Molecular and Cellular Biochemistry 254: 163–172, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Partial prevention of changes in SR gene expression in congestive heart failure due to myocardial infarction by enalapril or losartan Xiaobing Guo, Donald Chapman and Naranjan S. Dhalla Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada Received 15 January 2003; accepted 14 April 2003

Abstract Although activation of the renin-angiotensin system (RAS) is known to produce ventricular remodeling and congestive heart failure (CHF), its role in inducing changes in the sarcoplasmic reticulum (SR) protein and gene expression in CHF is not fully understood. In this study, CHF was induced in rats by ligation of the left coronary artery for 3 weeks and then the animals were treated orally with or without an angiotensin converting enzyme inhibitor, enalapril (10 mg/kg/day) or an angiotensin II receptor antagonist, losartan (20 mg/kg/day) for 4 weeks. Sham-operated animals were used as control. The animals were hemodynamically assessed and protein content as well as gene expression of SR Ca2+-release channel (ryanodine receptor, RYR), Ca2+-pump ATPase (SERCA2), phospholamban (PLB) and calsequestrin (CQS) were determined in the left ventricle (LV). The infarcted animals showed cardiac hypertrophy, lung congestion, depression in LV +dP/dt and –dP/dt, as well as increase in LV end diastolic pressure. Both protein content and mRNA levels for RYR, SERCA2 and PLB were decreased without any changes in CQS in the failing heart. These alterations in LV function as well as SR protein and gene expression in CHF were partially prevented by treatment with enalapril or losartan. The results suggest that partial improvement in LV function by enalapril and losartan treatments may be due to partial prevention of changes in SR protein and gene expression in CHF and that these effects may be due to blockade of the RAS. (Mol Cell Biochem 254: 163–172, 2003) Key words: congestive heart failure, renin-angiotensin system, sarcoplasmic reticulum proteins, cardiac gene expression, ACE inhibitors, angiotensin II receptor antagonists

Introduction By virtue of its ability to release and accumulate Ca2+, the sarcoplasmic reticulum (SR) is not only known to regulate the intracellular concentration of Ca2+ in cardiomyocytes but is also involved in the process of contraction and relaxation of the cardiac muscle [1–3]. While Ca2+-release channel (ryanodine receptor, RYR) and Ca2+-pump ATPase (SERCA2) are involved in SR Ca2+-release and Ca2+-accumulation, phospholamban (PLB) and calsequestrin (CQS) have been shown to regulate the SR Ca2+-pump activity and bind Ca2+ in lumen of the SR tubules, respectively [2–4]. Several investigators

[3–11] have reported varying degrees of defects in SR Ca2+release and Ca2+-uptake activities as well as SR protein content and gene expression in failing human hearts as well as in different types of experimental heart failure. Alterations in SR protein content and associated changes in the SR Ca2+release and Ca2+-uptake activities are considered to result in Ca2+-handling abnormalities in cardiomyocytes and subsequent cardiac dysfunction in the failing heart [3–5, 12]. Although changes in SR gene expression have been suggested to alter the molecular structure of SR membrane proteins in heart failure [4], the mechanisms of SR remodeling in the failing heart are poorly understood.

Address for offprints: N.S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada R2H 2A6 (E-mail: [email protected])

