompe Ca2+ et de la libération de Ca2+ dans le RS des cœurs

Received 10 February 2004. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 6 January 2005. B. Ren, Q. Shao, P.K. Ganguly, P.S. Tappia, and N.S. Dhalla.1 Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Departments of Physiology and Human Nutritional Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, MB R2H 2A6, Canada. N. Takeda. Aoto Hospital, Department of Internal Medicine, Jikei University, Tokyo, Japan. 1

Corresponding author (e-mail: [email protected]).

Can. J. Physiol. Pharmacol. 82: 1118–1127 (2004)

doi: 10.1139/Y04-115

© 2004 NRC Canada

Ren et al.

1119 insuffisants ont été partiellement prévenues par l’imidapril. Bien que les variations de l’expression génique pour les isoformes de la Na+/K+ ATPase du SL ainsi que pour l’échangeur Na+–Ca2+ et le phospholamban du RS aient été atténuées par le traitement à l’imidapril, aucune modification des taux d’ARNm pour les protéines des pompes Ca2+ et des canaux de libération du Ca2+ du RS n’a été observée chez les rats en insuffisance cardiaque traités ou non traités. Ces résultats donnent à penser que les effets bénéfiques de l’imidapril durant l’ICC pourraient être liés aux améliorations des performances du coeur et aux modifications de l’expression génique dans le SL. Mots clés : Na+/K+ ATPase sarcolemmale, échangeur Na+–Ca2+, réticulum sarcoplasmique, insuffisance cardiaque, inhibition de l’ECA. [Traduit par la Rédaction]

Ren et al.

Introduction It has become clear that Ca2+ is a critical physiological regulator of contractile and metabolic processes in cardiac muscle where both sarcolemma (SL) and sarcoplasmic reticulum (SR) are intimately involved in the regulation of intracellular Ca2+ (Carafoli 1988; Dhalla et al. 1982). For example, SL Na+/K+ ATPase plays a major role in the excitation–contraction coupling of the myocardium by maintaining the electrochemical gradient across the SL membrane and by indirectly regulating the concentration of intracellular Ca2+ via the Na+–Ca2+ exchanger. The changes in Na+/K+ ATPase and Na+–Ca2+exchange are implicated in cardiac dysfunction and in fact have been suggested to form one of the salient features of the subcellular basis of heart failure (Dhalla et al. 1998). Similarly, SR Ca2+ pump ATPase, Ca2+-binding protein (calsequestrin (CQS)), Ca2+ regulatory protein (phospholamban (PLB)), and Ca2+ release channels (ryanodine receptors (RYRs)) are known to participate in the regulation of intracellular Ca2+ concentration for the excitation–contraction coupling of the heart (Dhalla et al. 1998). There are several reports that indicate that altered Ca2+ transport activities in the SR are implicated in cardiac dysfunction in failing hearts (Dhalla et al. 1998; Afzal and Dhalla 1992; Suko et al. 1970). In view of the crucial role played by different membrane systems in regulating intracellular Ca2+ and heart function, it has been considered that the inability of the failing heart to generate contractile force is due to remodeling of both SL and SR membranes (Dhalla et al. 1998). Although there are reports indicating alterations in SR Ca2+ transport genes and proteins in heart failure (Arai et al. 1993; Zarain-Herzberg et al. 1996a), relatively little information concerning gene expression for SL Na+/K+ ATPase and the Na+–Ca2+ exchanger in heart failure due to myocardial infarction (MI) is available in the literature. The beneficial effects of angiotensin-converting enzyme (ACE) inhibitors on mortality and survival in congestive heart failure (CHF) are widely accepted (Pfeffer et al. 1985); however, the mechanism underlying their protective effect remains unknown. Early treatment of rats with imidapril, a long-acting ACE inhibitor (Yamada et al. 1992), has been shown to prevent changes in cardiac function due to MI (Shao et al. 1996). Furthermore, early treatment with imidapril was also found to decrease mortality and prevent arrhythmias due to MI in rats (Ren et al. 1998). These data provide information regarding the acute effects of imidapril, but virtually nothing is known concerning the long-term action of imidapril on CHF due to MI. Since ACE inhibitors

are considered to improve cardiac function by influencing subcellular mechanisms (Dhalla et al. 1998), the present study was undertaken to test the hypothesis if changes in cardiac function and gene expression for SL Na+/K+ ATPase isoforms and the Na+–Ca2+ exchanger in CHF are prevented by long-term treatment with imidapril. The effects of longterm imidapril treatment were also examined on SR gene expression for Ca2+ pump ATPase (SERCA2), PLB, CQS, and the Ca2+ release channel or RYR in heart failure for the purpose of comparison. In addition, the mortality of infarcted animals as well as morphological alterations in the heart and lung were investigated with or without long-term imidapril treatment. Alterations in cardiac performance and biochemical activities of SL and SR membranes obtained from the failing heart were also monitored.

