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H+ exchanger inhibition at the onset of reperfusion decreases myocardial infarct size: role of reactive oxygen species
Cardiovascular Pathology 15 (2006) 179 – 184
Original Article
Na+/H+ exchanger inhibition at the onset of reperfusion decreases myocardial infarct size: role of reactive oxygen species Juliana C. Fantinellia, Horacio E. Cingolanib, Susana M. Moscab,4 a
Fellowship of Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Centro de Investigaciones Cardiovasculares, 1900 La Plata, Argentina b Established Investigators of CONICET, Centro de Investigaciones Cardiovasculares, La Plata, Argentina Received 25 November 2005; received in revised form 22 February 2006; accepted 27 April 2006
Abstract Background: A burst of reactive oxygen species and activation of Na+/H+ exchanger take place at the beginning of reperfusion. The aim of this study was to assess the possible interrelation of the inhibition of Na+/H+ exchanger and reactive oxygen species about the determination of myocardial infarct size. Methods: Isolated rat hearts were submitted to 40 min of coronary occlusion and 2 h of reperfusion. Infarct size was determined through triphenyltetrazolium chloride staining technique and was expressed as a percentage of risk area. Lipid peroxidation, as a marker of oxidative stress, was estimated by the concentration of thiobarbituric reactive substances. Results: Treatment during the first 20 min of reperfusion with a selective inhibitor of Na+/H+ exchanger 1 isoform, HOE 642 (cariporide; 10 AM), significantly diminished infarct size (15.1F2.4% vs. 31F2% in untreated hearts). The administration of a bscavengerQ of hydroxyl radical, N-(2mercaptopropionyl)-glycine (2 mM), decreased infarct size in an extent similar to that of cariporide (18F3%). The combination cariporide+N-(2-mercaptopropionyl)-glycine did not produce additional protection (17F1.7%). Each intervention [HOE 642 or N-(2mercaptopropionyl)-glycine] and its combination improved the postischemic recovery of myocardial systolic and diastolic functions in a similar extent. The content of the thiobarbituric reactive substances of untreated hearts (1012F144 nmol/g) decreased to 431F81, 390F82, and 433F41 after cariporide, N-(2-mercaptopropionyl)-glycine, and cariporide+N-(2-mercaptopropionyl)-glycine treatments, respectively. Conclusions: The present data support the conclusion that the cardioprotective effect of cariporide is associated with diminution of oxidative stress. D 2006 Elsevier Inc. All rights reserved. Keywords: NHE; ROS; Cariporide; MPG; Ischemia–reperfusion injury
1. Introduction Since the seminal papers by Karmazyn [1], Mentzer et al. [2], Avkiran et al. [3], and Kusumoto et al. [4], it has been known that Na+/H+ exchanger 1 (NHE-1) inhibition protects an ischemic myocardium. The classic explanation for the mechanism of this protection is as follows: during ischemia, cytosolic acidosis occurs in about 10 min. This cytosolic acidosis stimulates NHE-1, increasing its activity and augmenting cytosolic Na+ (Na+i ) and Ca2+ levels [5,6]. 4 Corresponding author. Centro de Investigaciones Cardiovasculares, Universidad Nacional de La Plata, 60 y 120, 1900 La Plata, Argentina. Tel.:+54 221 425 5861; fax: +54 221 425 5861. E-mail address: [email protected] (S.M. Mosca). 1054-8807/06/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2006.04.005
Although other mechanisms could contribute to the increase in Na+i [7] during ischemia, it has been shown that blockade of the NHE-1 before ischemia abolishes the increase in Na+i during this period [5,8] and diminishes the increase in Na+i and Ca2+ during reperfusion [5,6]. The increase in Ca2+ secondary to the increase in Na+i seems to be caused by a Na+–Ca2+ exchanger (NCX) working in reverse mode [9,10]. An increase in cytosolic Ca2+ is a well-known necrotic and apoptotic signal. Despite some contradictory results, it has been possible to obtain protection from ischemia–reperfusion by blocking NHE-1 or NCX only after the onset of reperfusion [11–13], suggesting that the phenomenon taking place at the beginning of reperfusion mediated by NHE-1 and NCX activation is a determinant of infarct size.
