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Ann Thorac Surg 2001;72:836-843
© 2001 The Society of Thoracic Surgeons

Original article: cardiovascular

Effects of NHE-1 inhibition on cardioprotection and impact on protection by K/Mg cardioplegia

Yoshiya Toyoda, MDa, Shaher Khan, BSa, Weimin Chen, MDa, Robert A. Parker, ScDa, Sidney Levitsky, MDa, James D. McCully, PhDa

a Division of Cardiothoracic Surgery and Biometrics Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA

Accepted for publication May 3, 2001.

Address reprint requests to Dr McCully, Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Rm 144, 77 Ave Louis Pasteur, Boston, MA 02115, USA
e-mail: james_mccully{at}hms.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Cardiac sodium hydrogen exchanger isoform-1 (NHE-1) activity during ischemia/reperfusion contributes to myocardial injury. The effects of NHE-1 inhibition during ischemia or reperfusion and on the protection afforded by K/Mg cardioplegia was unknown.

Methods. Rabbit hearts were used for Langendorff perfusion. Control hearts were perfused for 180 minutes. Global ischemia (GI) hearts received 30 minutes normothermic global ischemia and 120 minutes reperfusion. K/Mg hearts received cardioplegia 5 minutes before ischemia. Separate groups of GI and K/Mg hearts received the NHE-1 inhibitor, HOE-642, before ischemia (HOE-642-I), at the immediate start of reperfusion (HOE-642-R), or both before ischemia and at the immediate start of reperfusion (HOE-642-IR).

Results. Left ventricular peak developed pressure was significantly increased in HOE-I, HOE-R, and HOE-IR throughout reperfusion (p < 0.05 versus GI). Infarct size was significantly decreased (p < 0.05 versus GI) in all groups, but was significantly increased in HOE-R as compared with HOE-IR (p < 0.05). NHE-1 inhibition with K/Mg cardioplegia significantly decreased left ventricular peak developed pressure after 90 minutes of reperfusion (p < 0.05 versus K/Mg), with no significant effect on infarct size.

Conclusions. NHE-1 inhibition used alone provides cardioprotection with optimal effects being observed with HOE-IR. NHE-1 inhibition with K/Mg cardioplegia decreases postischemic functional recovery during late reperfusion.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Previously, we have shown that magnesium-supplemented potassium (K/Mg) cardioplegia (K+: 20 mmol/L; Mg2+:20 mmol/L) significantly decreases infarct size and significantly enhances postischemic functional recovery [1–3]. The mechanisms by which K/Mg cardioplegia affords cardioprotection include the modulation of cytosolic calcium overload, enhanced preservation and resynthesis of high-energy phosphates, modulation of nuclear and mitochondrial function, and the attenuation of intracellular acidosis [3–8].

Recent reports have shown that sodium-hydrogen exchangers (NHE) play a central role in the regulation of intracellular Na+, Ca2+, and pH homeostasis, and contribute to ischemia and reperfusion injury [9–11]. At least six isoforms of NHE have been identified in the mammalian plasma membrane and mitochondria. Recent reports have indicated that specific inhibition of the NHE isoform 1 (NHE-1), located on the plasma membrane of the cardiac myocyte, with HOE-642 (4-isopropyl-3-methylsulphonylbenzoyl-guanidine methanesulphonate; cariporide) reduces myocardial infarct size and improves postischemic functional recovery after ischemia and reperfusion [12–14].

The purpose of this study was to determine if NHE-1 inhibition provided similar cardioprotection as that achieved with K/Mg cardioplegia and to determine if NHE-1 inhibition provided additive cardioprotection when used in concert with K/Mg cardioplegia. In addition, we examined the efficacy of NHE-1 inhibition when inhibition was induced before surgically induced normothermic global ischemia (GI) or during reperfusion.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Animals
New Zealand White rabbits (n = 64, 15 to 20 weeks old; 3 to 4 kg) were obtained from Millbrook Farm (Amherst, MA). All animals were housed individually and provided with laboratory chow and water ab libitum. All experiments were approved by the Beth Israel Deaconess Medical Center Animal Care and Use Committee and the Harvard Medical Area Standing Committee on Animals (Institutional Animal Care and Use Committee) and conformed to the U.S. National Institutes of Health guidelines regulating the care and use of laboratory animals (National Institutes of Health publication 5377-3, 1996).

