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Deletion of Endothelial Nitric Oxide Synthase Exacerbates Myocardial Stunning in an Isolated Mouse Heart Model
Journal of Surgical Research 93, 127–132 (2000) doi:10.1006/jsre.2000.5953, available online at http://www.idealibrary.com on
Deletion of Endothelial Nitric Oxide Synthase Exacerbates Myocardial Stunning in an Isolated Mouse Heart Model R. L. Hannan, M.D.,* ,1 M. C. John, B.A., P. C. Kouretas, M.D., Ph.D., B. D. Hack, M.D.,† G. P. Matherne, M.D.,† and V. E. Laubach, Ph.D. †Departments of Pediatrics and Surgery and the Cardiovascular Research Center University of Virginia, Charlottesville, Virginia; and *Division of Cardiovascular Surgery, Miami Children’s Hospital, Miami, Florida Submitted for publication January 21, 2000
Background. While endothelial nitric oxide synthase (eNOS) is an important regulator of vascular tone, it is also constitutively expressed in cardiac myocytes and contributes to the regulation of myocardial function. The role of eNOS in ischemia–reperfusion is uncertain, however, with some studies showing beneficial effects while other studies demonstrate increased cardiac injury. We hypothesized that the beneficial effects of eNOS would predominate, and thus that targeted deletion of eNOS would exacerbate myocardial dysfunction following ischemia–reperfusion. Materials and methods. ENOS knockout and wildtype mouse hearts were Langendorff-perfused using Krebs bicarbonate buffer and subjected to 20 min of global normothermic ischemia followed by 30 min of reperfusion. Myocardial function was measured using a ventricular balloon to determine time to onset of contracture, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and rate–pressure product (RPP). Results. Heart rate and coronary resistance were similar in both groups during baseline and reperfusion periods. Diastolic function as determined by peak LVEDP during ischemia and final LVEDP after reperfusion were worse in the eNOS knockout group vs wild-type (114 and 31 mmHg vs 92 and 18 mmHg, P < .05). Although RPP (heart rate ⴛ LVDP), measured as an index of systolic function, was initially better in eNOS knockouts (24216 vs 16353), wild-type hearts recovered more function than did eNOS knockout hearts by the end of 30 min of reperfusion (30892 vs 20522, P < .05). Conclusions. These data suggest that the deletion of eNOS results in increased myocardial dysfunction 1 To whom correspondence should be addressed at Division of Cardiovascular Surgery, Miami Children’s Hospital, 3200 SW 60 th Court Suite 102, Miami, FL 33155. Fax: 305-669-6574. E-mail: [email protected].
following ischemia–reperfusion in an isolated heart model. © 2000 Academic Press Key Words: endothelial nitric oxide synthase; ischemia-reperfusion; myocardial stunning. INTRODUCTION
Brief episodes of myocardial ischemia followed by reperfusion (ischemia–reperfusion) are common clinical events, occurring in cardiac surgery, interventional cardiology, and in coronary artery disease, and frequently result in some degree of myocardial dysfunction. The impaired release of nitric oxide (NO) has been implicated as one of the various mechanisms contributing to this ischemia–reperfusion injury. Endothelial nitric oxide synthase (eNOS) is the primary nitric oxide synthase (NOS) isoform that has been shown to be constitutively expressed not only in the endothelial cells of arteries, veins and capillaries, but in cardiac myocytes as well [1–3]. Newly emerging evidence suggests a role of endogenous NO in the regulation of myocardial function, in addition to the widely understood function of NO as an endothelium-derived relaxant of vascular smooth muscle. eNOS express has been documented in cardiac sinoatrial, atrioventricular and ventricular myocytes from several rodent species [1– 4]. Compelling evidence has also demonstrated eNOS expression in atrial and ventricular myocytes from human hearts [5, 6]. Recent studies suggest that NO in the myocyte functions as an important counterregulator of various biological processes. NO’s regulation of Ca 2⫹ homeostasis [7, 8], inhibition of adrenoreceptor agonists [9], and suppression of the toxic effects of endothelin-1 [10] have all been shown to improve myocardial function. Moreover, NO has been shown to reduce ischemic contracture and