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The activation of NHE-1 at the onset of reperfusion has been linked to an increase in reactive oxygen species (ROS) [14 –16]. This exchanger reaches its maximal activity early after reperfusion. It has been proposed that the increase in ROS leads to activation of the mitogen-activated protein kinase pathway, phosphorylation of ERK1/2, and phosphorylation of the cytosolic tail of NHE-1, thus increasing exchanger activity [17]. The interest in decreasing myocardial infarct size by interventions performed at the onset of reperfusion is obvious: they can be performed in patients undergoing reperfusion by direct angioplasty. The aim of this experimental study is to focus on protection from ischemia–reperfusion injury by acting only during the reperfusion period, comparing NHE-1 inhibition with ROS scavenging.
Fig. 1. Scheme of the protocols performed.
1. 2.
2. Materials and methods 2.1. Isolated heart preparation This investigation conformed 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 in 1996). Wistar rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight). The heart was rapidly excised and perfused by nonrecirculating Langendorff technique with Ringer’s solution containing (in mmol/l): NaCl, 118; KCl, 5.9; MgSO4, 1.2; CaCl2, 1.35; NaCO3H, 20; dextrose, 11.1. The buffer was saturated with a mixture of 95% O2–5% CO2 (pH 7.4) and maintained at 378C. The conductive tissue in the atrial septum was damaged with a fine needle to achieve atrioventricular block, and the right ventricle was paced at 280F10 beats/min. A latex balloon tied to the end of a polyethylene tube was passed into the left ventricle through the mitral valve; the opposite end of the tube was then connected to a Statham P23XL pressure transducer. The balloon was filled with water to provide a left ventricular end-diastolic pressure (LVEDP) of 8 –12 mmHg, and this volume was unchanged for the rest of the experiment. Coronary perfusion pressure was monitored at the point of cannulation of the aorta and was adjusted to approximately 60 –70 mmHg. Coronary flow, which was controlled with a peristaltic pump, was 11F2 ml/min. Left ventricular pressure (LVP) and its first derivative (dP/dt) were recorded with a direct writing recorder. 2.2. Experimental protocols After 20 min of stabilization, the left anterior descending (LAD) artery was occluded for 40 min and the myocardium was reperfused, releasing the ligature by 120 min. Four experimental protocols were performed, as follows (Fig. 1):
3.
4.
Ischemic control group (n=12): Hearts were reperfused with preischemic solution. NHEb group: To examine the effects of the inhibition of NHE-1 on reperfusion, a specific NHE-1 blocker, HOE 642 (cariporide) [1 AM (n=5), 5 AM (n=6), or 10 AM (n=7)] was administered during the first 20 min of reperfusion. N-(2-mercaptopropionyl)-glycine (MPG) group: A hydroxyl (d OH) scavenger, MPG [1 mM (n=6) or 2 mM (n=4)], was administered during the initial 20 min of reperfusion. NHEb+MPG group (n=8): To determine the link between NHE and ROS, 10 AM HOE 642 and 2 mM MPG were coadministered at the onset of reperfusion.
In these hearts, infarct size and myocardial function were assessed. Lipid peroxidation was measured in additional hearts (ischemic control: 9; 10 AM HOE 642: 7; 2 mM MPG: 5; HOE+MPG: 5). 2.3. Infarct size determination Infarct size was assessed by the widely validated triphenyltetrazolium chloride (TTC) staining technique [18–20]. At the end of reperfusion, the LAD artery was occluded again and the myocardium was perfused for 1 min with a 0.1% solution of blue dye. This procedure delineated the nonischemic myocardium as dark blue. The frozen heart was cut into six transverse slices, which were incubated for 5 min at 378C in a 1% solution of TTC. All atrial and right ventricular tissues were excised. To measure myocardial infarction, the slices were weighed and scanned. The infarcted (pale), viable ischemic/reperfused (red), and nonischemic (blue) areas were measured by computed planimetry (Scion Image 1.62; Scion Corp., Frederick, MD, USA). Noninfarcted viable myocardium containing dehydrogenase stained brick red after reacting with TTC, whereas the infarcted tissue remained unstained due to lack of enzyme. The reperfusion time used in this study, according to previous data [21], is enough to diminish the presence of pink-and-white area patches, which made difficult the accurate measurement of infarct size.