Langendorff perfusion
All rabbits were anesthetized with ketamine (33 mg/kg) and xylazine (16 mg/kg), and heparin (200 U/kg) was given intravenously through the marginal ear vein. The heart was excised and placed in a 4°C bath of Krebs-Ringer solution equilibrated with 95% O2 and 5% CO2 (pH 7.4 at 37°C, Table 1), in which spontaneous beating ceased within a few seconds. Langendorff retrograde perfusion was performed as described previously [1, 4, 8]. In brief, a latex balloon containing a catheter-tip transducer (Millar Instruments, Inc, Houston, TX) was inserted into the left ventricle. The volume of the balloon was maintained using a calibrated microsyringe to provide a constant physiologic end-diastolic pressure of 5 to 10 mm Hg during equilibrium, and this balloon volume was maintained for the duration of the experiment. The aorta was cannulated with a metal cannula and the heart underwent Langendorff retrograde perfusion at a constant pressure of 75 cm H2O at 37°C. Hearts were paced through the right atrium at 180 ± 3 beats/min throughout the experiment using a Medtronic, Model 5330 stimulator (Medtronic, Minneapolis MN). Hemodynamic variables were acquired using the PO-NE-MAH digital data acquisition system (Gould, Valley View, OH), with an Acquire Plus processor board, and left ventricular pressure analysis software.


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  		Table 1. Composition of Krebs-Ringer Solution and K/Mg Cardioplegia

 
Experimental protocol
Hearts were perfused for 30 minutes to establish preischemic base line hemodynamics (equilibrium). Equilibrium was ceased when heart rate, coronary flow, left ventricular peak developed pressure (LVPDP), and left ventricular end-diastolic pressure (LVEDP) were maintained at the same level for three continuous measurement periods timed 5 minutes apart. Control hearts (n = 8) were perfused without GI at 37°C for 180 minutes. Global ischemia hearts (n = 8) underwent 30 minutes normothermic (37°C) GI and 120 minutes reperfusion. Global ischemia was achieved by cross-clamping the perfusion line. K/Mg hearts (n = 8) were perfused with normothermic (37°C) K/Mg cardioplegia (Table 1) for 5 minutes before ischemia.

Effect of NHE-1 inhibition before normothermic global ischemia or at the immediate start of reperfusion on myocardial hemodynamics and infarct size
To investigate the effects of NHE-1 inhibition on infarct size and postischemic functional recovery in GI hearts during normothermic GI and reperfusion, GI hearts were perfused separately with HOE-642 (1 µmol/L in Krebs-Ringer solution; Hoechst, Frankfurt, Germany) for 15 minutes before GI (HOE-I; n = 6) or for 15 minutes at the immediate start of reperfusion (HOE-R; n = 6) or during both periods (HOE-IR; n = 6). The concentration and perfusion time for HOE-642 were based on previous reports [15–19].

Effect of NHE-1 inhibition before normothermic global ischemia or at the immediate start of reperfusion on the cardioprotection afforded by K/Mg cardioplegia
To investigate the effects of NHE-1 inhibition on the cardioprotection afforded by K/Mg cardioplegia during normothermic GI and reperfusion, K/Mg hearts were perfused separately with HOE-642 (1 µmol/L) for 10 minutes before K/Mg cardioplegia infusion and for the 5 minutes of K/Mg cardioplegia infusion before GI (K/Mg + HOE-I; n = 6), for 15 minutes at the immediate start of reperfusion (K/Mg + HOE-R; n = 6), or during both periods (K/Mg + HOE-IR; n = 6).

Measurement of infarct size
Infarct size was determined after 120 minutes of reperfusion as described previously using 1% triphenyl tetrazolium chloride (Sigma Chemical Co, St. Louis, MO) in phosphate buffer (pH 7.4) [1, 16]. The area of the left ventricle and the area of infarcted tissue were measured by an independent, blinded observer using computer planimetry as described previously [1, 16].

Wet weight/dry weight ratios
Wet/dry weight ratios were determined as described previously [1, 16].

Statistical analysis
Statistical analysis was performed using SAS (version 6.12) software package (SAS Institute, Cary, NC). The mean ± standard error of the mean (SEM) for all data were calculated for all variables. Statistical significance was assessed using repeated-measures analysis of variance (ANOVA) with group as a between-subjects factor and time as a within-subjects factor. If this overall test was significant, then one-way ANOVA was performed at individual times and when significant, post hoc comparisons were made between groups. The Dunnett test was used for comparisons between control and other groups to adjust for the multiplicity of tests. Tukey correction was used for comparisons between groups other than the control. A one-way ANOVA was used for infarction size.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Equilibrium hemodynamics
At the end of equilibrium, LVPDP was 106 ± 3.9 mm Hg, LVEDP was 6.8 ± 0.8 mm Hg, the positive first derivative of pressure over time (+dP/dt) was 1429 ± 67 mm Hg/s, and coronary flow was 44 ± 2.6 mL/min in control hearts. No significant differences in LVPDP, LVEDP, +dP/dt, or coronary flow were observed within or between groups at the end of equilibrium.