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reperfusion injury by virtue of its negative inotropic properties, possibly through myofilament desensitization to Ca 2⫹ [11] and stimulation of myocardial relaxation through the elevation of cGMP levels [12]. In other experiments, blockade of NO improved myocardial function following ischemia–reperfusion. NO can react with superoxide to produce peroxynitrite, a toxic oxidant that has been shown to induce cellular damage in cultured myocardial tissue [13]. NO can also cause myocardial dysfunction by impeding myocardial energetics. Relevant mechanisms capable of myocardial damage include S-nitrosylation of thiols, formation of nitrotyrosines [14], and inactivation of glyceraldehyde-3-phosphate dehydrogenase by covalent linkage to NAD or NADH [15]. Moreover, it has been suggested that NO can hinder glycolysis by binding to heme-containing proteins in the mitochondrial respiratory chain [16, 17], thereby reducing the amount of ATP available for contractile work. Thus, the precise role of NO in myocardial function, much less myocardial ischemia–reperfusion, remains unclear. Although a significant number of studies have examined the role of eNOS and NO in the myocyte, many of them either involved the administration of supraphysiological levels of NO or NO precursors. Those that sought to examine myocardial function in the absence of NO usually did so by inhibiting NO synthases using substances like N G-nitro-L-arginine methyl ester (L-NAME), which can themselves cause adverse side effects [18]. The engineering of a mouse with the engineered deletion of the eNOS gene (eNOS knockout) has afforded us a unique approach to studying the effect of NO on the myocardium following ischemia–reperfusion. Our protocol allows us to compare the function of a heart exhibiting physiological levels of NO against one which is exhibiting absolutely no eNOS-derived NO. Differences in function could then be directly linked to the presence or absence of physiological levels of eNOS-derived NO in the myocyte. Maintaining constant coronary flow throughout the experiment also minimizes the interference of vascular effects of eNOS deletion. The aim of the present study was to examine the effects of targeted deletion of the eNOS gene on myocardial functional recovery following ischemia–reperfusion. It was our hypothesis that the beneficial effects of NO on myocardial function would outweigh the possible deleterious effects at physiological levels, and that in comparison to wildtype hearts, deletion of the eNOS gene would exacerbate the injury associated with myocardial ischemia– reperfusion. MATERIALS AND METHODS eNOS knockout mice. Gene targeting was employed to engineer mice homozygous (⫺/⫺) for disruption of the eNOS gene [19]. Immunohistochemical staining with anti-eNOS antibodies showed an ab-
sence of eNOS proteins in (⫺/⫺) mice. These eNOS knockout mice have undergone seven generations of backcrossing onto the C57BL/6 mouse strain. At present, these mice are essentially an inbred strain comprising a homogeneous genetic background. Therefore the eNOS knockout mice can be directly compared to each other as well as to control wild-type C57BL/6 mice. Langendorff heart model. Male and female mice were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg), a thoracotomy was performed, and the hearts were excised into ice-cold buffer. The aorta was rapidly cannulated with a 20-gauge needle and retrograde coronary perfusion initiated with a modified Krebs bicarbonate buffer containing (in mM): NaCl, 