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The area at risk (AAR), the portion of the left ventricle supplied by the previously occluded coronary artery, was identified by the absence of blue dye. Infarct weights were calculated as: (A 1 W 1 )+(A 2 W 2 ) +(A 3 W 3 )+(A 4 W 4 )+ (A 5W 5)+(A 6W 6), where A is the area of infarct for the slice and W is the weight of the respective section. The weight of the AAR was calculated in similar fashion. Infarct size was expressed as a percentage of AAR [22]. 2.4. Systolic and diastolic functions Myocardial contractility was assessed by the left ventricular developed pressure (LVDP), which is obtained by subtracting LVEDP values from the LVP peak values and the maximal rise velocity of LVP (+dP/dt max) values. Data were expressed as the percentage of their respective preischemic values. Diastolic function was evaluated by isovolumetric LVEDP. 2.5. Assessment of lipid peroxidation At the end of reperfusion, the hearts were homogenized in physiological saline solution. After that, the samples were
Fig. 3. Infarct size (expressed as a percentage of risk area) in ischemic control hearts (IC) and in hearts treated with the highest dose of HOE 642, MPG, and the combination of HOE 642 (10 AM)+MPG (2 mM). Note that the combined treatment of NHE blockade and ROS scavenging did not produce protection further than that produced separately by each one. *Pb.05, ANOVA, with respect to IC.
centrifuged; in the supernatant, the concentration of thiobarbituric reactive substances (TBARS) was determined by spectroscopic technique [23]. The absorbance at 535 nm was measured and TBARS was expressed in nanomoles per gram of tissue weight, using an extinction coefficient of 1.56105 M 1 cm 1. TBARS were also measured in hearts perfused for 3 h without ischemia, in untreated hearts, and in hearts treated with HOE 642 or MPG. 2.6. Statistical analysis Data are given as meanFS.E. The analysis of infarct size and TBARS content was performed using repeated-measures one-way analysis of variance (ANOVA), with the Newman–Keuls test for multiple comparisons among groups. Pb.05 was considered significant. 3. Results
Fig. 2. Infarct size (expressed as a percentage of risk area) in ischemic control hearts (IC) and in hearts treated with HOE 642 (upper panel) and MPG (lower panel). Note that 5 and 10 AM HOE 642 and both doses of MPG decrease the infarct size detected in IC hearts. *Pb.05, ANOVA, with respect to IC.
Fig. 2 shows infarct size in ischemic control hearts and in hearts treated with HOE 642 and MPG. In ischemic control hearts, at the end of the reperfusion period after 40 min of coronary occlusion, the infarct size was 31F2% of the risk area. Treatment with 1 AM HOE 642 at the onset of reperfusion did not modify the infarct size with respect to ischemic control hearts (33F4%). However, higher doses of HOE 642 (5 and 10 AM) produced a significant decrease of infarct size with respect to that obtained from ischemic control hearts (19.6F3.3% and 15.1F2.4%, respectively). Infarct size was also diminished after treatment at the onset of reperfusion with 1 or 2 mM of a d OH scavenger MPG (17.6F1.9% and 18F3%, respectively). Fig. 3 shows that, when both treatments were combined, the decrease in infarct size was not different from that
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Fig. 4. Values of LVDP and +dP/dt max at the end of the reperfusion period (expressed as a percentage of preischemic values) in ischemic control hearts (IC) and after HOE 642, MPG, and HOE 642 (10 AM)+MPG (2 mM) treatments. All interventions significantly improved the postischemic recovery of myocardial systolic function. *Pb.05, ANOVA, with respect to IC.
detected separately with each one (17F1.7%). These results suggest that both treatments, NHE-1 blockade and scavenging therapy, use common pathways to protect the myocardium against reperfusion injury. As depicted in Fig. 4, the recovery of systolic function was improved by treatment with HOE 642, MPG, or the combination of both treatments. At the end of the reperfusion period, LVDP and +dP/dt max reached values higher than those obtained in ischemic control hearts. The increase in LVEDP (a reflection of an increase in diastolic stiffness) detected during reperfusion after ischemia did not occur after each of the treatments (Fig. 5). NHE1 blockade, ROS scavenging, and the combination of both treatments prevented the increase in diastolic stiffness. Our data also show an inverse correlation between infarct size and myocardial function, in accordance with recent data [24]. Thus, a reduction of infarct size produced by the
Fig. 5. Changes of LVEDP at the end of reperfusion with respect to preischemic values in ischemic control hearts (IC) and in hearts treated with HOE 642, MPG, and HOE 642 (10 AM)+MPG (2 mM). The treatments did not produce the increase of LVEDP detected in IC hearts. *Pb.05.