Methods Experimental model Animals used in this study were handled according to the guidelines of the Canadian Council on Animal Care and the experimental protocol was approved by the Animal Care Committee of the University of Manitoba. MI was induced in male Sprague-Dawley rats (175–200 g) by occluding the left coronary artery as described in Afzal and Dhalla (1992). The animals were anesthetized with a gas mixture of 1%– 5% isoflurane in 2 L of oxygen, the skin was incised along the left sternal border, the third and fourth ribs were cut open, and a retractor was inserted. Subsequent to exposing the heart, the pericardial sac was gently torn, the left coronary artery was tightly ligated 2–3 mm from its origin with a suture of 6-0 silk, and the heart was quickly repositioned into the chest. The air in the chest was sucked out with a 10-mL syringe just before closing the wound with a pursestring suture. Animals were allowed to recover in an incubator with sufficient oxygen supply for 6–12 h. The mortality of all animals operated on in this manner was about 35% within 48 h. Sham-operated animals were treated in the same way except that the coronary artery was not tied. Imidapril treatment Animals were housed in cages and received rat chow and water ad libitum. Room temperature and humidity were controlled and had a 12-h day–night cycle. These animals were randomly divided into four groups: sham, sham plus imidapril treatment, MI, and MI plus imidapril treatment. Imidapril (1 mg·kg·–1·day–1) was administered orally by gavage and started at the end of the third week after surgery © 2004 NRC Canada

1120

and the treatment was continued for up to 37 weeks or until the animals died. The number of animal deaths was counted during the period from the third week to the 40th week after MI. Hemodynamic studies The animals were anesthetized with an intraperitoneal injection of a mixture of ketamine (60 mg·kg–1) and xylazine (10 mg·kg–1). The right carotid artery was exposed and cannulated with a microtip pressure transducer (model SPR249; Millar Instruments, Houston, Texas) introduced through a proximal arteriotomy (Afzal and Dhalla 1992). The arterial blood pressure was measured at this point. The catheter was advanced carefully through the lumen of the carotid artery until it entered the left ventricle. The catheter was secured with a silk ligature around the artery, and readings were taken with a computer program (AcqKnowledge for Windows 3.0; Harvard, Montreal, Que.). The following measurements were made: left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), heart rate, maximum rate of pressure development (+dP/dt), and pressure decay (–dP/dt). At the end of the hemodynamic assessment, organs such as the heart, liver, lung, kidney, and spleen were dissected out as quickly as possible. Some tissue samples were processed, paraffin embedded, and stained with hematoxylin and eosin or Masson’s Trichrome for histological examination. For other hearts, the left ventricle (including septum but excluding scar tissue) and right ventricle were separated and frozen at –70 °C for isolation of RNA or SL and SR preparations. SL and SR fractions and measurements of activities Purified SL and SR membranes were prepared from the viable left ventricle tissue without scar but including septum according to the methods described in Dixon et al. (1992a) and Afzal and Dhalla (1992), respectively. Marker enzyme studies (Dixon et al. 1992a; Afzal and Dhalla 1992) revealed that each membrane fraction from control and experimental hearts was minimally (2%–3%) but equally contaminated with fragments of other subcellular organelles. The activities of Na+/K+ ATPase and Na+–Ca2+ exchange in the SL fractions were determined by methods used earlier (Dixon et al. 1992a, 1992b). SR Ca2+ uptake and Ca2+ stimulated ATPase activities were measured by methods employed previously (Afzal and Dhalla 1992). Isolation of total RNA and Northern blot analysis Total RNA was isolated from left ventricles of sham and MI rats with or without imidapril treatment by using the guanidine thiocyanate method (Chomczynski and Sacchi 1987). The final RNA pellet was suspended in sterile distilled water containing 0.1% diethyl pyrocarbonate and stored at –70 °C. Twenty micrograms of total RNA was denatured at 65 °C for 10 min and size fractioned on a 1.2% agarose gel containing 1.2 mol·L–1 formaldehyde. The blotted samples were transferred onto nylon membrane (Schleicher & Schuell, Keene, N.H.), UV cross-linked, and hybridized to randomly primed cDNA probes. The membrane was washed with standard saline citrate and 0.1% sodium dodecyl sulfate at 42 °C for 20 min and exposed to Kodak X-Omat-AR film using an intensifying screen at

Can. J. Physiol. Pharmacol. Vol. 82, 2004 Fig. 1. Graph showing influence of imidapril (IMP) (1 mg·kg–1·day–1) on 37-week mortality in myocardial infarcted (MI) rats. Treatment started 3 weeks after surgery and rats were followed for a further 37 weeks.

–70 °C. After autoradiography, the individual mRNA bands were quantitated using a densitometer (GS-670; Bio-Rad, Mississauga, Ont.). The optical density of each of the mRNA bands was divided by that for the GAPDH band. The relative level of these messages was corrected by the GAPDH value in each sample and calculated as a percentage of the mean value for the corresponding message level in the sham control group. These methods for RNA gel electrophoresis, blotting, and analysis were similar to those performed earlier by Zarain-Herzberg et al. (1996b). The cDNA probes used, Na+/K+ ATPase isoforms α1, α2, α3, and β1, and GAPDH were obtained from the American Type Cell Culture (Rockville, Md.); the Na+–Ca2+ exchanger was a kind gift from Dr. Kenneth D. Philipson (Los Angeles, Calif.). cDNA for SERCA2a, RYR, PLB, and CQS were the same as described earlier (Zarain-Herzberg et al. 1996a, 1996b). Statistical analysis The data are presented as means ± SE. Differences between the control and experimental and drug-treated and untreated groups were calculated with analysis of variance followed by Duncan’s new multiple test. The data on mortality were analyzed by using the χ2 method and Kaplan Meier curves. A value of p < 0.05 was considered significant.

