TY - JOUR
AU - Unger,, Thomas
AB - Abstract Objective: We investigated the effect of chronic treatment with the new Na+/H+-exchange inhibitor, cariporide, on cardiac function and remodelling 6 weeks after myocardial infarction (MI) in rats. Methods: Treatment with cariporide was commenced either 1 week pre or 30 min, 3 h, 24 h or 7 days after ligation of the left ventricular artery and was continued until haemodynamic parameters were obtained 6 weeks after MI in conscious rats. Results: Compared to sham animals, untreated MI-controls developed pronounced heart failure after 6 weeks. Basal left ventricular end-diastolic pressure (in mmHg) was reduced in the groups in which cariporide was started 1 week pre (16.0±1.7) or 30 min (12.5±1.1), 3 h (11.8±1.0) and 24 h (13.0±2.5) after MI compared to untreated MI-controls (22.4±1.5; P<0.01). Basal myocardial contractility (in 1000 mmHg/s) was only increased when treatment was initiated after 30 min (9.0±0.7), 3 h (8.5±0.3) and 24 h (8.0±0.7) compared to untreated MI-controls (5.8±0.7; P<0.05–0.01). Infarct size (in % of left ventricular circumference) was 40.0±2.1 in MI-controls and was decreased when treatment was begun after 30 min (32.6±2.7) or 3 h (32.4±2.3) (P<0.05). In animals, in which cariporide was started 3 h after induction of MI, heart weight/body weight ratio was significantly decreased, indicating reduced cardiac hypertrophy. When treatment started 7 days after MI, cariporide did not exert any beneficial actions on structural and functional cardiac parameters. Conclusion: Our results show for the first time that chronic treatment with the Na+/H+-exchange inhibitor cariporide engendered marked cardioprotective effects when commenced before and up to 24 h after MI. The optimal time for the start of treatment was between 30 min and 3 h post MI. Heart failure, Infarction, Na/H-exchanger, Remodelling, Ventricular function NHE, Na+/H+-exchanger, MI, myocardial infarction, LVP, left ventricular pressure, LVEDP, left ventricular end-diastolic pressure, dP/dtmax, myocardial contractility, MAP, mean arterial pressure, HR, heart rate, Car, cariporide, THW/BW, total heart weight to body weight ratio, IS, infarct size, nIML, non infarcted muscle length, mLVC, mean left ventricular circumference, dLV, inner left ventricular diameter, aS, average septal thickness Time for primary review 22 days. 1 Introduction The mammalian Na+/H+-exchanger (NHE) can be found in virtually all tissues. At least five different isoforms have been described and designated NHE-1 to NHE-5. The most important factor regulating the Na+/H+-exchange under physiological conditions is intracellular pH. An increase in intracellular H+ concentration dramatically stimulates the exchange activity. In addition to intracellular pH regulation, the Na+/H+-exchanger is involved in cell volume regulation and is also rapidly activated in response to a variety of mitogenic and nonmitogenic signals (for review, see Wakabayashi et al., 1997) [1]. Over the past years increasing evidence has been provided demonstrating that inhibition of the sarcolemmal Na+/H+-exchanger (NHE-1) can protect the heart during ischemia and reperfusion thus suggesting that the regulation of internal myocardial pH is of critical importance in clinical situations with ischemic episodes such as angina, heart surgery, myocardial infarction (MI) and following reperfusion. The NHE-1 inhibitor-induced cardiac protection observed in experimental studies includes increased functional recovery [2–6], decreased incidence of arrhythmias both in vitro and in vivo [3,7–10], attenuated 45Ca2+ uptake [11,12], preserved ultrastructure [13], reduced release of intracellular enzymes [8,9], preservation of high energy phosphates [8,9], and reduced cellular necrosis [14–16]. While pronounced cardioprotective effects were observed when the Na+/H+-exchanger was already inhibited during the ischemic period divergent results ranging from full protection [3,5,15] to lack of effects [3,4,14] have been reported when the inhibition was only present at the time of reperfusion. Thus far, the effect of long term inhibition of the Na+/H+-exchanger in ischemia–reperfusion related diseases of the cardiovascular system has not been studied, probably because of the lack of specific, well tolerated compounds. Amiloride and its analogues, which are up to now the most widely used substances to investigate the role of Na+/H+-exchange in ischemia/reperfusion injury have been shown to produce time and dose-dependent adverse effects on the heart in a concentration range which is relevant for Na+/H+-exchange inhibition [17]. In recent years, new specific Na+/H+-exchange inhibitors with an amino guanidine structure have been developed which are well tolerated even at high blood levels [8,9,18]. This prompted us to investigate the effect of long term Na+/H+-exchange inhibition on cardiac function and morphology in a chronic model of MI in rats. In addition, we addressed the question as to the best time point to commence therapy. The Na+/H+-exchange inhibitor cariporide (4-isopropyl-3-methylsulphonylbenzoyl-guanidine methansulphonate; HOE 642) used in the present experiments has been demonstrated to be a highly selective NHE-1 subtype inhibitor in different tissues and species including rats and to have no or very slight effects on transport systems other than the Na+/H+-exchanger [8]. 