Fig. 6. TBARS concentration (expressed in nmol/g heart weight) measured at the end of reperfusion in ischemic control hearts (IC) and in hearts treated with HOE 642 (10 AM), MPG (2 mM), and HOE 642 (10 AM)+MPG (2 mM). Note that all treatments produced a similar decrease of TBARS concentration detected in IC hearts. *Pb.05, ANOVA, with respect to IC.
treatments studied is associated with an improvement of myocardial dysfunction during reperfusion. TBARS concentration, an index of damage by lipid peroxidation, significantly increased after ischemia and reperfusion, reaching a value of 1012F144 nmol/g. Nonischemic hearts showed a TBARS content of 396F25 nmol/g. NHE-1 blockade decreased the increase in TBARS after reperfusion to a value of 431F81 nmol/g. MPG treatment also decreased lipid peroxidation, reaching a value similar to that of cariporide (TBARS=390F82 nmol/g). The combination of both treatments (10 AM cariporide+2 mM MPG) produced a similar decrease in TBARS (433F41 nmol/g) (Fig. 6). The addition of maximal doses of cariporide and MPG to nonischemic hearts did not modify TBARS concentration (374F54 and 321F14 nmol/g, respectively).
4. Discussion The protection of the ischemic myocardium by NHE-1 inhibition after the onset of reperfusion has been described [11,12,25]. The decrease in myocardial infarct size, through interference with the action of ROS burst at the beginning of
Fig. 7. Proposed mechanism of cardioprotection by cariporide.
J.C. Fantinelli et al. / Cardiovascular Pathology 15 (2006) 179 – 184
reperfusion, has also been reported [26 –29]. The data presented herein show that both pharmacological interventions (ROS scavenging and NHE-1 inhibition), when applied together and at their maximal effective concentration, did not induce protection further than that obtained separately by each one. This finding suggests that both interventions act through a common pathway. A plausible classic explanation for this finding would be that the release of ROS at the beginning of reperfusion activates NHE-1, increasing Na+i and subsequently Ca2+ through the NCX operating in its reverse mode. In this regard, it has been reported by Sabri et al. [14] and Snabaitis et al. [16] that ROS can activate NHE-1. Rothstein et al. [17] reported that approximately 50% of myocardial Ca2+ overload after reperfusion was due to ROS-mediated NHE-1 activation through an ERK1/2 MAP kinase signaling pathway. If this were the case, we should expect an increase in lipoperoxidation in ischemic control hearts and a decrease in this parameter when the scavenger MPG is present, but not after NHE-1 inhibition. Therefore, the interesting finding of this paper is that cariporide appears to be an intervention that decreases the damage induced by ROS, which seem to be generated downstream of NHE-1 activation. We do not deny that the ROS released during reperfusion activate NHE-1 and that NHE-1 inhibition blunts the increase in Na+i . We are proposing that, in addition to this effect of preventing Na+i increase, NHE-1 inhibition by cariporide decreases ROS-induced damage. Recent experiments in isolated myocytes exposed to simulated ischemia and reperfusion showed that Ca2+influx-dependent Ca2+ overload can be blunted by antioxidants and that ROS are required for the rapid activation of NCX after reoxygenation [30]. If this were the case and if ROS were directly involved in NCX activation, cariporide may be probably acting at two different levels: first, by preventing NHE-1-mediated Na+i increase (the classic proposed mechanism) and, second, by preventing a direct stimulation of ROS upon NCX in reverse mode. However, in the presence of cariporide, lipid peroxidation diminished to the same extent as MPG, suggesting that the NHE-1 blocker could exert its cardioprotective action by inhibiting ROS production. Infarct size was decreased to the same extent by ROS scavenging, NHE-1 blockade, or the combination of both pharmacological interventions. The mitochondrion is the main source of ROS release in reperfusion. A role played by the mitochondria permeability transition pore [31] and the ROS induce–ROS release mechanism by the mitochondria have been identified [32]. Furthermore, an action of cariporide on mitochondria has been suggested [33,34]. Whether the mitochondria is involved in the mechanism proposed is not apparent to us at this moment. In summary, we presented evidence that, in the protection brought about by NHE-1 inhibition at the onset of reperfusion, the action of ROS upon NCX plays an important role. Fig. 7 schematizes our proposed mechanism.