164 Previous work has shown that the left ventricular (LV) dysfunction in congestive heart failure (CHF) due to myocardial infarction (MI) is associated with abnormalities in Ca2+-handling by cardiomyocytes, defects in SR Ca2+-uptake and Ca2+release activities as well as changes in SR protein and gene expression for SERCA2 and PLB [13–18]. On the other hand, some investigators did not observe any change in the cardiomyocyte function and Ca 2+-handling despite altered SERCA2 gene expression in the infarcted animals [19, 20]. In fact, even no changes in SERCA2 or PLB gene expression were detected in cardiomyocytes from the infarcted rats [21, 22]. Since changes in SR function as well as mRNA levels for SR proteins were found to be region- and time-dependent following the coronary occlusion [14, 23], it is possible that some of the conflicting results with respect to SR abnormalities are due to differences in the duration of MI and the exact portion of the uninfarcted viable myocardium employed in various studies. Furthermore, such conflicting results could be due to differences in the degree of heart failure as a consequence of variation in infarct size in MI animals. Some attempts have been made to understand the mechanisms of changes in cardiac performance, cardiomyocyte Ca2+-handling, SR function as well as SR protein and gene expression upon treatment of the infarcted animals with different interventions. In this regard, improvement in hemodynamic function in the infarcted animals was associated with normalization of SERCA2 protein content upon treatment with 3-(2,2,2-trimethyl-hydrazinium) propionate [24] or by exercise training [25]. Although treatment of infarcted animals with thyroid hormones (T3) or its analogue (3,5-diiodothyropropionic acid) improved cardiac function and prevented changes in SERCA2, PLB and RYR protein contents in the SR membrane, the depression in SERCA2 gene expression was not corrected [26, 27]. On the other hand, treatment of infarcted animals with trandolapril, an angiotensin converting enzyme (ACE) inhibitor, improved cardiac function and prevented changes in SERCA2, PLB and RYR protein contents as well as depression in SR Ca2+-release activity [28]. Likewise, cilazapril, an ACE inhibitor, or candesartan, an angiotensin II receptor antagonist, prevented cardiac dysfunction and changes in SERCA2 gene expression in the infracted animals [29, 30]. Captopril, an ACE inhibitor, has also been reported to prevent MI-induced alterations in cardiac function [31, 32], Ca2+ regulation in cardiomyocytes [33] and SR Ca2+-pump activity as well as SERCA2 and PLB protein and gene expression [32]. Although enalapril, an ACE inhibitor, and losartan, an angiotension II receptor antagonist, have been reported to produce beneficial effects on cardiac remodeling and heart failure [34–36], no report regarding the effects of enalapril or losartan on changes in cardiac SR Ca2+regulating proteins and gene expression in the infarcted animals has appeared in the literature. In view of such a scattered and insufficient information on the role of renin-angiotensin

system (RAS) in changing the SR function in infarcted animals, the present study was undertaken to investigate if the beneficial effects of enalapril and losartan on cardiac function are associated with prevention of changes in SR protein and gene expression due to CHF induced by coronary occlusion in rats.

Materials and methods Experimental model MI was produced in male Sprague-Dawley rats (175–200 g) by occlusion of the left coronary artery as described earlier [14, 37, 38]. After rats were anesthetized with isofluorane, the hearts were exposed through left thoracotomy. The left coronary artery was ligated about 2–3 mm from the origin of the aorta with 6-0 silk suture and the heart was repositioned into the chest. The air in the thoracic cavity was removed by using a syringe after closing the chest with a purse-string suture. A mixture of 95% O2 and 5% CO2 was supplied to the animals under positive pressure during the operation. Shamoperated animals were treated similarly except that the coronary suture was not tied. Mortality rate of the ligated animals was about 35% within first 48 h. All experimental protocols were approved by the Animal Care Committee of the University of Manitoba following guidelines established by the Canadian Institutes of Health Research. Treatment with enalapril or losartan All rats received standard care, kept at 12 h day/night cycle and were fed rat chow and water ad libitum. The animals were randomly divided into 6 groups in this project: sham control (sham), sham-treated with enalapril (sham + ENP), shamtreated with losartan (sham + LOS), infarcted (MI), infarctedtreated with enalapril (MI + ENP) and infarcted-treated with losartan (MI + LOS). Three weeks after the operation, enalapril (10 mg/kg/day), losartan (20 mg/kg/day) or tap water was given orally via a gastric tube to untreated-sham and infarcted groups, respectively for 4 weeks. Preliminary experiments in our laboratory showed optimal beneficial effects of enalapril and losartan on heart function in the infracted animals at the doses used in this study. It should be pointed out that the infarcts in this experimental model are completely healed at 3 weeks whereas early and moderate stages of CHF become evident at 4 and 8 weeks after the operation, respectively [14, 31, 32, 37]. This study was undertaken to test the effects of enalapril and losartan on SR gene expression in the failing heart where the drug treatments were started at 3 weeks after inducing MI, i.e. just before the development of CHF. Enalapril and losartan were supplied by Merck Research

165 Laboratories (Rahway, NJ, USA). Although animals receiving drugs via gastric gavage daily for several weeks can be considered to be under stress, the hemodynamic performance of sham control animals receiving drugs was not different from those receiving tap water via the same route. Furthermore, the experimental design for this study was similar to that used in earlier investigations [32, 38].