2 Methods 2.1 Animals Experiments were performed in male Wistar rats (300–320 g, Charles River Viga GmbH, Sulzfeld, Germany), housed individually at controlled temperature, humidity and light/dark cycle. Animals had free access to a pellet rat diet (0.6% salt) and tap water. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was performed in accordance with the German law on animal protection as released in the new version in 1993. 2.2 Experimental protocol Rats were recruited randomly into seven groups until 15 animals in the MI control group (MI-control) and nine to ten in the remaining groups, respectively, survived the protocol: (1) sham surgery group, n=11, (2) MI-control, n=42, (3) cariporide treatment, beginning 7 days prior to the induction of MI, n=21, (4) 30 min, n=19, (5) 3 h, n=20, (6) 24 h, n=19, and (7) 7 days after induction of MI, n=26. The compound cariporide was provided in the rat chow pelleted at 3000 ppm. Treatment was continued throughout the experiment (Fig. 1). Cariporide was additionally administered by subcutaneous injection of 3×50 mg/kg/day in groups 3–6 until the individual food intake was at least 30% of the mean food intake calculated during 1 week before induction of MI for each animal. This was usually necessary for 3 days after surgery. In the sham and MI-control groups, rats received 3×1 ml/kg/day physiological saline s.c. Fig. 1 Open in new tabDownload slide Time table of experimental protocol. Fig. 1 Open in new tabDownload slide Time table of experimental protocol. After 1 week in single cages, the rats in group 1 underwent sham operation. In the remainder of rats, MI was induced by permanent ligation of the left coronary artery. Chronic arterial and venous catheters as well as a catheter in the left ventricle were implanted 6 weeks after MI or sham operation. Haemodynamic studies were performed 24 h later in conscious animals. At the end of the experiments, the rats were sacrificed and hearts subjected to morphological examination. Four animals with an IS less than 21% were excluded from the study (one rat of groups 2 and 4, two rats of group 6), since substantial haemodynamic changes are only apparent in animals with an infarction larger than 21% as shown by Schoemaker et al. [19] 2.3 Surgical procedures 2.3.1 Induction of myocardial infarction and sham surgery After induction of anaesthesia with ether and i.v. injection of methohexital-sodium (initially 10 mg/kg), rats were intubated, artificially ventilated and connected to an ECG recorder and left thoracotomy and ligation of the left coronary artery was performed as previously described [20–22]. Successful ligation was verified by the occurrence of arrhythmias and, visually, by the colour change of the ischemic area. In rats, which underwent sham surgery, the ligation was placed beside the coronary artery. The thoracic cavity was closed during respiration hold, and analgesia was induced by a subcutaneous injection of buprenorphin–HCl (0.2 mg/kg). 2.3.2 Arterial, venous and left ventricular catheters Anaesthesia was induced with ether and continued with methohexital-sodium. Arterial, venous [21] and left ventricular [20] catheters were chronically implanted using a procedure described elsewhere [22]. 2.4 Haemodynamic measurements Haemodynamic measurements were performed in conscious animals 24 h after the implantation of the catheters as previously described [23]. Baseline measurements of MAP, HR and LVP were recorded after rats had been acquainted with the recording procedures for 30 min. The animals then received an i.v. infusion of the α1-adrenoceptor agonist, methoxamine (1 mg/ml) to increase cardiac afterload. The rate of infusion was increased steadily until MAP was elevated by 20 mmHg; an infusion rate of 1.5 ml/h was usually necessary to attain the desired pressure response. This infusion rate was maintained, and a second recording was obtained. LVEDP and myocardial contractility were calculated off-line from the LVP signal using MEGA. MAP, HR, LVEDP and dP/dtmax under basal conditions and during increased afterload were averaged over 5-min periods to be used in the statistical analysis. 