Results A total of 180 animals were involved in the mortality study, which did not include animals that died within 3 days after the coronary artery ligation surgery. At the end of the third week after inducing MI, surviving rats were randomly divided into four groups as mentioned in the Methods section. Figure 1 shows that mortality was 9.9% and 6.6% in sham and sham plus imidapril treatment groups, respectively. On the other hand, mortality was 63.8% in the heart failure group; imidapril treatment produced a significant decrease in mortality (31.2%). The results for general characteristics of animals in each group are shown in Table 1. The mass of the failing heart was significantly increased when compared with that of sham heart (2.31 ± 0.02 vs. 1.51 ± 0.1 g); imidapril reduced the heart mass of the MI group to © 2004 NRC Canada

Ren et al.

1121 Table 1. General characteristics and hemodynamic parameters of myocardial infarcted (MI) rats with or without imidapril treatment.

Body mass (g) Heart mass (g) Viable left ventricular mass (mg) Right ventricular mass (mg) Scar mass (mg) Ascites (mL) Heart rate (beats·min–1) LVEDP (mmHg) LVSP (mmHg) +dP/dt (mmHg·s–1) –dP/dt (mmHg·s–1)

Sham control

Sham plus imidapril treatment

MI untreated

MI plus imidapril treatment

712±8 1510±10 1200±10 310±20 nd nd 288±13 3.2±0.8 142±8.2 5512±413 4939±483

689±15 1490±10 1190±60 300±30 nd nd 271±16 2.9±0.3 136±5.9 4993±289 4885±349

719±14 2310±20* 1500±50* 530±20* 280±32 10.7±0.74 203±19* 18.7±2* 95±7.8* 2686±326* 2233±176*

690±12 1910±50** 1307±60** 350±40** 253±27 2.6±0.52** 287±18** 8.5±0.4** 137±7.1** 4132±436** 4065±448**

Note: Values are means ± SE of six animals (1 mmHg = 133.322 Pa); nd, not detectable. Imidapril was given orally (1 mg·kg·–1·day–1) for 37 weeks starting at 3 weeks after surgery. Viable left ventricular mass did not include scar tissue. *p < 0.05 compared with the sham control; **p < 0.05 compared with MI untreated.

1.9 ± 0.05 g. It can also be seen that the left and right ventricular masses were significantly increased in the heart failure group and this increase was markedly attenuated by imidapril treatment. There was no difference in scar mass between the heart failure groups with or without imidapril treatment. The amount of ascites was reduced by the imidapril treatment to a quarter of that in the heart failure group (2.64 ± 0.52 vs. 10.75 ± 1.74 mL). Hemodynamic parameters in rats of all groups are also shown in Table 1. Significant changes in hemodynamic characteristics have been observed in the heart failure group compared with the sham group, including increased LVEDP and depressed LVSP, +dP/dt, and –dP/dt; heart failure decreased both +dP/dt and –dP/dt by more than 50%. All of these changes were attenuated by imidapril treatment. A marked depression in heart rate in the heart failure group was also prevented by imidapril treatment. It can also be seen from Table 1 that imidapril did not affect the hemodynamic parameters in sham animals. The heart, liver, lung, kidney, and spleen in all groups were examined histologically. The organs of animals from the sham group with or without imidapril treatment showed normal histology, indicating that imidapril itself did not cause any morphologic change in sham animals. The enlarged and dilated hearts from the 40-week infarcted animals showed fibrosis of the left ventricular free wall from the base to the apex and thickening of myofibrils in the right ventricular free wall and septum (Figs. 2 and 3). Except for a little surviving innermost myocardium, all layers of the failing left ventricle wall were fibrotic. The collagen in these failing hearts was abundant and thick (Fig. 2). On the other hand, treatment of infarcted animals with imidapril showed that collagen in most of the left ventricular free wall tended to be more sparse and thinner, even though thick collagen was still seen in the inner layer of the heart (Fig. 2). In addition, imidapril reduced the thickening of myofibrils in the right ventricle and septum (Figs. 2 and 3). Examination of lungs from animals with heart failure showed congestion of alveolar capillaries and proliferation of interstitial cells in the pulmonary tissue (Fig. 3). Intimal fibrosis of small pulmonary vein was also observed. Imidapril treatment mark-