Hemodynamic assessment The animals were anesthetized with an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (10 mg/kg) cocktail. A cannula with a microtip pressure transducer (model SPR-249, Millar Instruments, Houston, TX, USA) was introduced into the right carotid artery via proximal arteriotomy [14]. The systolic pressure, diastolic pressure and the mean arterial pressure (MAP) were measured in aorta and the catheter was advanced to enter the LV. Hemodynamic recordings were taken by AcqKnowledge for Windows 3.03 (MP100, BIOPAC Systems, Inc., Goleta, CA, USA). Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate, rate of pressure development (+dP/ dt) and pressure decay (–dP/dt) were measured in these anesthetized animals. After the hemodynamic measurements, the hearts were removed, ventricles were separated and weighed. The LV (including septum) as well as the scar tissue were dissected and weighed; the viable LV was frozen in liquid nitrogen and stored at –70°C. In addition, the lungs and liver from all the animals were removed and weighed for the wet weight; the lungs and liver were dried in the oven at 60°C for 48 h and weighed again. The scar weight/total LV weight (including septum and infarcted tissue) ratio was calculated.

Scar size measurement The scar size in the left ventricle was measured histologically according to the method used by Pfeffer et al. [39]. Briefly, the heart was fixed in 10% buffered formalin and cut from apex to base into five transverse. The ventricular sections (10 µM thick) were stained with Masson’s trichrome stain and the scar size (Fig. 1) was calculated as % of the LV wall area.

in 50% formamide, 7% formaldehyde, 20 mM MOPS (pH 7.4), 2 mM EDTA (pH 8.0), 0.1% SDS and electrophoresed in a 1.2% agarose/formaldehyde gel to size fractionate the mRNA transcripts. The fractionated RNA was transferred onto a 0.45 µm positively charge-modified nylon filter (NYTRAN® PLUS, Schleicher and Schuell, Keene, NH, USA) by capillary action. The filter was removed after 24 h and RNA was covalently crosslinked by UV radiation (UV Stratalinker 2400, Stratagene). Blots were prehybridized to random primed cDNA or oligonucleotide probes, in a mixture of 50% formamide, 10% Denhardt’s solution, 1% SDS, 0.2 mg/ml denatured salmon sperm DNA, 10 mM EDTA (pH 8.0), 25% ‘4 × RNA’ solution [3 M NaCl, 0.6 M Tris-HCl (pH 7.5), 0.18 M NaH2PO4, 0.24 M Na2PO4, 0.1 M Na4P2O7] at 42°C for 6–16 h. Membranes were hybridized for 16–24 h at 42°C in the presence of 32P-labelled specific probes. The following cDNA and oligonucleotide probes were utilized: (a) RYR: a 2.2 kb cDNA fragment of the rabbit cardiac RYR (courtesy of Dr. D.H. MacLennan, University of Toronto, Toronto, Canada); (b) SERCA2: a 0.762 kb cDNA fragment of the rabbit cardiac Ca 2+-ATPase (courtesy of Dr. A.K. Grover, McMaster University, Hamilton, Canada); (c) PLB: a 0.153 kb cDNA fragment of the rabbit cardiac PLB (courtesy of Dr. D.H. MacLennan, University of Toronto, Toronto, Canada); (d) CQS: a 2.5 kb cDNA fragment of the rabbit cardiac CQS (courtesy of Dr. A. Zilberman, University of Cincinnati, Cincinnati, OH, USA); (e) Glyceraldehyde-3phosphate dehydrogenase (GAPDH): a 1.2 kb cDNA fragment of the human GAPDH (American Type Culture Collection, Rockville, MD, USA) and (f) 18S: a 24 base oligonucleotide probe of rat 18S ribosomal RNA. Both GAPDH and 18S rRNA were used as an internal standard for correcting variations in loading and blotting efficiency of RNA. The filter was then washed in 1 × SSC/1% SDS inside an INNOVA 4000 incubator (New Brunswick Scientific, Edison, NJ, USA) oscillating at a rate of 64 rotations per min. After washing, the membrane was exposed to X-ray film (Kodak X-OMATAR) with two intensifying screens at –70°C. After autoradiography the mRNA bands were quantitated by scanning densitiometry (GS-670, Bio-RAD Company, Mississauga, Canada). All mRNA signals were normalized to the respective GAPDH mRNA signal and expressed as a percentage of the mean value observed for the sham control group.