2.5 Morphological examination After the haemodynamic measurements, rats were sacrificed and hearts were excised for morphological examination. Macroscopically, MI was found to be transmural in all infarcted rats. Hearts were stored in 4% phosphate buffered formalin in 0.15 M NaCl until further processing. 2.5.1 Tissue processing After removal of the atria and large vessels, the ventricles were cut in a standardized fashion into transvere slices from the apex to the base using a special plexiglass box adapted to rat hearts which contained slits for a microtome knife at 3 mm distance. Slice 5 represented the apex, slice 1, which was various in size, represented the base. The five slices obtained were weighed separately after removal of clotted blood from the ventricles. The weight of all five slices was summed up to obtain the total heart weight (THW). The slices were then transferred into 10% phosphate-buffered formalin, kept overnight, were subsequently dehydrated and embedded in paraffin via routine histological procedures. Serial 4-μm sections were cut and stained with haematoxilin–eosin or Goldner, respectively. 2.5.2 Measurement of infarct size, septum thickness and ventricular dilatation The histology of haematoxilin/eosin transections showed transmural MI normally extending from slice 2 to slice 5. For further morphometric analysis, the Goldner transection of the midsagittal level with the largest left ventricular circumference was used [24]. IS and ventricular dilatation, were determined in transections using a Quantimet 570 morphometer including morphometry software (Leica, Cambridge Instruments Ltd., Cambridge, UK) connected to a video camera [24,25]. In addition, the average septal thickness (aS) was determined and total infarct length (IS; in mm), non-infarcted muscle length (nIML), mean left ventricular circumference (mLVC) and the inner diameter of the left ventricle (dLV) of the transection were also calculated by the computer according to the formulae below: 2.6 Drugs Cariporide, as well as the rat chow were provided by Hoechst Marion Roussel AG (Frankfurt, Germany). Based on experiments in adult rats, a rat chow preparation with 3000 ppm cariporide was chosen (mean plasma levels of cariporide 450–800 ng/ml) although a mean concentration of 100 ng/ml cariporide is considered to be sufficient to inhibit Na+/H+-exchange (unpublished observations, U. Albus, Hoechst Marion Roussel, Frankfurt, Germany), because rats reveal reduced food intake after MI. In addition, 3×50 mg/kg/day cariporide were injected subcutaneously until the individual food intake was 30% of the value before induction of MI. In preliminary experiments, plasma levels of cariporide and metabolites were measured 1–8 h after subcutaneous injection of 50 mg/kg. The maximal concentration was 7988 ng/ml after 1 h, the minimum level was 119 ng/ml 8 h after injection. The elimination half life was 1.3 h. Adverse effects due to high plasma levels were not expected to occur, since the LD50 is more than 1000 mg/kg in rats. 2.7 Statistical analysis Data are presented as mean±S.E.M. Statistical analysis of the data obtained was performed using one-way ANOVA. When a P value was less than 0.05, differences between individual groups were evaluated using post-hoc Student's t-test. P<0.05 was considered statistically significant. 3 Results 3.1 Survival rate The occlusion of the left coronary artery resulted in a total mortality of 59%. In all infarcted groups, the highest mortality occurred within 24 h after surgery. Since the number of rats in each group was too low to yield statistically valid data no statistical analysis of survival was performed. Two out of 11 rats died after sham surgery. After MI, 34% in the MI-control group, 43% in the pre-treatment group and 44% in the 30 min, 50% in the 3 h, 47% in the 24 h and 38% in the 7 day post treatment groups survived the protocol. In general, more animals were required in the MI-control group and in the group in which treatment with cariporide was started 7 days after MI in order to obtain an adequate number of animals surviving the protocol (Table 1). Table 1 Haemodynamic and morphological results 6 weeks after myocardial infarction or sham-surgerya . Sham . MI-control . Car 7 days pre . Car 30 min post . Car 3 h post . Car 24 h post . Car 7 days post . . (n=9/11) . (n=14/41) . (n=9/21) . (n=8/18) . (n=10/20) . (n=8/17) . (n=10/26) . MAP (mmHg) 108±3 97±3 106±5 104±3 94±3 102±5 97±3 HR (beats/min) 382±6 383±13 378±12 376±14 385±10 378±9 391±12 IS (mm) – 10.94±0.63 9.46±0.88 9.32±0.82 8.82±0.58* 10.37±0.94 10.59±0.91 nIML (mm) – 16.49±0.77 17.37±1.04 19.22±0.83* 18.59±0.99 