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Acknowledgments This work was partly supported by a grant from the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (PIP 2208).
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Original Article
Na+/H+ exchanger inhibition at the onset of reperfusion decreases myocardial infarct size: role of reactive oxygen species Juliana C. Fantinellia, Horacio E. Cingolanib, Susana M. Moscab,4 a
Fellowship of Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Centro de Investigaciones Cardiovasculares, 1900 La Plata, Argentina b Established Investigators of CONICET, Centro de Investigaciones Cardiovasculares, La Plata, Argentina Received 25 November 2005; received in revised form 22 February 2006; accepted 27 April 2006
Abstract Background: A burst of reactive oxygen species and activation of Na+/H+ exchanger take place at the beginning of reperfusion. The aim of this study was to assess the possible interrelation of the inhibition of Na+/H+ exchanger and reactive oxygen species about the determination of myocardial infarct size. Methods: Isolated rat hearts were submitted to 40 min of coronary occlusion and 2 h of reperfusion. Infarct size was determined through triphenyltetrazolium chloride staining technique and was expressed as a percentage of risk area. Lipid peroxidation, as a marker of oxidative stress, was estimated by the concentration of thiobarbituric reactive substances. Results: Treatment during the first 20 min of reperfusion with a selective inhibitor of Na+/H+ exchanger 1 isoform, HOE 642 (cariporide; 10 AM), significantly diminished infarct size (15.1F2.4% vs. 31F2% in untreated hearts). The administration of a bscavengerQ of hydroxyl radical, N-(2mercaptopropionyl)-glycine (2 mM), decreased infarct size in an extent similar to that of cariporide (18F3%). The combination cariporide+N-(2-mercaptopropionyl)-glycine did not produce additional protection (17F1.7%). Each intervention [HOE 642 or N-(2mercaptopropionyl)-glycine] and its combination improved the postischemic recovery of myocardial systolic and diastolic functions in a similar extent. The content of the thiobarbituric reactive substances of untreated hearts (1012F144 nmol/g) decreased to 431F81, 390F82, and 433F41 after cariporide, N-(2-mercaptopropionyl)-glycine, and cariporide+N-(2-mercaptopropionyl)-glycine treatments, respectively. Conclusions: The present data support the conclusion that the cardioprotective effect of cariporide is associated with diminution of oxidative stress. D 2006 Elsevier Inc. All rights reserved. Keywords: NHE; ROS; Cariporide; MPG; Ischemia–reperfusion injury
1. Introduction Since the seminal papers by Karmazyn [1], Mentzer et al. [2], Avkiran et al. [3], and Kusumoto et al. [4], it has been known that Na+/H+ exchanger 1 (NHE-1) inhibition protects an ischemic myocardium. The classic explanation for the mechanism of this protection is as follows: during ischemia, cytosolic acidosis occurs in about 10 min. This cytosolic acidosis stimulates NHE-1, increasing its activity and augmenting cytosolic Na+ (Na+i ) and Ca2+ levels [5,6]. 4 Corresponding author. Centro de Investigaciones Cardiovasculares, Universidad Nacional de La Plata, 60 y 120, 1900 La Plata, Argentina. Tel.:+54 221 425 5861; fax: +54 221 425 5861. E-mail address: [email protected] (S.M. Mosca). 1054-8807/06/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2006.04.005
Although other mechanisms could contribute to the increase in Na+i [7] during ischemia, it has been shown that blockade of the NHE-1 before ischemia abolishes the increase in Na+i during this period [5,8] and diminishes the increase in Na+i and Ca2+ during reperfusion [5,6]. The increase in Ca2+ secondary to the increase in Na+i seems to be caused by a Na+–Ca2+ exchanger (NCX) working in reverse mode [9,10]. An increase in cytosolic Ca2+ is a well-known necrotic and apoptotic signal. Despite some contradictory results, it has been possible to obtain protection from ischemia–reperfusion by blocking NHE-1 or NCX only after the onset of reperfusion [11–13], suggesting that the phenomenon taking place at the beginning of reperfusion mediated by NHE-1 and NCX activation is a determinant of infarct size.