Isolation of SR membrane and Western blot analysis Isolation of total RNA and Northern blot analysis Total RNA was extracted from the viable LV (including the septum) in 6 different groups by the procedure of Chomczynski and Sacchi [40]. The final RNA pellet was suspended in 0.1% DEPC sterile distilled water and stored in –70°C refrigerator. Twenty µg of total RNA was denatured at 65°C for 10 min

SR membrane fraction was isolated according to the method described elsewhere [14]. In short, viable LV tissue was homogenized in a polytron (Kinematica, Switzerland) in a medium containing 10 mM NaHCO3, 5 mM NaN3, and 15 mM Tris-HCl (pH 6.8) at the speed of 12,000 rpm for 45 sec. The homogenate was centrifuged at 10,000 g for 20 min and the

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Fig. 1. Relationship of scar size and scar weight/LV weight ratio with total ventricular weight in rats at 7 weeks of coronary artery ligation. Histological sections of sham control and infarcted hearts are also shown. Blue staining represents scar. Thirty infarcted rats were used in each group. LV includes septum and scar tissue whereas total ventricular weight includes right ventricle and LV weight.

supernatant was centrifuged at 40,000 g for 30 min. The pellet thus obtained was suspended in 0.6 M KCl and 20 mM TrisHCl (pH 6.8) to solubilize the contractile proteins and again centrifuged at 40,000 g for 45 min. The final pellet was washed and suspended in 0.25 M sucrose and 10 mM histidine and stored at –70°C. The relative protein contents of SR RYR, SERCA2, PLB and CQS from control and experimental preparations were determined by running 5–12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) according to the method of Laemmli with a 4% stacking gel and Western blot analysis [32]. The SR vesicles (1 mg/ ml) were added to the SDS-PAGE loading buffer (0.25 M Tris-HCl (pH 6.8), 8% (w/v) SDS, 45% glycerol, 20% βmercaptoethanol and 0.006% bromophenol blue) in a ratio of 3:1. The sample loads for each group were of the same volume (10 µl in each well, 20 µl in RYR experiment). The SDS-PAGE was carried out at 200 voltage for 45–60 min. The separated proteins were then electroblotted to immobilon-P

transfer membrane (Millipore Corporation, Bedford, MA, USA) in transfer buffer containing 25 mM Tris-HCl, 120 mM glycine and 20% methanol (v/v) at 0.5 mA. The transferred membranes were shaken for 2 h in blocking buffer containing TBS (10 mM Tris, 150 mM NaCl) and 5% fat-free powdered milk. The membranes were incubated for 1 h at room temperature with either a monoclonal mouse anti-SR RYR antibody (1:1,000; Research Diagnostics Inc., NJ, USA), SERCA2 antibody (1:2,000; Affinity Bioreagents Inc., Golden, CO, USA), mouse anti-SR PLB antibody (1:5,000; Upstate Biotechnology, Lake Placid, NY, USA) or anti-SR CQS antibody (1:3,000; Upstate Biotechnology, Lake Placid, NY, USA). The membranes were subsequently incubated for 40 min with secondary antibody (IgG antibody 1:3,000; Amersham Corporation, Arlington Heights, IL, USA). Finally, the membranes were incubated with strepdavidin conjugated horseradish peroxidase (1:5,000; Amersham Corporation, Arlington Heights, IL, USA) for 30 min at room

167 temperature. The blots were rinsed in the TBST (TBS and 0.2% Tween 20) buffer 3 times (10 min each time) between each of the preceding steps. For chemiluminescent detection, the membrane sheets were dipped into the luminal substrate solution (Amersham Corporation, Arlington Heights, IL, USA) and the chemilumigrams were developed on ECL-Hyperfilm (Amersham Corporation) to visualize RYR, SERCA2, PLB and CQS bands. The bands were analyzed by the model GS670 Imaging Densitometer (Bio-Rad Company, Mississauga, Canada) with the Image Analysis Software Version 1.0 and were expressed in relation to control values.