17.96±0.93 16.70±0.98 mLVC (mm) 21.47±0.58 27.43±0.75# 26.82±0.81# 28.55±0.58# 27.41±0.76# 28.34±0.50# 27.29±0.80# dLV (mm) 5.13±0.20 7.70±0.24# 7.47±0.27# 8.17±0.29# 7.55±0.30# 7.69±0.18# 7.58±0.30# aS (mm) 2.02±0.10 1.76± 0.07 1.73±0.08 1.87± 0.06 1.85±0.07 1.87±0.11 1.86±0.08 . Sham . MI-control . Car 7 days pre . Car 30 min post . Car 3 h post . Car 24 h post . Car 7 days post . . (n=9/11) . (n=14/41) . (n=9/21) . (n=8/18) . (n=10/20) . (n=8/17) . (n=10/26) . MAP (mmHg) 108±3 97±3 106±5 104±3 94±3 102±5 97±3 HR (beats/min) 382±6 383±13 378±12 376±14 385±10 378±9 391±12 IS (mm) – 10.94±0.63 9.46±0.88 9.32±0.82 8.82±0.58* 10.37±0.94 10.59±0.91 nIML (mm) – 16.49±0.77 17.37±1.04 19.22±0.83* 18.59±0.99 17.96±0.93 16.70±0.98 mLVC (mm) 21.47±0.58 27.43±0.75# 26.82±0.81# 28.55±0.58# 27.41±0.76# 28.34±0.50# 27.29±0.80# dLV (mm) 5.13±0.20 7.70±0.24# 7.47±0.27# 8.17±0.29# 7.55±0.30# 7.69±0.18# 7.58±0.30# aS (mm) 2.02±0.10 1.76± 0.07 1.73±0.08 1.87± 0.06 1.85±0.07 1.87±0.11 1.86±0.08 a Indicated are animals that underwent sham-surgery (sham), untreated MI-control rats (MI-control) and rats treatment with cariporide (Car) was initiated 7 days before (7 days pre), 30 min, 3 h, 24 h and 7days after (post) induction of MI (n=number analysed/number in treatment groups). Data are presented as mean±S.E.M.: Basal mean arterial pressure (MAP) and heart rate (HR); length of the infarcted tissue (IS); non-infarcted muscle length (nIML); mean left ventricular circumference (mLVC); inner left ventricular diameter (dLV); average septal thickness (aS). # #P<0.001 compared with sham; *P<0.05 compared to control. Open in new tab Table 1 Haemodynamic and morphological results 6 weeks after myocardial infarction or sham-surgerya . Sham . MI-control . Car 7 days pre . Car 30 min post . Car 3 h post . Car 24 h post . Car 7 days post . . (n=9/11) . (n=14/41) . (n=9/21) . (n=8/18) . (n=10/20) . (n=8/17) . (n=10/26) . MAP (mmHg) 108±3 97±3 106±5 104±3 94±3 102±5 97±3 HR (beats/min) 382±6 383±13 378±12 376±14 385±10 378±9 391±12 IS (mm) – 10.94±0.63 9.46±0.88 9.32±0.82 8.82±0.58* 10.37±0.94 10.59±0.91 nIML (mm) – 16.49±0.77 17.37±1.04 19.22±0.83* 18.59±0.99 17.96±0.93 16.70±0.98 mLVC (mm) 21.47±0.58 27.43±0.75# 26.82±0.81# 28.55±0.58# 27.41±0.76# 28.34±0.50# 27.29±0.80# dLV (mm) 5.13±0.20 7.70±0.24# 7.47±0.27# 8.17±0.29# 7.55±0.30# 7.69±0.18# 7.58±0.30# aS (mm) 2.02±0.10 1.76± 0.07 1.73±0.08 1.87± 0.06 1.85±0.07 1.87±0.11 1.86±0.08 . Sham . MI-control . Car 7 days pre . Car 30 min post . Car 3 h post . Car 24 h post . Car 7 days post . . (n=9/11) . (n=14/41) . (n=9/21) . (n=8/18) . (n=10/20) . (n=8/17) . (n=10/26) . MAP (mmHg) 108±3 97±3 106±5 104±3 94±3 102±5 97±3 HR (beats/min) 382±6 383±13 378±12 376±14 385±10 378±9 391±12 IS (mm) – 10.94±0.63 9.46±0.88 9.32±0.82 8.82±0.58* 10.37±0.94 10.59±0.91 nIML (mm) – 16.49±0.77 17.37±1.04 19.22±0.83* 18.59±0.99 17.96±0.93 16.70±0.98 mLVC (mm) 21.47±0.58 27.43±0.75# 26.82±0.81# 28.55±0.58# 27.41±0.76# 28.34±0.50# 27.29±0.80# dLV (mm) 5.13±0.20 7.70±0.24# 7.47±0.27# 8.17±0.29# 7.55±0.30# 7.69±0.18# 7.58±0.30# aS (mm) 2.02±0.10 1.76± 0.07 1.73±0.08 1.87± 0.06 1.85±0.07 1.87±0.11 1.86±0.08 a Indicated are animals that underwent sham-surgery (sham), untreated MI-control rats (MI-control) and rats treatment with cariporide (Car) was initiated 7 days before (7 days pre), 30 min, 3 h, 24 h and 7days after (post) induction of MI (n=number analysed/number in treatment groups). Data are presented as mean±S.E.M.: Basal mean arterial pressure (MAP) and heart rate (HR); length of the infarcted tissue (IS); non-infarcted muscle length (nIML); mean left ventricular circumference (mLVC); inner left ventricular diameter (dLV); average septal thickness (aS). # #P<0.001 compared with sham; *P<0.05 compared to control. Open in new tab 3.2 Infarct size and cardiac remodelling IS as percentage of mLVC is shown in Fig. 2. In addition, the absolute length of the infarcted tissue of the slices with the largest left ventricular circumference is given in Table 1. Compared to the non-treated infarcted control group, IS tended to decrease in pretreated animals (35.3±3.0 vs. 40.0±2.1% in MI-controls) and was significantly reduced in animals in which treatment with cariporide was initiated 30 min and 3 h after induction of MI (32.6±2.7 and 32.4±2.3% vs. 40.0±2.1%, respectively, P<0.05). When treatment was started 24 h or 7 days after induction of MI, no difference in IS was detected compared to the MI-control group. The absolute length of the infarcted tissue was also reduced in the early treated groups, and this reduction was significant when medication started 3 h after induction of MI (Table 1). In contrast, IS in the late treatment groups were not different from those in