edly reduced the congestion of alveolar capillaries and proliferation of interstitial cells, indicating a large improvement in pulmonary derangement associated with heart failure (Fig. 3). Compared with heart and lung tissues, abnormal morphology was not evident in other organs such as liver, kidney, and spleen in the infarcted animals (data not shown). To test if SL and SR membranes are altered in the failing hearts, both Na+/K+ ATPase and Na+-dependent Ca2+ uptake activities in SL as well as Ca2+ pump and Ca2+ release activities in SR were determined in the sham and infarcted hearts. Furthermore, the SL and SR activities in these animals were measured upon imidapril treatment. The data in Table 2 indicate that the SL Na+/K+ ATPase activity was decreased whereas the SL Na+–Ca2+ exchange activity was increased in the failing heart. Treatment of infarcted animals with imidapril prevented the observed changes in SL Na+/K+ ATPase and Na+-dependent Ca2+ exchange activities whereas imidapril did not affect these activities in sham control animals. Both SR Ca2+ release and Ca2+ uptake activities as well as SR Ca2+ stimulated ATPase activity were markedly depressed in failing hearts; these changes were prevented by treatment with imidapril (Table 2). On the other hand, the SR Ca2+ pump and Ca2+ release activities in the sham control animals were not affected by imidapril treatment. The mRNA levels for Na+/K+ ATPase isoforms and Na+– Ca2+ exchanger were measured in the left ventricle by Northern blot analysis in MI and sham animals with or without imidapril (Fig. 4). The results showed that the relative level of Na+/K+ ATPase isoform α2 was markedly decreased in the MI group compared with the sham control, but mRNA levels for isoforms α1 and α3 were significantly increased in MI rats, especially the α3 level, which increased more than 12-fold (Fig. 5). Although mRNA level for Na+/K+ ATPase isoform β1 was increased slightly in the failing heart, this change was not significant. On the other hand, a significant increase in the relative mRNA level of the SL Na+–Ca2+ exchanger was observed in the failing hearts due to MI (Fig. 6). These changes in SL gene expression in the infarcted animals were prevented by imidapril treatment (Figs. 5 and 6). The mRNA levels for SR proteins were also determined by Northern blot analysis (Fig. 7). The relative © 2004 NRC Canada

1122

Can. J. Physiol. Pharmacol. Vol. 82, 2004

Fig. 2. Sections of the left ventricular wall and ventricular septum of the heart at the end of the 40-week experimental period. A normal left ventricular wall appearance is seen in sham animals (a) whereas fibrosis replacement is found in the whole layer in the MI group (b). The collagen is abundant and thick and surviving myocardial fibers are scattered in the inner layer. The left ventricular wall is replaced by fibrosis except for a little surviving myocardium in the innermost layer in the MI plus imidapril group (c). Noticeably, the collagen is considerably more sparse and thinner than that shown in the MI group. Masson’s Trichrome stain, 100× magnification. Longitudinal section of interventricular septum show normal size of myocardial fibers in sham group (d). The myocardial fibers in the MI group (e) are much thicker than those in sham whereas the myocardial fibers in MI plus imidapril group (f) are markedly thinner than those in the failing heart. Hematoxylin and eosin stain, 400× magnification.

levels of mRNA for SR proteins in Fig. 8 show that mRNA levels for RYR, SERCA2, and CQS remained unchanged whereas that for PLB was significantly decreased in the heart failure group. The depression in mRNA for PLB was prevented by the long-term treatment with imidapril.

Discussion In this study, we have shown that long-term treatment of the infarcted rats with an ACE inhibitor, imidapril, decreased mortality and attenuated clinical signs of CHF including

ascites and lung congestion. These results are in agreement with another report showing similar beneficial effects of long-term treatment of infarcted rats with captopril (Pfeffer et al. 1985). Chronic inhibition of ACE by enalapril has also been shown to improve the survival of patients with heart failure (CONSENSUS Trial Study Group 1987; SOLVD Investigators 1991, 1992; Acute Infarction Ramipril Efficacy Study Investigators 1993). Several other ACE inhibitors have also been reported to produce beneficial effects in CHF both in patients and in experimental models (Dhalla et al. 1998; Goldstein et al. 1995; Oldroyd et al. 1991; Kagaya et © 2004 NRC Canada

Ren et al.

1123

Fig. 3. Right ventricular wall of the heart and section of lung at the end of the 40-week experimental period. Normal thickness of the right ventricular wall is seen in the sham group (a). The right ventricular free wall is wider and the myocyte size is increased in the heart failure (MI) group (b). The right ventricular free wall due to the imidapril treatment (MI plus imidapril) group (c) is narrower compared with that in the failing heart, even though it is still wider than in the sham group. Hematoxylin and eosin stain, 100× magnification. Lung section showing normal alveolar size in sham group (d) and congestion of alveolar capillaries and intimal fibrosis in the MI group (e). Imidapril treatment reduces alveolar congestion and intimal fibrosis (f). Hematoxylin and eosin stain, 100× magnification.

al. 1996). Imidapril has also been shown to prevent pulmonary damage in chronic heart failure (Guazzi et al. 1997) and renal dysfunction in hypertensive rats (Fujiwara et al. 1994) and reduce mortality in a small coronary artery disease model in mice (Ogiku et al. 1994). Since angiotensin II type 1 receptor antagonists have been reported to produce beneficial effects in infarcted animals (Hanatani et al. 1998; Daniels et al. 2001; Guo et al. 2003), it is likely that the observed effects of imidapril are mediated through blockade of the renin–angiotensin system. On the other hand, the role of bradykinin accumulated as a consequence of ACE inhibition