118; NaHCO 3, 25; KCL, 4.7; KH 2PO 4, 1.2; CaCl 2, 2.5; MgSO 4, 1.2; glucose, 10.7; and EDTA, 0.6. Coronary flow was established during the baseline equilibration period to establish an afterload of approximately 80 mmHg using a Masterflex pump, and held constant thereafter. The perfusate was equilibrated with 95% O 2, 5% CO 2 at 37°C, giving a pH of 7.4. Hearts were bathed in perfusate within a water-jacketed bath maintained at 37°C. The left ventricle was vented with a polyethylene apical drain. Coronary perfusion was monitored using an ultrasonic flow probe (Transonic Systems, Inc., Ithaca, NY) in the aortic perfusion line. A small “cling film” isovolumic balloon was inserted into the left ventricle and attached to a pressure transducer. Apicobasal displacement was continuously recorded with a MacLab four-channel data acquisition unit (AD Instruments, Castle Hill, Australia) to yield heart rate and developed pressure. Ischemia–reperfusion protocol. All hearts were equilibrated, unpaced, for 30 min. Subsequently, hearts underwent 20 min of global normothermic ischemia. Arresting coronary flow and simultaneously bubbling 95% N 2 /5% CO 2 through the bathing perfusate to reduce pO 2 produced global ischemia. Reperfusion was achieved by reinitiating coronary flow and discontinuing the nitrogen bubbling. Hearts were then reperfused for 30 min. Cardiac parameters were monitored continuously and included coronary flow rate (CF), heart rate (HR), aortic pressure (AoP), peak systolic pressure (LVP), end diastolic pressure (EDP), left ventricular developed pressure (LVDP; difference between left ventricular peak systolic pressure and end diastolic pressure), and rate pressure product (RPP; HR ⫻ LVDP). Time to onset of ischemic contracture was assessed as the duration of time during ischemia to a 20 mmHg rise in EDP. Two experimental groups were examined, (1) wild-type control hearts and (2) eNOS knockout hearts. Determination of coronary effluent lactate dehydrogenase content. To assess degree of cell death, coronary venous effluent was collected from the pulmonary artery for quantitation of lactate dehydrogenase (LDH) release. To determine whether the preconditioning stimuli alone resulted in enzyme release, a sample was collected immediately before the onset of sustained global ischemia. Following 20 min of global ischemia and 30 min of reperfusion, two aliquots of coronary effluent were collected. Samples were stored at ⫺20°C until analysis. An enzymatic assay (Sigma, St. Louis) was optimized for sensitivity of detection and spectrophotometric analysis (Shimadzu) was used for determination of LDH concentration in each aliquot. An index of total LDH release during reperfusion was calculated by multiplying the enzyme concentration in each aliquot by the duration of its collection. Total LDH release was normalized for wet heart weight [20]. Data analysis and statistical comparisons. Baseline statistics refer to myocardial function just before the onset of ischemia, after 30 min normoxic perfusion. peak ischemic contracture refers to the peak diastolic pressure of the asystolic heart. All analyses were performed using two-way ANOVA for repeated measures with Student– Neuman–Keuls post hoc test. Statistical significance was accepted for P ⬍ 0.05.
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TABLE 1
CF (nL/min/g) AoP (mmHg) LVP (mmHg) EDP (mmHg) LVDP (mmHg) HR (BMP) RPP (mmHg/min) Body wt (g)
Wild-type (n ⫽ 8)
ENOS knockout (n ⫽ 10)
P value*
23.3 ⫾ 4.9 85 ⫾ 3 119 ⫾ 7 5⫾1 114 ⫾ 7 349 ⫾ 11 39806 ⫾ 3015 24.99 ⫾ 0.6
22 ⫾ 2 79 ⫾ 3 118 ⫾ 7 4⫾1 115 ⫾ 7 349 ⫾ 8 40038 ⫾ 2491 32.69 ⫾ 1.85
NS NS NS NS NS NS NS P ⬍ 0.003
Note. All means are ⫾ SEM. CF, coronary flow; AoP, aortic pressure; LVP, left ventricular (systolic) pressure; EDP, end diastolic pressure; LVDP, left ventricular developed pressure; HR, (intrinsic) heart rate; RPP, rate pressure product; Body wt, body weight of animal. *Unpaired t test.