Statistical analysis All values were expressed as mean ± S.E. The difference among sham and experimental groups were calculated by oneway analysis of variance (ANOVA) followed by the NewmanKeuls test. Significant differences among groups were defined by a probability of p < 0.05.

Results General and hemodynamic characteristics There was no significant change of body weight and liver wet/ dry weight ratio between sham control and infarcted animal group at 7 weeks after MI; however, the heart weight (including LV, RV and scar), heart/body weight ratio and the lung wet/dry weight ratio were significantly increased indicating the presence of cardiac hypertrophy and lung congestion in the untreated infarcted animals (Table 1). These changes in general characteristics in the infarcted animals were partially prevented upon treatment with enalapril or losartan. There was no difference for either the scar weight or the scar weight/ total LV weight ratio among the untreated, enalapril-treated

and losartan-treated animals (Table 1). A significant decrease in both LV +dP/dt and –dP/dt, and an increase in LVEDP were observed in the 7 weeks untreated infarcted animals; however, no difference in heart rate, LVSP or MAP between the infarcted and sham groups was observed (Table 2). Treatment of MI animals with enalapril or losartan partially normalized these hemodynamic alterations. Treatment of sham control animals with enalapril or losartan did not affect the cardiac performance and general characteristics significantly. Although the interpretation of hemodynamic data may be complicated because we have utilized anesthetized animals, it is pointed out that both sham control and infarcted animals were anesthetized under similar conditions. Furthermore, no improvement of heart function by drug treatments was observed in the sham control anesthetized animals, unlike the infarcted animals. Since changes in heart function due to MI are dependent upon scar size as measured histologically [39] and the viable LV fixed for scar measurements cannot be employed for biochemical studies, we have used scar weight/total LV (including septum and infarcted tissue) weight as a marker for the extent of scar size [38, 41]. In order to show that there exists a linear relationship between scar size and scar weight/LV weight, we measured scar size and scar weight in 7 weeks infarcted rats by using 30 animals in each group. Hearts from sham control rats used for histological examination (Fig. 1) showed no scar. The data in Fig. 1 show that both scar size and scar weight are linearly related to the total ventricular (right ventricle and LV including septum and scar tissue) weight. It is pointed out that 10–15% of the infarcted animals showed small infarct (scar weight/total LV ratio < 15% corresponding to scar size < 30% of the LV wall). Thus, the hemodynamic data from animals showing small infarct were not included and the cardiac tissue from these animals was discarded. The experimental rats with large infarct (17–27% of scar weight/LV weight ratio corresponding to 35–55% scar size) were employed in this study.

Table 1. General characteristics of infarcted rats with and without enalapril or losartan treatments for 4 weeks starting at 3 weeks after coronary occlusion

BW (g) Heart wt (mg) Viable LV wt (mg) Scar wt (mg) Scar wt/total LV wt (%) Heart wt/BW ratio (mg/g) Lung wet/dry wt ratio Liver wet/dry wt ratio

Sham

Sham + ENP

Sham + LOS

MI

MI + ENP

MI + LOS

495 ± 9 1158 ± 45 875 ± 41.4 ND ND 2.33 ± 0.12 4.28 ± 0.1 3.14 ± 0.04

479 ± 9 1183 ± 43 873 ± 11.9 ND ND 2.47 ± 0.08 4.71 ± 0.22 3.28 ± 0.06

491 ± 20 1091 ± 32 856 ± 19.9 ND ND 2.22 ± 0.1 4.37 ± 0.16 3.29 ± 0.11

495 ± 21 1537 ± 30* 819 ± 10.8* 221 ± 24 21.3 ± 1.9 3.10 ± 0.05* 5.39 ± 0.2* 3.28 ± 0.07

500 ± 25 1314 ± 17# 738 ± 25.3# 219 ± 23 22.3 ± 3.9 2.63 ± 0.06# 4.87 ± 0.29# 3.44 ± 0.03