(Gohlke et al. 1994) can not be ruled out for the imidapril effects in infarcted animals. Likewise, reduction of the activated sympathetic activity (Basu et al. 1996) as well as oxidative stress (Bagchi et al. 1989) by imidapril as mechanisms for preventing the CHF cannot be excluded at this time. Thus, it appears that the beneficial effects of imidapril in CHF may be associated with changes in hormonal profile and status of oxidative stress (Michel et al. 1998; Khaper and Singal 2001). Since imidapril was found to attenuate the increase in LVEDP as well as depressed heart rate, LVSP, +dP/dt, and © 2004 NRC Canada

1124

Can. J. Physiol. Pharmacol. Vol. 82, 2004

Table 2. SL ATPase and Na+–Ca2+ exchange and SR Ca2+ release and Ca2+ pump activities in sham and myocardial infarcted (MI) rats with or without imidapril treatment.

SL activities Mg2+ ATPase (µmol Pi·mg–1·h–1) Na+/K+ ATPase (µmol Pi·mg–1·h–1) Na+-dependent Ca2+ uptake (nmol·mg–1·2 s–1) SR activities Ca2+ release (nmol·mg–1·15 s–1) Ca2+ uptake (nmol·mg–1·min–1) Ca2+ stimulated ATPase (nmol Pi·mg–1·min–1)

Sham control

Sham plus imidapril treatment

MI untreated

MI plus imidapril treatment

75±4.2 17.6±1.5 6.4±0.33

77±5.1 18.4±0.9 6.1±0.27

71±3.4 7.6±0.4* 10.5±0.65*

72±4.4 13.2±1.2** 7.2±0.25**

8.9±0.51 90±5.8 118±7.9

8.6±0.49 86±6.4 108±8.8

3.2±0.14* 34±3.2* 45±4.1*

6.8±0.64** 76±4.5** 87±5.2**

Note: Each value is the mean ± SE of six experiments in each group. Imidapril (1 mg·kg–1·day–1) was given orally for 37 weeks starting 3 weeks after surgery to both sham control and infarcted (MI) animals. Viable left ventricular tissue including septum, but excluding scar tissue, was employed. *p < 0.05 compared with the sham control; **p < 0.05 compared with MI untreated.

Fig. 4. Typical Northern blots of Na+/K+ ATPase and the Na+– Ca2+ exchanger mRNA in left ventricles from 40-weeks sham and MI rats with or without imidapril (IMP) treatment. Blots for different isoforms of Na+/K+ ATPase (α 1, α 2, α 3, and β 1) were obtained whereas GAPDH mRNA was used as an internal standard for correcting loading variations in each sample.

–dP/dt in the infarcted animals, it can be argued that the beneficial effects of imidapril in CHF are due to improvement in cardiac performance. It is pointed out that the observed decrease in heart rate in the MI group may be due to down-regulation of β-adrenoceptor-mediated signal transduction mechanisms (Dhalla et al. 1997). Alterations in cardiac performance were also prevented by treatment of infarcted animals with imidapril for a shorter duration (Wang et al. 2002). The ability of imidapril to improve heart

Fig. 5. mRNA abundance of isoforms β 1 (a), α 1 (b), α 2 (c), and α 3 (d) of Na+/K+ ATPase in left ventricles from sham and MI rats with or without imidapril (IMP) treatment. Values are means ± SE of five samples in each group. *p < 0.05 compared with the sham control; #p < 0.05 compared with the MI group.

function was found not to be due to reduction in the infarct size because the scar mass in the treated and untreated animals was not different. On the other hand, it should be pointed out that cardiac hypertrophy, and increased ventricular wall thickness, fiber size, and collagen in the failing myocardium were reduced by long-term treatment with imidapril. Treatment of infarcted animals with angiotensin II type 1 receptor antagonists has also been reported to decrease myocardial fibrosis and collagen (Ju et al. 1997) and cardiac hypertrophy (Guo et al. 2003). These observations support the view that improvement in cardiac performance in the infarcted heart by imidapril may be due to ventricular © 2004 NRC Canada

Ren et al. Fig. 6. mRNA abundance of the Na+–Ca2+ exchanger in left ventricles from sham and MI rats with or without imidapril (IMP) treatment. Values are mean ± SE of five samples in each group. *p < 0.05 compared with the sham control; #p < 0.05 compared with the MI group.

1125 Fig. 8. mRNA abundance of Ca2+ channel or ryanodine receptor (RYR) (a), Ca2+ pump ATPase (SERCA2) (b), phospholamban (PLB) (c), and calsequestrin (CQS) (d) in left ventricles from sham and MI rats with or without imidapril (IMP) treatment. Values are means ± SE of five samples in each group. *p < 0.05 compared with the sham control; #p < 0.05 compared with the MI group.