RESULTS
Isolated Hearts Baseline parameters. Baseline functional data for wild-type hearts (n ⫽ 8, 24.99 ⫾ 0.6 g) and eNOS knockout hearts (n ⫽ 10, 32.69 ⫾ 1.85 g) are shown in Table 1. No differences in any baseline functional parameters were observed between wild-type and eNOS knockout hearts. Functional effects of ischemia–reperfusion. The effects on aortic pressure and heart rate resulting from deletion of eNOS in the post-ischemic isolated heart are shown in Figs. 1 and 2. At constant flow and unpaced, eNOS deficiency did not alter aortic pressure or heart rate during ischemia or reperfusion. Global normothermic ischemia abolished heart rate and contractile function in all hearts within 5 min and then caused a rapid increase in diastolic tone. Time to ischemic contracture (time to a 20 mmHg rise in dia-
FIG. 1. Aortic pressure during 20 min of global normothermic ischemia and 30 min of reperfusion. Time 30 marks the onset of ischemia. Wild-type control (n ⫽ 8), eNOS knockout (n ⫽ 10). Values are means ⫾ SEM. No significant differences were noted between groups.
FIG. 2. Heart rate during 20 min of global normothermic ischemia and 30 min of reperfusion. Time 30 marks the onset of ischemia. Wild-type control (n ⫽ 8), eNOS knockout (n ⫽ 10). Values are means ⫾ SEM. No significant differences were noted between groups.
stolic pressure) was 9.8 ⫾ 1.2 min in wild-type and 8.1 ⫾ 0.8 min in eNOS knockout hearts. Functional recovery expressed as rate pressure product (heart rate X developed pressure) is shown in Fig. 3. With the onset of global ischemia, all hearts demonstrated an immediate decline in rate pressure product and full arrest within 2 to 3 min. Upon reperfusion, hearts resumed spontaneous contraction within 1 to 2 min. After an initial increased recovery lasting 2 to 3 min, all hearts demonstrated a rapid decline in function followed by a progressive recovery of rate pressure product for the remainder of reperfusion. The eNOS knockout group (Fig. 3, open symbols), demonstrated an improved initial recovery of function compared to the wild-type group within 2 min of reperfusion (24217 ⫾ 2405 vs 17082 ⫾ 786). This recovery
FIG. 3. Rate pressure product (heart rate ⫻ left ventricular developed pressure) during 20 min of global normothermic ischemia and 30 min of reperfusion. Time 30 marks the onset of ischemia. Wild-type control (n ⫽ 8), eNOS knockout (n ⫽ 10). Values are means ⫾ SEM; *Significant differences from the wild-type group, P ⬍ 0.05.
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FIG. 4. End diastolic pressure at baseline (30 min perfusion), peak contracture (peak diastolic pressure of the asystolic heart), and final point. Values are means ⫾ SEM; *Significant differences from the wild-type group, P ⬍ 0.05.
was followed by a rapid decline in rate pressure product, with the end result of the wild-type group displaying a marked improvement in functional recovery at 10, 20, and 30 min reperfusion (Fig. 3). Baseline, peak, and final diastolic pressure is shown in Fig. 4. Baseline diastolic pressure was similar in both groups. Peak and final diastolic pressure in the eNOS knockout group was 114 ⫾ 5 and 29 ⫾ 7, significantly higher than that of the wild-type group, which was 95 ⫾ 8 and 18 ⫾ 3 mmHg (P ⬍ .05). Determinants of Tissue Viability Lactate dehydrogenase release. There were no significant differences in total efflux of LDH, and thus cell death, between the two groups (Fig. 5). DISCUSSION
In the present study, with the use of an isolated heart preparation that minimized vascular effects, we found that the deletion of eNOS resulted in significantly depressed LVDP and RPP following ischemia– reperfusion. The eNOS knockout model also suffered more pronounced levels of left ventricular end-diastolic pressure (LVEDP) dysfunction. These data provide compelling evidence that deletion of the major NOS isoform responsible for NO production in the heart (eNOS) results in a pronounced increase in myocardial stunning following ischemia–reperfusion. Moreover, this dysfunction occurs without affecting the rate of cardiac necrosis. The role of NO on myocardial contractility is controversial [21–27]. Gross et al. [22] have reported a decline in cardiac contractile reserve following infusion of the NO donor SNAC, possibly through depression of high energy phosphate metabolism. Hattler et al. [26] have