the MI-control group. Fig. 2 Open in new tabDownload slide Infarct size 6 weeks after induction of myocardial infarction. Infarct size is given as percentage of left ventricular circumference. Indicated are MI-control animals (control, n=14); black columns indicate animals treated with the Na+/H+-exchange inhibitor, cariporide, beginning 7 days before (pre, n=9), 30 min (n=8), 3 h (n=10), 24 h (n=8) and 7days (7d, n=10) after myocardial infarction. Data represent mean±S.E.M. *P<0.05 compared to control. Fig. 2 Open in new tabDownload slide Infarct size 6 weeks after induction of myocardial infarction. Infarct size is given as percentage of left ventricular circumference. Indicated are MI-control animals (control, n=14); black columns indicate animals treated with the Na+/H+-exchange inhibitor, cariporide, beginning 7 days before (pre, n=9), 30 min (n=8), 3 h (n=10), 24 h (n=8) and 7days (7d, n=10) after myocardial infarction. Data represent mean±S.E.M. *P<0.05 compared to control. As an index for left ventricular expansion, mLVC and the inner left ventricular diameter (dLV) were determined (Table 1). Compared to animals subjected to sham-surgery, mLVC and dLV were significantly increased in the MI-control group. In addition, in all cariporide treated groups, both parameters were significantly increased compared to the sham group. No differences in mLVC and dLV were detected among the six MI groups. The remaining left ventricular muscle tissue can be estimated using average septal thickness (aS) and non-infarcted muscle length (nIML) (Table 1). In the MI-control group, aS was not increased compared to animals that underwent sham-surgery. Treatment with cariporide started at any time point did not show any difference in aS compared to MI-control rats. On the other hand, commencing therapy with cariporide 30 min and 3 h after induction of MI increased nIML compared to the MI-control group. This increase was significant in animals in which treatment with cariporide was started 30 min after MI (P<0.05). 3.3 Cardiac weight Cardiac weight was examined by determining the ratio of total heart weight to body weight (THW/BW) (Fig. 3). THW/BW was significantly increased in MI-controls compared to animals that underwent sham-surgery (P<0.01). An increase in THW/BW compared to the sham group was also present in animals in which treatment with cariporide was started 24 h and 7 days after induction of MI (P<0.05, respectively). Compared to the MI-control group, THW/BW was significantly reduced when treatment with cariporide was initiated 3 h following MI (P<0.05). Fig. 3 Open in new tabDownload slide Total heart weight to body weight (THW/BW) 6 weeks after induction of myocardial infarction. Indicated are animals subjected to sham-surgery (sham, n=9); MI-control animals (control, n=14); black columns indicate animals treated with the Na+/H+-exchange inhibitor, cariporide, beginning 7 days before (pre, n=9), 30 min (n=8), 3 h (n=10), 24 h (n=8) and 7 days (7d, n=10) after myocardial infarction. Data represent mean±S.E.M. *P<0.05 compared to control; #P<0.05, and #P<0.01 compared to sham. Fig. 3 Open in new tabDownload slide Total heart weight to body weight (THW/BW) 6 weeks after induction of myocardial infarction. Indicated are animals subjected to sham-surgery (sham, n=9); MI-control animals (control, n=14); black columns indicate animals treated with the Na+/H+-exchange inhibitor, cariporide, beginning 7 days before (pre, n=9), 30 min (n=8), 3 h (n=10), 24 h (n=8) and 7 days (7d, n=10) after myocardial infarction. Data represent mean±S.E.M. *P<0.05 compared to control; #P<0.05, and #P<0.01 compared to sham. 3.4 Arterial blood pressure and heart rate Mean blood pressure and heart rate at week six after MI/sham surgery did not differ significantly among groups (Table 1). The elevation of mean arterial pressure with an infusion of methoxamine to increase afterload was not different among groups (data not shown). 3.5 Left ventricular end-diastolic pressure LVEDP was recorded at baseline (Fig. 4A) and during increased afterload. MI-control rats had a significantly higher LVEDP at baseline than those that underwent sham-surgery (P<0.01). In cariporide-pretreated animals, LVEDP at baseline was lower compared to MI-control, but was still significantly elevated compared to sham operated animals (P<0.01, respectively). When treatment with cariporide was initiated 30 min, 3 h or 24 h after induction of MI, LVEDP was significantly reduced at baseline (Fig. 4A) and during increased afterload (data not shown). In contrast, when treatment with cariporide was begun 7 days after MI, LVEDP was not reduced compared to the MI-control group (Fig. 4A). Fig. 4 Open in new tabDownload slide Left