Fig. 7. Typical Northern blots of Ca2+ channel or ryanodine receptor (RYR), Ca2+ pump ATPase (SERCA2), phospholamban (PLB), and calsequestrin (CQS) proteins in left ventricles from sham and MI rats with or without imidapril treatment.

remodeling. However, the effect of imidapril on changes in cardiomyocyte phenotype in the failing heart is suggested by our observation that treatment of imidapril prevented alterations in both SL and SR activities as well as cardiac gene expression. Thus, it is evident that improvement in heart function by long-term treatment of infarcted animals may be related to prevention in subcellular remodeling in the failing heart. The results described in this study reveal that depression in SL Na+/K+ ATPase activity in the 40-week infarcted heart was associated with alterations in gene expression for Na+/K+ ATPase isoforms. Depression in SL Na+/K+ ATPase

in failing hearts from both humans and cardiomyopathic hamsters has been reported to be due to alterations in the gene expression for different Na+/K+ ATPase isoforms (Schwinger et al. 1999; Kato et al. 2000). Furthermore, the increased SL Na+–Ca2+ exchange activity and mRNA levels were also observed in the failing heart. Although some investigators (Studer et al. 1994) have reported an increase in Na+–Ca2+ exchange and gene expression activity in the end stage of human heart failure, others (Schwinger et al. 1999) have failed to confirm the presence of such changes in the failing heart. Changes in SL activities and gene expression have been reported by other investigators (Dixon et al. 1992a, 1992b; Charlemagne et al. 1994). These conflicting results appear to depend on the stage and type of heart failure. This view is supported by the fact that the observed decrease in SR Ca2+ uptake in the 40-week infarcted heart was not associated with depressions in mRNA levels for SERCA2, RYR, and CQS whereas association of changes in SR Ca2+ uptake activity with those in SR gene expression in 8-week infarcted hearts was observed (Afzal and Dhalla 1992; Zarain-Herzberg et al. 1996a; Shao et al. 1999). Alterations in SR function and gene expression have also been observed in different types of failing hearts (Gupta et al. 1997; Lecarpentier et al. 1987; Arai et al. 1994). Nonetheless, our results concerning the effect of imidapril treatment to attenuate changes in SL and SR activities as well as in SL gene expression lend further support to the concept that improvement in cardiac function may be related to the prevention of cardiac remodeling in the failing heart. © 2004 NRC Canada

1126

It can be argued that the changes in SL Na+/K+ ATPase gene expression in the infarcted heart are the secondary effect of improvements in cardiac function after treatment with imidapril. Although it is difficult to rule out this possibility on the basis of the information at hand, we believe that this may not be the case. This view is based on our observations that the improved cardiac function of the imidapril-treated infarcted group was associated with both depressions in mRNA levels for isoforms α1 and α3 of Na+/K+ ATPase and the Na+–Ca2+ exchanger and increases in mRNA levels for isoform α2 of Na+/K+ ATPase and SR PLB. Furthermore, mRNA levels for isoform β1 of Na+/K+ ATPase as well as SR SERCA2 and RYR were unaffected by imidapril treatment. Such a differential effect of imidapril treatment on cardiac genes in the infarcted group is thus unlikely to be due to improved heart function but can be seen to be due to a depression in the angiotensin II mediated signal transduction as a consequence of ACE inhibition. However, it remains to be examined whether other ACE inhibitors produce effects similar to those observed with imidapril upon treating the infarcted group under chronic conditions. Since treatment of sham control animals with imidapril did not produce any changes in cardiac gene expression, it is unlikely that the observed alterations in the infarcted animals are due to a direct effect of imidapril. At any rate, the observed alterations in cardiac gene expression can be seen to result in corresponding changes in cardiac proteins as well as in both SL and SR activities in CHF. Thus, it appears that drugs such as imidapril may prevent alterations in cardiac gene expression as well as subcellular activities and improve cardiac performance in CHF.

Acknowledgements The work reported in this paper was supported by grants from the Canadian Institutes of Health Research (CIHR Group in Experimental Cardiology) and a grant from the CIHR Institute of Circulatory and Respiratory Health on Gene Environment Interaction in Heart Failure. N.S.D. holds the CIHR Research and Development Chair in Cardiovascular Research supported by Merck Frosst, Canada. P.K.G. was a visiting professor from the College of Medicine and Medical Sciences, Arabian Gulf University, Bahrain.

References Acute Infarction Ramipril Efficacy Study Investigators. 1993. Effect of ramipril on mortality and morbidity of survivors of acute myocardial infarction with clinical evidence of heart failure. Lancet, 342: 821–828. Afzal, N., and Dhalla, N.S. 1992. Differential changes in left and right ventricular SR Ca2+ transport in congestive heart failure. Am. J. Physiol. Heart Circ. Physiol. 262: H868–H874. Arai, M., Alpert, N.R., MacLennan, D.H., Barton, P., and Periasamy, M. 1993. Alterations in sarcoplasmic reticulum gene expression in human heart failure. A possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ. Res. 72: 463–469. Arai, M., Matsui, H., and Periasamy, M. 1994. Sarcoplasmic reticulum gene expression in cardiac hypertrophy and heart failure. Circ. Res. 74: 555–564.