demonstrated increased myocardial stunning with elevations in NO production in the human heart in the postpulmonary bypass interval, suggesting a link between NO production and myocardial depression. Moreover, Brady et al. [21] have shown that the NO donor sodium nitroprusside when perfused over guinea pig myocytes resulted in depressed myocardial contractility. This decrease in contraction amplitude was reversed by methylene blue, an inhibitor of cGMP (a mediator in the NO pathway). In contrast, Pabla et al. [24] previously reported that in the isolated rat model exogenous NO infusion significantly improved both LVDP and RPP, while preventing elevations in LVEDP. Inhibition of NO using L-NAME exacerbated contractile dysfunction. Furthermore, work by Hasebe et al. [23] showed that inhibition of endogenous NO synthesis by use of L-NAME enhances myocardial stunning. In another study, Weyrich et al. [25] investigated the effects of physiological concentrations of exogenous NO on myocardial contractility in isolated rat cardiomyocytes. These investigators found that physiologically relevant concentrations of NO do not induce physiologically significant negative inotropic effects in the cat or rat heart. These findings are in agreement with the present study, that in fact, endogenous NO synthesis in the myocyte does not induce a negative inotropic effect with ischemia–reperfusion and that endogenous eNOS-derived NO may be important for maintaining normal myocardial contractility. A recent study of ischemia–reperfusion in isolated mouse hearts using eNOS knockout mice by Flogel et al. [27] demonstrated increased ischemia–reperfusion injury in wild-type mice and a protective effect of eNOS deletion on ischemia–reperfusion injury in eNOS knockout mice. Significant differences in the protocol used in this study and that reported by Flogel include a significantly longer period of reperfusion (60 versus 30 min) and a slighter shorter period of ischemia (16 versus 30 min). It may be that the effect of eNOS deletion may vary during the time period of reperfusion: during early reperfusion, the protective effect of
FIG. 5. Determinants of tissue viability assessed by total efflux of LDH. Values are means ⫾ SEM. No significant differences were noted between groups.
HANNAN ET AL.: EFFECTS OF DELETION OF eNOS IN HEART MODEL
NO may predominate, while in late reperfusion NO may increase reperfusion injury. In our own data, function in the eNOS knockout group was better than control at 2 min reperfusion but decreased compared to control at all later points. Alternatively, it is possible that during pathological conditions where NO might be produced via the inducible NOS pathway, or when NO is administered at supraphysiological levels, high levels of NO might contribute to the cardiac dysfunction associated with myocardial stunning. Xie et al. [28] have speculated that elevated levels of NO may retard mitochondrial respiration, resulting in a decrease in the ATP synthesis needed for force development, resulting in a negative inotropic effect. It is possible that these opposing observations may be attributed to differential effects on the myocyte to NO at low and high concentrations. The worsening of diastolic function we witnessed in the eNOS knockout group is consistent with a number of studies, which correlated NO in the myocyte with improved myocardial relaxation. Grocott-Mason et al. [12] found that exogenous NO exerts direct myocardial relaxant effects in the isolated guinea pig heart, independent of NO’s known vasodilator activity. NO has been shown to raise intracellular cGMP levels and this, in turn, desensitizes the myofilament response to Ca 2⫹ [11]. Thus, the removal of eNOS-derived NO from the myocardium might eliminate an important cGMP counterregulator. If NO is not present to modulate cGMP elevations, CA 2⫹ influx may go unchecked, resulting in the degree of calcium loading that has been associated with myocardial stunning. It is possible that blocking this mechanism as we did in our experiment through the deletion of eNOS-derived NO contributed to enhanced myocardial stunning. NO may also be involved in optimizing O 2 consumption, as suggested by Xie et al. [28]. They found that exogenous NO infusion using SNAP attenuated O 2 consumption in myocardial tissue. In a study with similar findings, Shen