ventricular end-diastolic pressure (LVEDP) (A) and myocardial contractility (dP/dtmax) (B) under basal conditions 6 weeks after induction of myocardial infarction in conscious rats. Indicated are animals subjected to sham-surgery (sham, n=9); MI-control animals (control, n=14); black columns indicate animals treated with the Na+/H+-exchange inhibitor, cariporide, beginning 7 days before (pre, n=9), 30 min (n=8), 3 h (n=10), 24 h (n=8) and 7 days (7d, n=10) after myocardial infarction. Data represent mean±S.E.M. *P<0.05 and *P<0.01 compared to control; #P<0.05 and #P<0.01 compared to sham. Fig. 4 Open in new tabDownload slide Left ventricular end-diastolic pressure (LVEDP) (A) and myocardial contractility (dP/dtmax) (B) under basal conditions 6 weeks after induction of myocardial infarction in conscious rats. Indicated are animals subjected to sham-surgery (sham, n=9); MI-control animals (control, n=14); black columns indicate animals treated with the Na+/H+-exchange inhibitor, cariporide, beginning 7 days before (pre, n=9), 30 min (n=8), 3 h (n=10), 24 h (n=8) and 7 days (7d, n=10) after myocardial infarction. Data represent mean±S.E.M. *P<0.05 and *P<0.01 compared to control; #P<0.05 and #P<0.01 compared to sham. 3.6 Myocardial contractility Myocardial contractility (dP/dtmax) at baseline was impaired in MI-control rats, pretreated rats and in rats in which treatment with cariporide was initiated 7 days after induction of MI (Fig. 4B). In contrast, when treatment with the Na+/H+-exchange inhibitor was initiated between 30 min and 24 h after induction of MI, dP/dtmax was significantly increased at baseline compared to the MI-control group (P<0.05–0.01). Similar results were obtained under increased afterload (data not shown). 4 Discussion Cariporide has been characterised as a highly specific Na+/H+-exchange subtype 1 (NHE-1) inhibitor in rabbit erythrocytes, human thrombocytes and rat cardiomyocytes showing cardioprotective and antiarrhythmic effects in regional ischemic and reperfused hearts [8]. To our knowledge, no experiments have been undertaken thus far to investigate long-term effects of NHE-1 inhibition on cardiac function and morphology after MI. The present study demonstrates that chronic treatment with cariporide in rats decreased LVEDP and improved myocardial contractility when initiated in the early phase of MI. In addition, myocardial IS was reduced in animals in which treatment with cariporide was initiated up to 3 h after induction of MI. The beneficial effect on cardiac remodelling was underlined by reduced cardiac hypertrophy in animals in which cariporide treatment was initiated 3 h following MI. A small effect on cardiac hypertrophy in the 30 min post treatment group may have been missed because of the small animal numbers used. No beneficial effects on myocardial function and morphology were seen when treatment with cariporide commenced 7 days after coronary artery ligation. The infarction model used in the present experiment results in pronounced heart failure 6 weeks after induction of MI with an increase in LVEDP, impaired myocardial contractility and development of an eccentric cardiac hypertrophy, evidenced by an increase in THW/BW ratio accompanied by left ventricular dilatation without an increase in septal thickness. LVEDP at baseline and during increased afterload was significantly lower in pretreated animals and in those in which treatment with cariporide was started within 24 h after MI. The effect on LVEDP was accompanied by increased dP/dtmax under basal conditions and during increased afterload in animals in which treatment was started 30 min up to 24 h after coronary artery ligation, indicating an improved myocardial contractility in animals with early-onset treatment. These data would suggest that the optimal time to commence therapy with a Na+/H+-exchange inhibitor is between 30 min and 24 h after the acute event. Since cardiac myocytes do not regenerate to any significant degree, the necrotic loss of contractile tissue produces a permanent loss of cardiac functional reserve. An initial salvage of viable myocardial tissue may result in improved cardiac function which is even more pronounced after several weeks, when overt heart failure has manifested itself. This is supported by decreased IS measured in animals in which treatment was commenced 30 min and 3 h following MI. It is also possible that improved cardiac function 6 weeks after MI could reflect an overall inotropic effect of cariporide. The Na+/H+-exchange inhibitor, amiloride, for instance, has been reported to inhibit Na+/K+-ATPase and, consequently, to exhibit a glycoside-like effect [26]. However, in isolated working rat heart preparations, cariporide had no influence