Effect of global ischemia and K/Mg cardioplegia on myocardial hemodynamics and infarct size
LVPDP and +dP/dt in GI hearts were decreased significantly and LVEDP was increased significantly (p < 0.05 versus control and K/Mg) throughout reperfusion (Fig 1, Table 2). Coronary flow in GI hearts was decreased significantly only at 120 minutes of reperfusion (180 minutes of perfusion, p < 0.05 versus control and K/Mg; Table 2). Myocardial infarct size was 1.9% ± 0.4% in control hearts, and was increased significantly to 29.4% ± 2.1% in GI hearts (p < 0.05 versus control; Fig 2). Myocardial cellular injury in K/Mg hearts was 3.7% ± 0.5% (p < 0.05 versus GI, NS versus control). No significant differences in LVPDP, LVEDP, coronary flow, +dP/dt, or myocardial cellular injury were observed between control and K/Mg hearts (Fig 1 and Fig 2, Table 1).



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  		Fig 1. Left ventricular peak developed pressure (LVPDP, mm Hg), during 30 minutes of equilibrium, 30 minutes of global ischemia (GI), and 120 minutes of reperfusion for control, GI, magnesium-supplemented potassium cardioplegia hearts (K/Mg), and GI hearts perfused with the cardiac Na+/H+ exchanger isoform 1 inhibitor, HOE-642, before GI (HOE-I) or at the immediate start of reperfusion (HOE-R) or during both periods (HOE-IR). All results are shown as the mean ± standard error of the mean for each group. Significant differences are shown as * for p < 0.05 versus control, §p < 0.05 versus GI.

 

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  		Table 2. LVEDP, +dP/dt, and Coronary Flow During Reperfusion for Control, GI, and K/Mg Hearts, and GI Hearts Perfused With HOE-642

 


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  		Fig 2. Infarct size after 180 minutes of perfusion for control, global ischemia (GI), magnesium-supplemented potassium cardioplegia hearts (K/Mg), GI hearts perfused with the cardiac Na+/H+ exchanger isoform 1 inhibitor, HOE-642, before GI (HOE-I) or at the immediate start of reperfusion (HOE-R) or during both periods (HOE-IR), and K/Mg hearts perfused with HOE-642 before GI (K/Mg + HOE-I) or at the immediate start of reperfusion (K/Mg + HOE-R) or during both periods (K/Mg + HOE-IR). All results are shown as the mean ± standard error of the mean for each group. Significant differences are shown as * for p < 0.05 versus control, # for p < 0.05 versus K/Mg, and § for p < 0.05 versus GI.

 
Effect of HOE-642 on myocardial hemodynamics and cellular injury
To determine the effect of HOE-642 on hemodynamics and myocardial cellular injury, control hearts received HOE-642 for two 15-minute periods during perfusion: at 15 to 30 minutes of perfusion and at 60 to 75 minutes of perfusion as a sham treatment (n = 4). No significant differences in LVPDP, LVEDP, +dP/dt, coronary flow, or myocardial cellular injury as determined by triphenyl tetrazolium chloride (TTC) staining or myocardial wet weight/dry weight ratio were observed as compared with control hearts (data not shown).

Effect of NHE-1 inhibition before normothermic global ischemia or at the immediate start of reperfusion on myocardial hemodynamics and infarct size
LVPDP in GI hearts in which HOE-642 was perfused for 15 minutes before GI (HOE-I), perfused for 15 minutes at the immediate start of reperfusion (HOE-R), or in which HOE-642 was perfused both before ischemia and at the immediate start of reperfusion (HOE-IR) was increased significantly (p < 0.05) as compared with GI hearts after 20 minutes of reperfusion (80 to 180 minutes of perfusion) (Fig 1). No significant difference in LVPDP was observed between HOE-I, HOE-R, HOE-IR, and K/Mg hearts after 30 minutes of reperfusion (90 to 180 minutes of perfusion) (NS versus K/Mg).