et al. [29] demonstrated that NO inhibition using NLA caused marked elevations in O 2 consumption and decreases in cardiac output in conscious dogs. However, possible adverse actions of this mechanism are likely under pathological conditions where elevated and sustained NO levels (such as might be produced by inducible NOS) could impede the O 2dependent production of ATP. Thus, NO’s effect on O 2 consumption at high concentrations might well result in the negative inotropic effect witnessed in some studies. It is our speculation that at the onset of reperfusion and in the period up to approximately 5 min thereafter, the rapid influx of O 2 into the myocyte causes the rapid and excessive production of NO via eNOS. This increase in NO concentration to well above the physiological threshold causes a transient period of myocardial depression, which dissipates after a prolonged
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period of reperfusion. This mechanism would explain the apparent enhanced myocardial contractility of the eNOS knockout group during the brief initial stage of reperfusion. The eNOS knockouts, lacking the NOS isoform capable of producing such rapid NO elevations in the myocardium, would be unable to experience the pathological levels of NO that might be detrimental to post-ischemic cardiac function. Nevertheless, as reperfusion continues the response to sudden influx O 2 (increased rate of NO synthesis) disappears, and physiological levels of NO synthesis are restored, as are more physiological levels of myocardial contractility. In future studies the rate of NO synthesis in the myocardium as a function of reperfusion duration might yield data that would further elucidate NO’s role on contractility. Chief among NO’s effects believed to be harmful to myocardial function is the possible formation of peroxynitrite, a toxic free radical species. In an investigation by Schulz and Wambolt [30], some free radical species, possibly peroxynitrite, was found to contribute to the myocardial dysfunction associated with ischemia–reperfusion. In contrast with these and other reports, some studies have found that NO may be beneficial to certain physiological processes. In fact, Lefer et al. [31] showed that peroxynitrite reduced the degree of neutrophil-induced myocardial dysfunction after ischemia and reperfusion of the myocardium. However, it is important to note that peroxynitrite is the reaction product of NO and superoxide at equimolar concentrations of these reactants, limiting the maximally achievable concentration of ONOO ⫺ to the highest amount of NO or superoxide present in the myocardium. Basal concentrations of NO occur in the 1–20 nM range [32] and are believed to increase to maximal levels in the low micromolar range (2–5 M) in pathological states where inducible NOS is activated [31]. Consequently, the maximal achievable levels of peroxynitrite that can be formed in vivo would also be in the low micromolar range (2–5 M). Yet the concentration of peroxynitrite necessary to produce lipid peroxidation is well above this level (100 M–1 mM) [33]. Along with its short life span (⬇1.9 s) [13] and the supraphysiological levels of peroxynitrite necessary to exert significant toxic effects, it is highly unlikely for endogenous NO-mediated production of peroxynitrite to play a significant role in myocardial stunning. In conclusion, deletion of eNOS caused impaired diastolic function during ischemia and significant depression of myocardial contractility following ischemia–reperfusion in the isolated mouse heart. Furthermore, this occurred without altering cell viability. Previous reports have for the most part suggested a negative inotropic effect from NO. In that case, eliminating NO should result in improved, not worsened, contractility following ischemia–reperfusion. We suggest that high, nonphysiologic levels of NO might elicit
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the negative inotropic effects found in these studies, but that lower, physiologic endogenous levels do not. Indeed, in light of the depressed contractility of the eNOS knockout animals, our data suggest that eNOSderived NO is in fact cardioprotective at endogenous levels. The lack of eNOS-derived NO appears to increase myocardial dysfunction following ischemia– reperfusion in this isolated mouse model.