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The activation of NHE-1 at the onset of reperfusion has been linked to an increase in reactive oxygen species (ROS) [14 –16]. This exchanger reaches its maximal activity early after reperfusion. It has been proposed that the increase in ROS leads to activation of the mitogen-activated protein kinase pathway, phosphorylation of ERK1/2, and phosphorylation of the cytosolic tail of NHE-1, thus increasing exchanger activity [17]. The interest in decreasing myocardial infarct size by interventions performed at the onset of reperfusion is obvious: they can be performed in patients undergoing reperfusion by direct angioplasty. The aim of this experimental study is to focus on protection from ischemia–reperfusion injury by acting only during the reperfusion period, comparing NHE-1 inhibition with ROS scavenging.
Fig. 1. Scheme of the protocols performed.
1. 2.
2. Materials and methods 2.1. Isolated heart preparation This investigation conformed 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 in 1996). Wistar rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight). The heart was rapidly excised and perfused by nonrecirculating Langendorff technique with Ringer’s solution containing (in mmol/l): NaCl, 118; KCl, 5.9; MgSO4, 1.2; CaCl2, 1.35; NaCO3H, 20; dextrose, 11.1. The buffer was saturated with a mixture of 95% O2–5% CO2 (pH 7.4) and maintained at 378C. The conductive tissue in the atrial septum was damaged with a fine needle to achieve atrioventricular block, and the right ventricle was paced at 280F10 beats/min. A latex balloon tied to the end of a polyethylene tube was passed into the left ventricle through the mitral valve; the opposite end of the tube was then connected to a Statham P23XL pressure transducer. The balloon was filled with water to provide a left ventricular end-diastolic pressure (LVEDP) of 8 –12 mmHg, and this volume was unchanged for the rest of the experiment. Coronary perfusion pressure was monitored at the point of cannulation of the aorta and was adjusted to approximately 60 –70 mmHg. Coronary flow, which was controlled with a peristaltic pump, was 11F2 ml/min. Left ventricular pressure (LVP) and its first derivative (dP/dt) were recorded with a direct writing recorder. 2.2. Experimental protocols After 20 min of stabilization, the left anterior descending (LAD) artery was occluded for 40 min and the myocardium was reperfused, releasing the ligature by 120 min. Four experimental protocols were performed, as follows (Fig. 1):
3.
4.
Ischemic control group (n=12): Hearts were reperfused with preischemic solution. NHEb group: To examine the effects of the inhibition of NHE-1 on reperfusion, a specific NHE-1 blocker, HOE 642 (cariporide) [1 AM (n=5), 5 AM (n=6), or 10 AM (n=7)] was administered during the first 20 min of reperfusion. N-(2-mercaptopropionyl)-glycine (MPG) group: A hydroxyl (d OH) scavenger, MPG [1 mM (n=6) or 2 mM (n=4)], was administered during the initial 20 min of reperfusion. NHEb+MPG group (n=8): To determine the link between NHE and ROS, 10 AM HOE 642 and 2 mM MPG were coadministered at the onset of reperfusion.
In these hearts, infarct size and myocardial function were assessed. Lipid peroxidation was measured in additional hearts (ischemic control: 9; 10 AM HOE 642: 7; 2 mM MPG: 5; HOE+MPG: 5). 2.3. Infarct size determination Infarct size was assessed by the widely validated triphenyltetrazolium chloride (TTC) staining technique [18–20]. At the end of reperfusion, the LAD artery was occluded again and the myocardium was perfused for 1 min with a 0.1% solution of blue dye. This procedure delineated the nonischemic myocardium as dark blue. The frozen heart was cut into six transverse slices, which were incubated for 5 min at 378C in a 1% solution of TTC. All atrial and right ventricular tissues were excised. To measure myocardial infarction, the slices were weighed and scanned. The infarcted (pale), viable ischemic/reperfused (red), and nonischemic (blue) areas were measured by computed planimetry (Scion Image 1.62; Scion Corp., Frederick, MD, USA). Noninfarcted viable myocardium containing dehydrogenase stained brick red after reacting with TTC, whereas the infarcted tissue remained unstained due to lack of enzyme. The reperfusion time used in this study, according to previous data [21], is enough to diminish the presence of pink-and-white area patches, which made difficult the accurate measurement of infarct size.