489 ± 24 1333 ± 27# 751 ± 43.4# 235 ± 15 24.0 ± 2.3 2.72 ± 0.08# 4.92 ± 0.31# 3.34 ± 0.08

Values are mean ± S.E. of 7 animals in each group except there were 14 rats in sham group. BW – body weight 7 weeks after the operation; LV – left ventricle; ND – not detected; MI – myocardial infarction; ENP – enalapril (10 mg/kg/day); LOS – losartan (20 mg/kg/day). The viable LV included septum (without scar tissue). *p < 0.05 compared with sham group; #p < 0.05 compared with MI group. The values for scar weight/total LV (including septum and scar) weight ratio corresponded to 44.1 ± 1.6, 45.4 ± 2.8 and 47.4 ± 2.7% scar size (of free LV wall) for MI, MI + ENP and MI + LOS groups, respectively.

168 Table 2. Hemodynamic parameters in infarcted rats with or without enalapril or losartan treatments for 4 weeks starting at 3 weeks after coronary occlusion

Heart rate (beats/min) LVSP (mm Hg) LVEDP (mm Hg) +dP/dt (mm Hg/sec) –dP/dt (mm Hg/sec) MAP (mm Hg)

Sham

Sham + ENP

Sham + LOS

MI

MI + ENP

MI + LOS

231 ± 31 121 ± 5.6 4.3 ± 0.4 7988 ± 745 8216 ± 834 104 ± 9

262 ± 9 113 ± 7.3 3.9 ± 0.3 6844 ± 566 7945 ± 722 94 ± 9

287 ± 23 107 ± 8.2 3.8 ± 0.2 6683 ± 633 7490 ± 450 93 ± 8

250 ± 31 112 ± 12 18.9 ± 3.1* 5194 ± 650* 6069 ± 717* 101 ± 11

287 ± 17 110 ± 2.8 7.2 ± 0.6# 6235 ± 318# 7799 ± 335# 93 ± 8

279 ± 10 120 ± 11 7.5 ± 0.5# 6117 ± 685# 7093 ± 427# 92 ± 8

Values are mean ± S.E. of 7 animals in each group except there were 14 rats in sham group. The animals indicated in Table 1 were used for obtaining these data. LVSP – left ventricular systolic pressure; LVEDP – left ventricular end diastolic pressure; MI – myocardial infarction; MAP – mean arterial pressure; ENP – enalapril (10 mg/kg/day); LOS – losartan (20 mg/kg/day); *p < 0.05 compared with sham group; #p < 0.05 compared with MI group.

SR Ca2+-transport protein contents

mRNA levels of SR Ca2+-transport proteins

Western blots of SR membrane proteins were obtained by employing specific antibodies for RYR, SERCA2, PLB and CQS proteins from sham and infarcted animals with or without enalapril or losartan therapy (Fig. 2). Densitometric analysis of the immunoblots revealed 42, 49 and 31% reductions in the relative protein contents for RYR, SERCA2 and PLB in the infarcted rats in comparison to the sham group, respectively (Figs 3 and 4). Furthermore, treatment with enalapril or losartan partially attenuated these reductions (Figs 2–4). In contrast, there was no significant change in the relative values for CQS content in the infarcted groups with or without treatment (Fig. 4). Treatment of sham animals with enalapril or losartan had no effect on any of the SR Ca2+-regulating protein contents.

To examine the effects of RAS blockade on gene expression of SR Ca2+-transport protein, the relative levels of RYR, SERCA2, PLB and CQS mRNA in control and infarcted groups were determined by Northern blot analysis (Fig. 5). The results showed that the levels of RYR, SERCA2 and PLB

Fig. 2. Typical Western blots of sarcoplasmic reticular (SR) proteins in left ventricle (LV) from sham and infarcted (MI) rats with or without enalapril (ENP) and losartan (LOS) treatments. ENP (10 mg/kg/day) or LOS (20 mg/ kg/day) was given orally for 4 weeks starting at 3 weeks of the surgical operation. Blots for ryanodine receptor (RYR), Ca2+-stimulated ATPase (SERCA2), phospholamban (PLB) and calsequestrin (CQS) were obtained by using specific antibodies.