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Deletion of Endothelial Nitric Oxide Synthase Exacerbates Myocardial Stunning in an Isolated Mouse Heart Model R. L. Hannan, M.D.,* ,1 M. C. John, B.A., P. C. Kouretas, M.D., Ph.D., B. D. Hack, M.D.,† G. P. Matherne, M.D.,† and V. E. Laubach, Ph.D. †Departments of Pediatrics and Surgery and the Cardiovascular Research Center University of Virginia, Charlottesville, Virginia; and *Division of Cardiovascular Surgery, Miami Children’s Hospital, Miami, Florida Submitted for publication January 21, 2000
Background. While endothelial nitric oxide synthase (eNOS) is an important regulator of vascular tone, it is also constitutively expressed in cardiac myocytes and contributes to the regulation of myocardial function. The role of eNOS in ischemia–reperfusion is uncertain, however, with some studies showing beneficial effects while other studies demonstrate increased cardiac injury. We hypothesized that the beneficial effects of eNOS would predominate, and thus that targeted deletion of eNOS would exacerbate myocardial dysfunction following ischemia–reperfusion. Materials and methods. ENOS knockout and wildtype mouse hearts were Langendorff-perfused using Krebs bicarbonate buffer and subjected to 20 min of global normothermic ischemia followed by 30 min of reperfusion. Myocardial function was measured using a ventricular balloon to determine time to onset of contracture, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and rate–pressure product (RPP). Results. Heart rate and coronary resistance were similar in both groups during baseline and reperfusion periods. Diastolic function as determined by peak LVEDP during ischemia and final LVEDP after reperfusion were worse in the eNOS knockout group vs wild-type (114 and 31 mmHg vs 92 and 18 mmHg, P < .05). Although RPP (heart rate ⴛ LVDP), measured as an index of systolic function, was initially better in eNOS knockouts (24216 vs 16353), wild-type hearts recovered more function than did eNOS knockout hearts by the end of 30 min of reperfusion (30892 vs 20522, P < .05). Conclusions. These data suggest that the deletion of eNOS results in increased myocardial dysfunction 1 To whom correspondence should be addressed at Division of Cardiovascular Surgery, Miami Children’s Hospital, 3200 SW 60 th Court Suite 102, Miami, FL 33155. Fax: 305-669-6574. E-mail: [email protected].
following ischemia–reperfusion in an isolated heart model. © 2000 Academic Press Key Words: endothelial nitric oxide synthase; ischemia-reperfusion; myocardial stunning. INTRODUCTION
Brief episodes of myocardial ischemia followed by reperfusion (ischemia–reperfusion) are common clinical events, occurring in cardiac surgery, interventional cardiology, and in coronary artery disease, and frequently result in some degree of myocardial dysfunction. The impaired release of nitric oxide (NO) has been implicated as one of the various mechanisms contributing to this ischemia–reperfusion injury. Endothelial nitric oxide synthase (eNOS) is the primary nitric oxide synthase (NOS) isoform that has been shown to be constitutively expressed not only in the endothelial cells of arteries, veins and capillaries, but in cardiac myocytes as well [1–3]. Newly emerging evidence suggests a role of endogenous NO in the regulation of myocardial function, in addition to the widely understood function of NO as an endothelium-derived relaxant of vascular smooth muscle. eNOS express has been documented in cardiac sinoatrial, atrioventricular and ventricular myocytes from several rodent species [1– 4]. Compelling evidence has also demonstrated eNOS expression in atrial and ventricular myocytes from human hearts [5, 6]. Recent studies suggest that NO in the myocyte functions as an important counterregulator of various biological processes. NO’s regulation of Ca 2⫹ homeostasis [7, 8], inhibition of adrenoreceptor agonists [9], and suppression of the toxic effects of endothelin-1 [10] have all been shown to improve myocardial function. Moreover, NO has been shown to reduce ischemic contracture and