LVEDP in HOE-I, HOE-R, and HOE-IR hearts was decreased significantly throughout reperfusion (p < 0.05 versus GI; NS versus control) (Table 2). Coronary flow in HOE-R hearts was increased significantly after 60 minutes of reperfusion (120 to 180 minutes of perfusion) (p < 0.05 versus GI; NS versus control) (Table 2). +dP/dt in HOE-R and HOE-IR hearts was increased significantly after 30 minutes of reperfusion (90 to 180 minutes of perfusion; p < 0.05 versus GI; NS versus control; Table 2). No significant differences in LVEDP, coronary flow, or +dP/dt were observed between HOE-I, HOE-R, HOE-IR, and K/Mg hearts.

Infarct size was decreased significantly to 9.7% ± 1.9% in HOE-I, 14.9% ± 0.7% in HOE-R, and 6.5% ± 2.3% in HOE-IR (p < 0.05 versus GI) as compared with 29.4% ± 2.1% in GI hearts (Fig 2). It should be noted that infarct size in HOE-R hearts was increased significantly (p < 0.05) as compared with HOE-IR hearts. No significant difference in infarct size was observed between HOE-I and HOE-IR hearts (Fig 2). Infarct size in both HOE-I and HOE-R hearts were increased significantly (p < 0.05) as compared with control and K/Mg hearts, but no significant difference in infarct size was observed between HOE-IR and control and K/Mg hearts.

Effect of NHE-1 inhibition before normothermic global ischemia or at the immediate start of reperfusion on the cardioprotection afforded by K/Mg cardioplegia
LVPDP in K/Mg + HOE-I, K/Mg + HOE-R, and K/Mg + HOE-IR hearts was decreased significantly as compared with K/Mg hearts (p < 0.05 versus K/Mg) after 90, 60, and 90 minutes of reperfusion, respectively (150, 120, and 150 minutes of perfusion, respectively; Fig 3). No significant difference in LVEDP, coronary flow, or +dP/dt was observed between K/Mg + HOE-I, K/Mg + HOE-R, K/Mg + HOE-IR, and K/Mg hearts throughout reperfusion (Table 3).



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  		Fig 3. Left ventricular peak developed pressure (LVPDP, mm Hg), during 30 minutes of equilibrium, 30 minutes of global ischemia (GI), and 120 minutes of reperfusion for control, GI, magnesium-supplemented potassium cardioplegia hearts (K/Mg), and K/Mg hearts perfused with the cardiac Na+/H+ exchanger isoform 1 inhibitor, HOE-642, before GI (K/Mg + HOE-I) or at the immediate start of reperfusion (K/Mg + HOE-R) or during both periods (K/Mg + HOE-IR). All results are shown as the mean ± standard error of the mean for each group. Significant differences are shown as * for p < 0.05 versus control, and # for p < 0.05 versus K/Mg.

 

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  		Table 3. LVEDP, +dP/dt, and Coronary Flow During Reperfusion for Control, GI, and K/Mg Hearts, and K/Mg Hearts Perfused With HOE-642

 
In comparison with GI hearts, LVPDP was increased significantly and LVEDP was decreased significantly in K/Mg + HOE-I, K/Mg + HOE-R, and K/Mg + HOE-IR hearts (p < 0.05 versus GI; Table 3). Coronary flow in K/Mg + HOE-I and K/Mg + HOE-IR hearts was significantly increased after 90 minutes of reperfusion (150 to 180 minutes of perfusion) as compared with GI hearts (p < 0.05 versus GI; Table 3).

Infarct size was 6.3% ± 0.5% in K/Mg + HOE-I, 7.4% ± 0.6% in K/Mg + HOE-R, and 3.9% ± 0.4% in K/Mg + HOE-IR hearts. No significant difference in infarct size was observed between K/Mg + HOE-I, K/Mg + HOE-R, K/Mg + HOE-IR, and K/Mg hearts (NS versus control) (Fig 2).

Wet weight/dry weight ratios
Wet weight/dry weight ratio after 180 minutes of perfusion was 6.0 ± 0.3 in Control, 6.9 ± 0.4 in GI, 5.6 ± 0.2 in K/Mg, 6.0 ± 0.3 in HOE-I, 6.6 ± 0.7 in HOE-R, 5.4 ± 1.1 in HOE-IR, 5.8 ± 0.3 in K/Mg + HOE-I, 5.8 ± 0.7 in K/Mg + HOE-R, and 5.8 ± 0.3 in K/Mg + HOE-IR hearts, respectively. No significant difference in wet/dry weight ratio was observed between groups.