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The area at risk (AAR), the portion of the left ventricle supplied by the previously occluded coronary artery, was identified by the absence of blue dye. Infarct weights were calculated as: (A 1 W 1 )+(A 2 W 2 ) +(A 3 W 3 )+(A 4 W 4 )+ (A 5W 5)+(A 6W 6), where A is the area of infarct for the slice and W is the weight of the respective section. The weight of the AAR was calculated in similar fashion. Infarct size was expressed as a percentage of AAR [22]. 2.4. Systolic and diastolic functions Myocardial contractility was assessed by the left ventricular developed pressure (LVDP), which is obtained by subtracting LVEDP values from the LVP peak values and the maximal rise velocity of LVP (+dP/dt max) values. Data were expressed as the percentage of their respective preischemic values. Diastolic function was evaluated by isovolumetric LVEDP. 2.5. Assessment of lipid peroxidation At the end of reperfusion, the hearts were homogenized in physiological saline solution. After that, the samples were
Fig. 3. Infarct size (expressed as a percentage of risk area) in ischemic control hearts (IC) and in hearts treated with the highest dose of HOE 642, MPG, and the combination of HOE 642 (10 AM)+MPG (2 mM). Note that the combined treatment of NHE blockade and ROS scavenging did not produce protection further than that produced separately by each one. *Pb.05, ANOVA, with respect to IC.
centrifuged; in the supernatant, the concentration of thiobarbituric reactive substances (TBARS) was determined by spectroscopic technique [23]. The absorbance at 535 nm was measured and TBARS was expressed in nanomoles per gram of tissue weight, using an extinction coefficient of 1.56105 M 1 cm 1. TBARS were also measured in hearts perfused for 3 h without ischemia, in untreated hearts, and in hearts treated with HOE 642 or MPG. 2.6. Statistical analysis Data are given as meanFS.E. The analysis of infarct size and TBARS content was performed using repeated-measures one-way analysis of variance (ANOVA), with the Newman–Keuls test for multiple comparisons among groups. Pb.05 was considered significant. 3. Results
Fig. 2. Infarct size (expressed as a percentage of risk area) in ischemic control hearts (IC) and in hearts treated with HOE 642 (upper panel) and MPG (lower panel). Note that 5 and 10 AM HOE 642 and both doses of MPG decrease the infarct size detected in IC hearts. *Pb.05, ANOVA, with respect to IC.
Fig. 2 shows infarct size in ischemic control hearts and in hearts treated with HOE 642 and MPG. In ischemic control hearts, at the end of the reperfusion period after 40 min of coronary occlusion, the infarct size was 31F2% of the risk area. Treatment with 1 AM HOE 642 at the onset of reperfusion did not modify the infarct size with respect to ischemic control hearts (33F4%). However, higher doses of HOE 642 (5 and 10 AM) produced a significant decrease of infarct size with respect to that obtained from ischemic control hearts (19.6F3.3% and 15.1F2.4%, respectively). Infarct size was also diminished after treatment at the onset of reperfusion with 1 or 2 mM of a d OH scavenger MPG (17.6F1.9% and 18F3%, respectively). Fig. 3 shows that, when both treatments were combined, the decrease in infarct size was not different from that
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Fig. 4. Values of LVDP and +dP/dt max at the end of the reperfusion period (expressed as a percentage of preischemic values) in ischemic control hearts (IC) and after HOE 642, MPG, and HOE 642 (10 AM)+MPG (2 mM) treatments. All interventions significantly improved the postischemic recovery of myocardial systolic function. *Pb.05, ANOVA, with respect to IC.
detected separately with each one (17F1.7%). These results suggest that both treatments, NHE-1 blockade and scavenging therapy, use common pathways to protect the myocardium against reperfusion injury. As depicted in Fig. 4, the recovery of systolic function was improved by treatment with HOE 642, MPG, or the combination of both treatments. At the end of the reperfusion period, LVDP and +dP/dt max reached values higher than those obtained in ischemic control hearts. The increase in LVEDP (a reflection of an increase in diastolic stiffness) detected during reperfusion after ischemia did not occur after each of the treatments (Fig. 5). NHE1 blockade, ROS scavenging, and the combination of both treatments prevented the increase in diastolic stiffness. Our data also show an inverse correlation between infarct size and myocardial function, in accordance with recent data [24]. Thus, a reduction of infarct size produced by the
Fig. 5. Changes of LVEDP at the end of reperfusion with respect to preischemic values in ischemic control hearts (IC) and in hearts treated with HOE 642, MPG, and HOE 642 (10 AM)+MPG (2 mM). The treatments did not produce the increase of LVEDP detected in IC hearts. *Pb.05.