Can. J. Physiol. Pharmacol. Vol. 82, 2004 Bagchi, D., Prasad, R., and Das, D.K. 1989. Direct scavenging of free radicals by captopril, an angiotensin converting enzyme inhibitor. Biochem. Biophys. Res. Commun. 158: 52–57. Basu, S., Sinha, S.K., Shao, Q., Ganguly, P.K., and Dhalla, N.S. 1996. Neuropeptide Y modulation of sympathetic activity in myocardial infarction. J. Am. Coll. Cardiol. 27: 1796–1803. Carafoli, E. 1988. Membrane transport of calcium: an overview. Methods Enzymol. 157: 3–11. Charlemagne, D., Orlowski, J., Oliviero, P., Rannou, F., SainteBeuve, C., Swynghedauw, B., and Lane, L.K. 1994. Alteration of Na+-K+ ATPase subunit mRNA and protein levels in hypertrophied rat heart. J. Biol. Chem. 269: 1541–1547. Chomczynski, P., and Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate – phenol – chloroform extraction. Anal. Biochem. 162: 156–159. CONSENSUS Trial Study Group. 1987. Effects of enalapril on mortality in severe congestive heart failure: results of the Cooperative North Scandinavian Enalapril Survival Study (CONSENSUS). N. Engl. J. Med. 316: 1429–1435. Daniels, M.C., Keller, R.S., and de Tombe, P.P. 2001. Losartan prevents contractile dysfunction in rat myocardium after left ventricular myocardial infarction. Am. J. Physiol. Heart Circ. Physiol. 281: H2150–H2158. Dhalla, N.S. Pierce, G.N., Panagia, V., Singal, P.K., and Beamish, R.E. 1982. Calcium movements in relation to heart function. Basic Res. Cardiol. 77: 117–139. Dhalla, N.S., Wang, X., Sethi, R., Das, P.K., and Beamish, R.E. 1997. β-Adrenergic linked signal transduction mechanisms in failing hearts. Heart Failure Rev. 2: 55–65. Dhalla, N.S., Shao, Q., and Panagia, V. 1998. Remodeling of cardiac membranes during the development of congestive heart failure. Heart Failure Rev. 2: 261–272. Dixon, I.M.C., Hata, T., and Dhalla, N.S. 1992a. Sarcolemmal Na+-K+ ATPase activity in congestive heart failure due to myocardial infarction. Am. J. Physiol. Cell Physiol. 262: C664– C671. Dixon, I.M.C., Hata, T., and Dhalla, N.S. 1992b. Sarcolemmal calcium transport in congestive heart failure due to myocardial infarction in rats. Am. J. Physiol. Heart Circ. Physiol. 262: H1387–H1394. Fujiwara, T., Yuasa, H., Ogiku, N., and Kawai, Y. 1994. Histopathological investigation on salt-loaded stroke-prone spontaneously hypertensive rats, whose biochemical parameters of renal dysfunction were ameliorated by administration of imidapril. Jpn. J. Pharmacol. 66: 231–240. Gohlke, P., Linz, W., Scholkens, B.A., Kuwer, I., Bartenbach, S., Schnell, A., and Unger, T. 1994. Angiotensin-converting enzyme inhibition improves cardiac function: role of bradykinin. Hypertension, 23: 411–418. Goldstein, S., Sharov, V.G., Cook, J.M., and Sabbah, H.N. 1995. Ventricular remodeling: insights from pharmacologic interventions with angiotensin-converting enzyme inhibitors. Mol. Cell. Biochem. 147: 51–55. Guazzi, M., Marenzi, G., Alimento, M., Contini, M., and Agostoni, P. 1997. Improvement of alveolar-capillary membrane diffusing capacity with enalapril in chronic heart failure and counteracting effect of aspirin. Circulation, 95: 1930–1936. Guo, X., Chapman, D., and Dhalla, N.S. 2003. Partial prevention of changes in SR gene expression in congestive heart failure due to myocardial infarction by enalapril or losartan. Mol. Cell. Biochem. 254: 163–172. Gupta, R.C., Shimoyama, H., Tanimura, M., Nair, R., Lesch, M., and Sabbah, H.N. 1997. SR Ca2+ ATPase activity and expression © 2004 NRC Canada