Fig. 3. Relative protein content of sarcoplasmic reticular (SR) ryanodine receptor (RYR, panel A) and calsequestrin (CQS, panel B) in left ventricle from sham and infarcted (MI) rats with or without enalapril (ENP) or losartan (LOS) treatments. ENP (10 mg/kg/day) or LOS (20 mg/kg/day) was given orally as indicated in Fig. 1. Values are mean ± S.E. of 7 samples in each group as indicated in Tables 1 and 2. *p < 0.05 compared with sham. # p < 0.05 compared with MI group.

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Fig. 4. Relative protein content of sarcoplasmic reticular Ca2+-stimulated ATPase (SERCA2, panel A) and phospholamban (PLB, panel B) in left ventricle from sham and infarcted (MI) rats with or without enalapril (ENP) or losartan (LOS) treatments. ENP (10 mg/kg/day) or LOS (20 mg/kg/day) was given orally as indicated in Fig. 1. Values are mean ± S.E. of 7 samples in each group as indicated in Tables 1 and 2. *p < 0.05 compared with sham. #p < 0.05 compared with MI group.

mRNA were significantly reduced by 35, 26 and 22% in the untreated infarcted group in comparison with those in the sham groups, respectively (Figs 6 and 7). Enalapril or losartan treatment significantly prevented the decrease in RYR, SERCA2 and PLB mRNA levels in the infarcted animals (Figs 5–7). The results did not show any difference in the CQS mRNA level among different groups. Treatment with enalapril or losartan in sham group did not show any alterations of mRNA levels for SR proteins.

Discussion We have shown that cardiac hypertrophy, lung congestion and heart dysfunction in the infarcted animals were associated with depressed SR RYR, SERCA2 and PLB contents and gene expression. Our results regarding SR protein and gene expression are in agreement with earlier reports in MI rat model [16, 17, 29, 30] and other types of CHF [4, 6–11]. The observed decrease in SR RYR, SERCA2 and PLB protein and

Fig. 5. Typical Northern blots of sarcoplasmic reticular protein mRNA in left ventricle from sham and infarcted (MI) rats with or without enalapril (ENP) or losartan (LOS) treatments. ENP (10 mg/kg/day) or LOS (20 mg/ kg/day) was given orally for 4 weeks starting at 3 weeks of the surgical operation. Blots for ryanodine receptor (RYR), Ca 2+-stimulated ATPase (SERCA2), phospholamban (PLB) and calsequestrin (CQS) mRNA were obtained by using specific molecular probes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level was used as internal standard for correcting loading variations in each group. The quality of mRNA preparation is evident from the ethidium bromide staining of the 28S and 18S ribosomal RNA.

gene expression was of specific nature since the expression of SR CQS protein content as well as SR CQS mRNA level were not altered in the MI-induced failing heart. Furthermore, the protein contents and mRNA levels for protein kinase isoforms [41] as well as mRNA level for atrial natriuretic peptide [22] were elevated in this experimental model. Differential alterations in protein contents in mRNA levels for α- and β-myosin heavy chains were also reported to occur in the infarcted hearts [38]. Although the exact mechanisms for changes in cardiac gene expression and subcellular protein contents are not clear at present, increased RAS and sympathetic nervous system activities and subsequent alterations in signal transduction mechanisms in heart failure have been suggested to play an important role in the genesis of these molecular changes [4]. A depression in SR RYR and SERCA2 gene expression can be seen to result in a decrease in SR RYR and SERCA2

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Fig. 6. mRNA abundance for sarcoplasmic reticular ryanodine receptor (RYR, panel A) and calsequestrin (CQS, panel B) in left ventricle from sham and infarcted (MI) rats with or without enalapril (ENP) or losartan (LOS) treatments. ENP (10 mg/kg/day) or LOS (20 mg/kg/day) was given orally as indicated in Fig. 4. The values were normalized with respect to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels and were expressed as a percentage of values for sham. Each value is a mean ± S.E. of 7 samples in each group as indicated in Tables 1 and 2. *p < 0.05 compared with sham control. #p < 0.05 compared with MI group.