Fig. 6. TBARS concentration (expressed in nmol/g heart weight) measured at the end of reperfusion in ischemic control hearts (IC) and in hearts treated with HOE 642 (10 AM), MPG (2 mM), and HOE 642 (10 AM)+MPG (2 mM). Note that all treatments produced a similar decrease of TBARS concentration detected in IC hearts. *Pb.05, ANOVA, with respect to IC.
treatments studied is associated with an improvement of myocardial dysfunction during reperfusion. TBARS concentration, an index of damage by lipid peroxidation, significantly increased after ischemia and reperfusion, reaching a value of 1012F144 nmol/g. Nonischemic hearts showed a TBARS content of 396F25 nmol/g. NHE-1 blockade decreased the increase in TBARS after reperfusion to a value of 431F81 nmol/g. MPG treatment also decreased lipid peroxidation, reaching a value similar to that of cariporide (TBARS=390F82 nmol/g). The combination of both treatments (10 AM cariporide+2 mM MPG) produced a similar decrease in TBARS (433F41 nmol/g) (Fig. 6). The addition of maximal doses of cariporide and MPG to nonischemic hearts did not modify TBARS concentration (374F54 and 321F14 nmol/g, respectively).
4. Discussion The protection of the ischemic myocardium by NHE-1 inhibition after the onset of reperfusion has been described [11,12,25]. The decrease in myocardial infarct size, through interference with the action of ROS burst at the beginning of
Fig. 7. Proposed mechanism of cardioprotection by cariporide.
J.C. Fantinelli et al. / Cardiovascular Pathology 15 (2006) 179 – 184
reperfusion, has also been reported [26 –29]. The data presented herein show that both pharmacological interventions (ROS scavenging and NHE-1 inhibition), when applied together and at their maximal effective concentration, did not induce protection further than that obtained separately by each one. This finding suggests that both interventions act through a common pathway. A plausible classic explanation for this finding would be that the release of ROS at the beginning of reperfusion activates NHE-1, increasing Na+i and subsequently Ca2+ through the NCX operating in its reverse mode. In this regard, it has been reported by Sabri et al. [14] and Snabaitis et al. [16] that ROS can activate NHE-1. Rothstein et al. [17] reported that approximately 50% of myocardial Ca2+ overload after reperfusion was due to ROS-mediated NHE-1 activation through an ERK1/2 MAP kinase signaling pathway. If this were the case, we should expect an increase in lipoperoxidation in ischemic control hearts and a decrease in this parameter when the scavenger MPG is present, but not after NHE-1 inhibition. Therefore, the interesting finding of this paper is that cariporide appears to be an intervention that decreases the damage induced by ROS, which seem to be generated downstream of NHE-1 activation. We do not deny that the ROS released during reperfusion activate NHE-1 and that NHE-1 inhibition blunts the increase in Na+i . We are proposing that, in addition to this effect of preventing Na+i increase, NHE-1 inhibition by cariporide decreases ROS-induced damage. Recent experiments in isolated myocytes exposed to simulated ischemia and reperfusion showed that Ca2+influx-dependent Ca2+ overload can be blunted by antioxidants and that ROS are required for the rapid activation of NCX after reoxygenation [30]. If this were the case and if ROS were directly involved in NCX activation, cariporide may be probably acting at two different levels: first, by preventing NHE-1-mediated Na+i increase (the classic proposed mechanism) and, second, by preventing a direct stimulation of ROS upon NCX in reverse mode. However, in the presence of cariporide, lipid peroxidation diminished to the same extent as MPG, suggesting that the NHE-1 blocker could exert its cardioprotective action by inhibiting ROS production. Infarct size was decreased to the same extent by ROS scavenging, NHE-1 blockade, or the combination of both pharmacological interventions. The mitochondrion is the main source of ROS release in reperfusion. A role played by the mitochondria permeability transition pore [31] and the ROS induce–ROS release mechanism by the mitochondria have been identified [32]. Furthermore, an action of cariporide on mitochondria has been suggested [33,34]. Whether the mitochondria is involved in the mechanism proposed is not apparent to us at this moment. In summary, we presented evidence that, in the protection brought about by NHE-1 inhibition at the onset of reperfusion, the action of ROS upon NCX plays an important role. Fig. 7 schematizes our proposed mechanism.
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Acknowledgments This work was partly supported by a grant from the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (PIP 2208).
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