on pre-ischemic cardiac function as well as on LVP, dP/dtmax, heart rate and coronary flow during 15 min of regional ischemia and 30 min of reperfusion compared to untreated control hearts [8]. In the same study cariporide had no substantial influence on circulatory indices in anaesthetised rats [8]. Therefore, it seems unlikely that acute haemodynamic effects following drug administration, such as a reduction in preload and/or afterload or a direct effect on coronary flow, contributed to the improved cardiac function measured 6 weeks after MI in animals with early-onset cariporide treatment. In addition, in the present study, LVEDP and dP/dtmax, a parameter for myocardial contractility, were not different in animals in which Na+/H+-exchange inhibition was initiated 7 days following MI when compared to MI-control animals. Together with the finding that mean arterial pressure and heart rate were not different among the six infarcted groups, a chronic haemodynamic effect of cariporide can be excluded. Since it has been shown that cariporide has no or only very slight effects on transport systems other than the Na+/H+-exchanger [8] our data most likely imply that the observed cardioprotective effects were due to inhibition of the Na+/H+-exchanger isoform NHE-1 in the early phase of MI. In view of previous results from ischemia/reperfusion experiments, most effective cardioprotection by Na+/H+-exchange inhibitors could have been expected in the pre-treatment group. However, in the present study, the cardioprotective effects were more pronounced in early-onset treated rats since, in addition to improved cardiac function, IS was significantly reduced in these animals. Cariporide has qualified as potent inhibitor of ischemia [8] and ischemia/reperfusion [10] induced ventricular arrhythmias in rats. This was accompanied by reduced mortality in pretreated animals and those treated during ischemia [10]. Taking into account that most deaths occurred within 24 h after MI in the present experiments and that a possible antiarrhythmic effect of cariporide will be most effective if initiated before the onset of ischemia, one could speculate that pre-treatment allowed more animals with large infarctions to survive. This may have been the reason why no significant differences in IS were detected in pretreated animals compared to MI-control rats. Another possible explanation for the observation that pre-treatment was less effective could be that a blockade of the Na+/H+-exchanger for 7 days before the acute ischemic event may have evoked some yet unidentified compensatory mechanisms, that were acting during and after ischemia in this group lessening the effect of Na+/H+-exchange inhibition. It is interesting to note that 40 min of short-term Na+/H+-exchange inhibition with ethyl-isopropyl amiloride (EIPA) followed by a 10-min washout period altered pH-regulation in response to global ischemia in isolated perfused rat hearts with limitation of acidosis during ischemia and slowed recovery of pH on reperfusion [27]. Since these changes were distinct from those observed with inhibition of the exchanger during ischemia, the authors suggested that short-term treatment may have effects during myocardial ischemia different from Na+/H+-exchange inhibition. Further investigations on the effect on cardiac function and morphology in the first few days following MI, including a larger number of animals, are necessary to clarify the finding that pre-treatment with cariporide did not have a better effect than treatment after the ischemic event. Although this study clearly demonstrates that early-onset treatment of MI with cariporide is cardioprotective, our findings do not allow for an exact definition of the mechanisms involved in myocardial protection. Since the present study was not designed to focus on the extent of ischemic cell death we cannot rule out the possibility that reduced IS measured 6 weeks after induction of MI reflects reduced scar enlargement during the healing process rather than initial salvage of cardiomyocytes. mLVC, the inner left ventricular diameter and average septal thickness were not different among all infarcted groups indicating similar left ventricular expansion. On the other hand, cardiac non-infarcted muscle length was increased while total infarct length was reduced in animals in which cariporide was started 30 min and 3 h after the acute event. This supports the hypothesis that an initial salvage of cardiomyocytes was responsible for the reduced IS after 6 weeks. It has been shown that inhibition of the sarcolemmal Na+/H+-exchanger allowed long term structural and functional recovery in a model of ischemia/reperfusion applied to cultured neonatal cardiac