   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
Our results indicate that the selective cardiac NHE-1 inhibitor HOE-642 (1 µmol/L; cariporide) significantly improved postischemic systolic and diastolic functional recovery (LVPDP and LVEDP, p < 0.05 versus GI) and significantly decreased infarct size (p < 0.05 versus GI) after 30 minutes normothermic GI, when used either before ischemia (HOE-I) or at the immediate start of reperfusion (HOE-R, HOE-IR). However, infarct size in hearts in which HOE-642 was perfused both before ischemia and at the immediate start of reperfusion (HOE-IR) was significantly decreased (p < 0.05) as compared with hearts in which HOE-642 was perfused only during reperfusion (HOE-R). No significant difference in infarct size was observed between HOE-IR and control hearts. These results indicate that for maximal cardioprotection, HOE-642 should be perfused both before ischemia and at the immediate start of reperfusion.

Previously, we had shown that K/Mg cardioplegia significantly decreased infarct size and significantly enhanced postischemic functional recovery (p < 0.05 versus GI), with no significant difference being observed as compared with control hearts [1]. In a series of experiments, we investigated the mechanisms of cardioprotection afforded by K/Mg cardioplegia [3–8]. We speculated that NHE-1 inhibition with HOE-642 used in concert with K/Mg cardioplegia would provide for additive cardioprotection by further decreasing infarct size more than that achieved by K/Mg cardioplegia alone. However, our results indicate that the addition of the cardiac-specific NHE-1 inhibitor, HOE-642, before GI or at the immediate start of reperfusion (K/Mg + HOE-I, K/Mg + HOE-R, and K/Mg + HOE-IR) had no significant effect on K/Mg infarct size reduction, and significantly decreased systolic functional recovery (LVPDP) (p < 0.05 versus K/Mg) after 90 minutes of reperfusion (150 to 180 minutes of perfusion).

These results are at variance with previous reports indicating that HOE-642 provided enhanced cardioprotection when used in combination with St. Thomas solution [19, 20]. Shipolini and coworkers [19] reported that HOE-642 when used as an adjunct to St. Thomas solution significantly improved aortic flow and significantly decreased creatine kinase leakage in the isolated rat heart as compared with St. Thomas solution alone. Tritto and coworkers [20] also reported that in the isolated rat heart that the addition of HOE-642 to St. Thomas solution significantly increased left ventricular developed pressure and significantly decreased lactate dehydrogenase release. The differences in the data in our report and those of the other studies [19, 20] may reflect species differences; for example, the isolated rat heart has been shown previously to respond differently to cardioprotective protocols as compared with the rabbit, dog, and pig heart [21–24]. In addition, differences in reperfusion times (45 and 90 minutes used in these earlier reports as compared with our 120 minutes of reperfusion) may also contribute to the differences observed [19, 20].

Investigations using the isolated rabbit heart are limited, but Koike and coworkers [25] reported that the use of the NHE inhibitor, amiloride, with St. Thomas solution significantly increased LVPDP at 30 minutes of reperfusion as compared with St. Thomas solution alone. They used a 2-minute perfusion of St. Thomas solution before 45 minutes of GI and 50 minutes of reperfusion. In contrast, we used a 5-minute perfusion of K/Mg cardioplegia before 30 minutes of GI and 120 minutes of reperfusion. These times were chosen so that infarct size could be properly determined and for the optimal maintenance of the isolated rabbit heart preparation. Near maximal protection is afforded with K/Mg cardioplegia in this protocol [1–4]. Our data indicated that LVPDP in hearts treated with K/Mg cardioplegia was 105 mm Hg (99% of equilibrium) at 30 minutes of reperfusion as compared with 55 mm Hg (76% of equilibrium) as reported by Koike and coworkers [25] with St. Thomas solution. Our data also showed that LVPDP in K/Mg hearts in which HOE-642 was perfused for 15 minutes before ischemia was 90 mm Hg (90% of equilibrium) at 30 minutes of reperfusion as compared with 71 mm Hg (99% of equilibrium) at 30 minutes of reperfusion with amiloride in combination with St. Thomas solution [25].

Controversy exists as to whether NHE-1 inhibition affords greater cardioprotection during ischemia or during reperfusion [26, 27]. Karmazyn [26] reported that in the isolated perfused rat heart cardioprotection afforded by NHE-1 inhibition is greater when used before ischemia than during reperfusion. Hurtado and Pierce [27], however, recently showed that in the isolated beating rat cardiomyocyte, NHE-1 inhibition during reperfusion affords superior cardioprotection as compared with ischemia and reperfusion. Our results indicated that maximal cardioprotection was achieved when HOE-642 was used both before ischemia and during reperfusion. These differences may be associated with species differences or the effects of in vivo versus in vitro cardiomyocyte function.