Fig. 7. mRNA abundance for sarcoplasmic reticular Ca2+-stimulated ATPase (SERCA2, panel A) and phospholamban (PLB, panel B) in left ventricle from sham and infarcted (MI) rats with or without enalapril (ENP) or losartan (LOS) treatments. ENP (10 mg/kg/day) or LOS (20 mg/kg/day) was given orally as indicated in Fig. 4. The values were normalized with respect to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA levels and were expressed as percentage of values for sham. Each value is a mean ± S.E. of 7 samples in each group as indicated in Tables 1 and 2. *p < 0.05 compared with sham control. #p < 0.05 compared with MI group.

protein content, which defects would produce abnormalities in SR Ca2+-release and Ca2+-uptake activities reported previously in CHF subsequent to MI [14, 15, 32]. In view of the critical role of SR function in the regulation of intracellular Ca2+ and cardiac performance [1–6], it is likely that Ca2+-handling abnormalities in cardiomyocytes [13, 33, 42] as well as the occurrence of cardiac dysfunction in CHF due to MI [31, 32, 39, 43–46] may partly be due to observed changes in SR gene expression. It should be pointed out that since the unphosphorylated PLB is known to produce an inhibitory effect on SR SERCA2 [47], the observed depression in SR PLB content in the MI-induced heart failure may produce an increase in Ca2+-uptake in the failing heart SR. Thus, the decrease in SR PLB protein content may serve as an adaptive mechanism in this type of failing heart. However, since the depression in PLB content was smaller than that in SERCA2 content in the infarcted heart, it is possible that the relative functional contribution of PLB in the regulation of SERCA2 activity may not change in the failing heart. Nonetheless, it should be noted that transfer of SERCA2 gene into failing cardiomyocytes has been reported to improve cardiac function in the failing heart [48] and thus it is likely that the ob-

served changes in SR protein and gene expression play a crucial role in cardiac dysfunction in CHF due to MI. In this study, treatment of infarcted animals with enalapril or losartan was found to partially prevent cardiac hypertrophy, lung congestion and heart dysfunction due to MI. These beneficial effects of enalapril or losartan are in agreement with those reported with other ACE inhibitors and angiotensin receptor antagonists [28, 32, 34, 36, 49–51]. The partial attenuation of MI-induced depressions in SR RYR, SERCA2 and PLB mRNA levels and associated SR protein contents by enalapril and losartan treatments are also consistent with previous scattered observations regarding the effects of captopril, cilizapril and candesartan [29, 30, 32]. Although Ambrose et al. [22] did not observe any increase in mRNA levels for SERCA and PLB in hearts of the infarcted animals pretreated with an AT1 receptor antagonist, irbesartan their data are difficult to interpret because these investigators failed to detect any depression in mRNA levels for these SR proteins in the untreated infarcted hearts. The beneficial effects of enalapril and losartan on SR gene expression and protein contents can be seen to improve SR function and cardiomyocyte Ca2+-handling abnormalities in the infarcted heart

171 similar to those reported for captopril [28, 32, 33]. Since the treatment with enalapril or losartan was started in animals with healed MI, there was no difference in the scar weight or scar weight/total LV weight ratio between the untreated and treated MI groups and thus the effects of both these agents on SR protein and gene expression are associated with the prevention of progression in cardiac remodelling and CHF in the experimental model employed in this study. Since ACE inhibitors are also known to result in the accumulation of bradykinin, which produces beneficial effects in CHF [52], it is possible that the prevention of MI-induced changes in SR protein and gene expression by captopril [32] or enalapril (as reported here) may be due to this mechanism rather than through a depression in the level of angiotensin II. However, it is important to note that the actions of losartan were qualitatively similar to those produced by enalapril indicating the involvement of RAS in eliciting the observed changes in cardiac function as well as SR protein and gene expression in the failing heart. Furthermore, RAS has been shown to be activated and the expression of angiotensin II receptors is increased in the untreated infarcted animals [30, 53]. Accordingly, it is possible that the beneficial effects of enalapril and losartan on cardiac function as well as SR protein and gene expression in CHF due to occlusion of coronary artery are partly due to the blockade of RAS.

Acknowledgements The research work reported in this study was supported by a grant 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. NSD holds CIHR/ Pharmaceutical Development and Research Chair in Cardiovascular Research supported by Merck Frosst Canada. XG is supported by the Manitoba Health Research Council Studentship.

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