Ren et al. in ventricular myocardium of dogs with heart failure. Am. J. Physiol. Heart Circ. Physiol. 273: H12–H18. Hanatani, A., Yoshiyama, M., Takeuchi, K., Kim, S., Nakayama, K., Omura, T. et al. 1998. Angiotensin II type1-receptor antagonist candesartan cilexitil prevents left ventricular dysfunction in myocardial infarcted rats. Jpn. J. Pharmacol. 78: 45–54. Ju, H., Zhao, S., Jassal, D.S., and Dixon, I.M.C. 1997. Effect of AT1 receptor blockade on cardiac collagen remodeling after myocardial infarction. Cardiovasc. Res. 35: 223–232. Kagaya, Y., Hajjar, R.J., Gwathmey, J.K., Barry, W.H., and Lorell, B.H. 1996. Long-term angiotensin-converting enzyme inhibition with fosinopril improves depressed responsiveness to Ca2+ in myocyctes from aortic-banded rats. Circulation, 94: 2915–2922. Kato, K., Lukas, A., Chapman, D.C., and Dhalla, N.S. 2000. Changes in the expression of cardiac Na+-K+ ATPase subunits in the UM-X7.1 cardiomyopathic hamster. Life Sci. 67: 1175– 1183. Khaper, N., and Singal, P.K. 2001. Modulation of oxidative stress by a selective inhibition of angiotensin II type 1 receptors in MI rats. J. Am. Coll. Cardiol. 37: 1461–1466. Lecarpentier, Y., Waldenström, A., Clergue, M., Chemla, D., Oliviero, P., Martin, J.L., and Swynghedauw, B. 1987. Major alterations in relaxation during cardiac hypertrophy induced by aortic stenosis in guinea pigs. Circ. Res. 61: 107–116. Michel, J.B., Lattion, A.L., Salzmann, J.L., Cerol, M.L., Philippe, M., Camilleri, J.P., and Corvol, P. 1988. Hormonal and cardiac effects of converting enzyme inhibition in rat myocardial infarction. Circ. Res. 62: 641–650. Ogiku, N., Sumikawa, H., Nishimura, T., Narita, H., and Ishida, R. 1994. Reduction of the mortality rate by imidapril in a small coronary artery disease model (NZW × BXSB) F1 male mice. Jpn. J. Pharmacol. 64: 129–133. Oldroyd, K.G., Pye, M.P., Ray, S.G., Christie, J., Ford, I., Cobbe, S.M., and Dargie, H.J. 1991. Effect of early captopril administration on infarct expansion, left ventricular remodelling and exercise capacity after acute myocardial infarction. Am. J. Cardiol. 68: 713–718. Pfeffer, M.A., Pfeffer, J.M., Steinberg, C., and Finn, P. 1985. Survival after an experimental myocardial infarction: beneficial effects of long-term therapy with captopril. Circulation, 72: 406– 412. Ren, B., Lukas, A., Shao, Q., Guo, M., Takeda, N., Aitken, R.M., and Dhalla, N.S. 1998. Electrocardiographic changes and mortality due to myocardial infarction in rats with or without imidapril treatment. J. Cardiovasc. Pharmacol. Ther. 3: 11–22.

1127 Schwinger, R.H., Wang, J., Frank, K., Muller-Ehmsen, J., Brixius, K., McDonough, A.A., and Erdmann, E. 1999. Reduced sodium pump α 1, α 3, and β 1-isoform protein levels and Na+-K+ ATPase activity but unchanged Na+–Ca2+ exchanger protein levels in human heart failure. Circulation, 99: 2105–2112. Shao, Q., Takeda, N., Temsah, R., Dhalla, K.S., and Dhalla, N.S. 1996. Prevention of hemodynamic changes due to myocardial infarction by early treatment of rats with imidapril. Cardiovasc. Pathobiol. 1: 180–186. Shao, Q., Ren, B., Zarin-Herzberg, A., Ganguly, P.K., and Dhalla, N.S. 1999. Captopril treatment improves the sarcoplasmic reticular Ca(2+) transport in heart failure due to myocardial infarction. J. Mol. Cell. Cardiol. 31: 1663–1672. SOLVD Investigators. 1991. Effect of enalapril on survival in patients with reduced left ventricular ejection fractions and congestive heart failure. N. Engl. J. Med. 325: 293–302. SOLVD Investigators. 1992. Effect of enalapril on mortality and the development of heart failure in asymptomatic patients with reduced left ventricular ejection fraction. N. Engl. J. Med. 327: 685–691. Studer, R., Reinecke, H., Bilger, J., Eschenhagen, T., Böhm, M., Hasenfuss, G. et al. 1994. Gene expression of the cardiac Na+– Ca2+ exchanger in end-stage human heart failure. Circ. Res. 75: 443–453. Suko, J., Vogel, J.H., and Chidsey, C.A. 1970. Intracellular calcium and myocardial contractility. Reduced calcium uptake and ATPase of the sarcoplasmic reticular fraction prepared from chronically failing calf hearts. Circ. Res. 27: 235–247. Wang, J., Liu, X., Ren, B., Rupp, H., Takeda, N., and Dhalla, N.S. 2002. Modification of myosin gene expression by imidapril in failing heart due to myocardial infarction. J. Mol. Cell. Cardiol. 34: 847–857. Yamada, Y., Endo, M., Kohno, M., Otsuka, M., and Takaita, O. 1992. Metabolic fate of the new angiotensin-converting enzyme inhibitor imidapril in animals. 6th communication: interspecies comparison of pharmacokinetics and excretion of imidapril metabolites in rats, dogs and monkeys. Arzneim-Forsch. 4: 499– 506. Zarain-Herzberg, A., Afzal, N., Elimban, V., and Dhalla, N.S. 1996a. Decreased expression of cardiac sarcoplasmic reticulum Ca2+ pump ATPase in congestive heart failure due to myocardial infarction. Mol. Cell. Biochem. 163/164: 285–290. Zarain-Herzberg, A., Rupp, H., Elimban, V., and Dhalla, N.S. 1996b. Modification of sarcoplasmic reticulum gene expression in pressure overload cardiac hypertrophy by etomoxir. FASEB J. 10: 1303–1309.

© 2004 NRC Canada