The mechanism by which NHE-1 inhibition decreases LVPDP in K/Mg cardioprotection during late reperfusion remains to be elucidated. It has been proposed that NHE-1 modulates ischemia/reperfusion injury through the extracellular transport of H+ in exchange for Na+ influx. Intracellular Na+ is then exchanged with Ca2+ through the action of the sarcolemmal Na+-Ca2+ exchange, with Na+ being transported to extracellular space and Ca2+ being taken up into the cytosol, resulting in increased intracellular Ca2+ accumulation [9, 11, 17, 18]. Upon reperfusion, as extracellular H+ rapidly decreases, a large intracellular-to-extracellular H+ gradient activates NHE-1, and NHE-1 actively restores intracellular pH, which is accompanied by Na+ influx resulting in intracellular Ca2+ overload that contributes to myocardial contracture and reperfusion arrhythmias [28].

Previous reports suggest that decreased intracellular Ca2+ accumulation by NHE-1 inhibition is achieved by decreased intracellular pH. Hendrikx and associates [29] reported that NHE-1 inhibition significantly delayed the recovery of intracellular pH to 6.97 ± 0.02 as compared with 7.14 ± 0.05 in GI hearts during the initial 5 minutes of reperfusion, and that was associated with reduced Ca2+ overload examined by electron microscopy with Ca2+ staining in the isolated rabbit heart. NHE-1 inhibition decreases intracellular pH and decreases intracellular Ca2+ by blocking H+ efflux and Na+ influx, resulting in decreased pH and inactivation of Na+-Ca2+ exchanger leading to the prevention of Ca2+ influx [9].

We have shown previously that K/Mg cardioprotection is associated with the attenuation of intracellular acidosis, and that K/Mg cardioplegia significantly attenuates intracellular acidosis during ischemia and reperfusion with intracellular pH 6.64 ± 0.05 at 30 minutes ischemia and 7.26 ± 0.09 at 30 minutes reperfusion as compared with 6.46 ± 0.05 and 7.08 ± 0.04, respectively, in GI hearts [4]. In the present study, we did not measure intracellular pH; however, we speculate that the addition of HOE-642 in combination with K/Mg cardioplegia decreases intracellular pH during ischemia and reperfusion. Support for this speculation comes from Koike and associates [25], who showed that NHE-1 inhibition in combination with St. Thomas solution decreased intracellular pH both during ischemia and reperfusion as compared with St. Thomas solution alone. We further speculate that the decrease in intracellular pH with HOE-642 in combination with the decrease in intracellular Ca2+ with K/Mg cardioplegia would decrease ventricular contractile function as a result of decreased Ca2+ sensitivity of the contractile elements during acidosis [30–32]. Previous reports have shown that intracellular acidosis decreases myofilament Ca2+ sensitivity [31], Ca2+ affinity of troponin C [32], and absolute cross-bridge formation [31]. These data would support our results indicating that NHE-1 inhibition acts negatively on K/Mg-enhanced postischemic functional recovery.

In this study we investigated the effect of NHE-1 inhibition in the isolated buffer-perfused Langendorff heart model and therefore the effects of neutrophils and plasma-borne inflammatory components on cardioprotection were not assessed. Recently, NHE-1 inhibition has been shown to attenuate neutrophil accumulation and activation [33]. In earlier reports, we showed that the beneficial effects of K/Mg cardioplegia in reducing infarct size reduction and enhancing postischemic functional recovery are preserved in the in situ blood-perfused heart model [1, 2]. However, the role of NHE-1 inhibition in the cardioprotection afforded by K/Mg cardioplegia in a blood perfused model remains to be elucidated.

Our results show that NHE-1 inhibition when used alone provides cardioprotection with optimal effects being observed when used both before ischemia and during reperfusion (HOE-IR). NHE-1 inhibition with K/Mg cardioplegia decreases postischemic functional recovery during late reperfusion with no significant effect on infarct size being observed as compared with K/Mg cardioplegia alone.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
This study was supported by the National Institutes of Health (grant nos. HL29077, HL 59542) and the American Heart Association.

The authors thank Dr Andreas Weichert and Dr Jürgen Pünter of Aventis Pharma, Deutschland GmbH, Frankfurt, Germany for their efforts on our behalf in obtaining HOE 642.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

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