myocytes [28]. Few studies have been published that specifically examined the direct effect of the exchanger on cell death in intact cardiac tissue. In isolated perfused rat hearts subjected to regional ischemia for 30 min, a reduction of IS has been demonstrated after 120 min of reperfusion by pre-treatment with the Na+/H+-exchange inhibitor EIPA [14]. In addition, Klein et al. [16] observed an IS reduction in pigs after 45 min of left anterior descending coronary artery ligation followed by a prolonged reperfusion period of 24 h when the Na+/H+-exchange inhibitor HOE 694 was administered 10 min before ischemia. In both experiments, no significant reduction in IS was observed when EIPA or HOE 694 was administered before the onset of reperfusion [15,16], suggesting that myocardial protection was achieved primarily by inhibition of the Na+/H+-exchanger during ischemia. However, in isolated perfused rabbit papillary muscles subjected to 30 or 60 min of ischemia, reduced cell death was demonstrated 30 min following reperfusion with 5-(N,N-dimethyl)-amiloride containing buffer, indicating that inhibition of Na+/H+-exchange during reperfusion can salvage cardiomyocytes [15]. Also the findings of Klein et al. [16] are not entirely contradictory to the hypothesis that, in the present study, improved cardiac function after 6 weeks was the result of diminished cardiomyocyte loss in the early phase following MI. In contrast to the present experiments, Na+/H+-exchange was not inhibited throughout the experimental protocol since the compound HOE 694 could not be detected in the plasma after 24 h. These investigators, too, observed a small (insignificant) reduction in IS of 15% when Na+/H+-exchange inhibition was initiated before the onset of reperfusion [16]. Therefore, a protective effect of Na+/H+-exchange inhibition initiated after the onset of ischemia cannot be excluded. In the present study, no profound reduction of cardiomyocyte loss as revealed in ischemia reperfusion experiments [14–16] was to be expected because a possible protection of cardiomyocytes is restricted to the marginal zone of the infarct. Intracellular acidosis has been shown to increase intracellular Na+ through activation of the Na+/H+-exchanger [29,30]. This results in increased intracellular Ca2+ content because of reduced Ca2+ efflux through the Na+/Ca2+-exchanger [11,12,29]. Rapid correction of internal pH by activation of the Na+/H+-exchanger may, paradoxically, further increase cellular damage [15,28,31]. Both situations may account for additional myocyte loss in the marginal zone of the infarct since a heterogeneous metabolic situation can be expected between ischemic tissue and adjacent perfused tissue. Until the overall cardiovascular situation has been stabilised after a few days, a delay in cell death as shown by pre-treatment with cariporide in porcine hearts subjected to regional ischemia and reperfusion [32], may limit infarct extent in this region when Na+/H+-exchange inhibition is initiated within the first hours after MI. Saraste et al. [33] observed apoptotic cardiomyocytes, particularly in the border zones of infarcted human myocardium. This form of cell death can be rapidly induced after global ischemia in isolated rat hearts (maximum after 30 min) and is attenuated by pre-treatment with cariporide [34]. Together with the increase of the NHE-1 isoform in isolated rat hearts after 3 h of low flow ischemia [35], these findings further point to the involvement of the exchanger in the early pathophysiological events associated with cardiac ischemia. In summary, our data show for the first time that 6-week treatment with the Na+/H+-exchange inhibitor, cariporide, engendered marked cardioprotective effects when commenced before and up to 24 h after MI as evidenced by an improvement in cardiac function, even under increased afterload. The optimal time for the start of treatment appeared to be between 30 min and 3 h following MI since IS was significantly reduced in these treatment groups. When treatment was initiated as late as 7 days after coronary artery ligation, cariporide did not afford cardioprotection. Our data imply that Na+/H+ exchange inhibition may constitute a new concept in the early therapy of MI. Acknowledgements The authors thank Dr R. Schulz and Professor D. 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TI - Cardioprotective effects of the Na+/H+-exchange inhibitor cariporide in infarct-induced heart failure
JF - Cardiovascular Research
DO - 10.1016/S0008-6363(99)00428-9
DA - 2000-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/cardioprotective-effects-of-the-na-h-exchange-inhibitor-cariporide-in-vgxGyDPCb5
SP - 102
EP - 110
VL - 46
IS - 1
DP - DeepDyve
ER -