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reperfusion injury by virtue of its negative inotropic properties, possibly through myofilament desensitization to Ca 2⫹ [11] and stimulation of myocardial relaxation through the elevation of cGMP levels [12]. In other experiments, blockade of NO improved myocardial function following ischemia–reperfusion. NO can react with superoxide to produce peroxynitrite, a toxic oxidant that has been shown to induce cellular damage in cultured myocardial tissue [13]. NO can also cause myocardial dysfunction by impeding myocardial energetics. Relevant mechanisms capable of myocardial damage include S-nitrosylation of thiols, formation of nitrotyrosines [14], and inactivation of glyceraldehyde-3-phosphate dehydrogenase by covalent linkage to NAD or NADH [15]. Moreover, it has been suggested that NO can hinder glycolysis by binding to heme-containing proteins in the mitochondrial respiratory chain [16, 17], thereby reducing the amount of ATP available for contractile work. Thus, the precise role of NO in myocardial function, much less myocardial ischemia–reperfusion, remains unclear. Although a significant number of studies have examined the role of eNOS and NO in the myocyte, many of them either involved the administration of supraphysiological levels of NO or NO precursors. Those that sought to examine myocardial function in the absence of NO usually did so by inhibiting NO synthases using substances like N G-nitro-L-arginine methyl ester (L-NAME), which can themselves cause adverse side effects [18]. The engineering of a mouse with the engineered deletion of the eNOS gene (eNOS knockout) has afforded us a unique approach to studying the effect of NO on the myocardium following ischemia–reperfusion. Our protocol allows us to compare the function of a heart exhibiting physiological levels of NO against one which is exhibiting absolutely no eNOS-derived NO. Differences in function could then be directly linked to the presence or absence of physiological levels of eNOS-derived NO in the myocyte. Maintaining constant coronary flow throughout the experiment also minimizes the interference of vascular effects of eNOS deletion. The aim of the present study was to examine the effects of targeted deletion of the eNOS gene on myocardial functional recovery following ischemia–reperfusion. It was our hypothesis that the beneficial effects of NO on myocardial function would outweigh the possible deleterious effects at physiological levels, and that in comparison to wildtype hearts, deletion of the eNOS gene would exacerbate the injury associated with myocardial ischemia– reperfusion. MATERIALS AND METHODS eNOS knockout mice. Gene targeting was employed to engineer mice homozygous (⫺/⫺) for disruption of the eNOS gene [19]. Immunohistochemical staining with anti-eNOS antibodies showed an ab-
sence of eNOS proteins in (⫺/⫺) mice. These eNOS knockout mice have undergone seven generations of backcrossing onto the C57BL/6 mouse strain. At present, these mice are essentially an inbred strain comprising a homogeneous genetic background. Therefore the eNOS knockout mice can be directly compared to each other as well as to control wild-type C57BL/6 mice. Langendorff heart model. Male and female mice were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg), a thoracotomy was performed, and the hearts were excised into ice-cold buffer. The aorta was rapidly cannulated with a 20-gauge needle and retrograde coronary perfusion initiated with a modified Krebs bicarbonate buffer containing (in mM): NaCl, 118; NaHCO 3, 25; KCL, 4.7; KH 2PO 4, 1.2; CaCl 2, 2.5; MgSO 4, 1.2; glucose, 10.7; and EDTA, 0.6. Coronary flow was established during the baseline equilibration period to establish an afterload of approximately 80 mmHg using a Masterflex pump, and held constant thereafter. The perfusate was equilibrated with 95% O 2, 5% CO 2 at 37°C, giving a pH of 7.4. Hearts were bathed in perfusate within a water-jacketed bath maintained at 37°C. The left ventricle was vented with a polyethylene apical drain. Coronary perfusion was monitored using an ultrasonic flow probe (Transonic Systems, Inc., Ithaca, NY) in the aortic perfusion line. A small “cling film” isovolumic balloon was inserted into the left ventricle and attached to a pressure transducer. Apicobasal displacement was continuously recorded with a MacLab four-channel data acquisition unit (AD Instruments, Castle Hill, Australia) to yield heart rate and developed pressure. Ischemia–reperfusion protocol. All hearts were equilibrated, unpaced, for 30 min. Subsequently, hearts underwent 20 min of global normothermic ischemia. Arresting coronary flow and simultaneously bubbling 95% N 2 /5% CO 2 through the bathing perfusate to reduce pO 2 produced global ischemia. Reperfusion was achieved by reinitiating coronary flow and discontinuing the nitrogen bubbling. Hearts were then reperfused for 30 min. Cardiac parameters were monitored continuously and included coronary flow rate (CF), heart rate (HR), aortic pressure (AoP), peak systolic pressure (LVP), end diastolic pressure (EDP), left ventricular developed pressure (LVDP; difference between left ventricular peak systolic pressure and end diastolic pressure), and rate pressure product (RPP; HR ⫻ LVDP). Time to onset of ischemic contracture was assessed as the duration of time during ischemia to a 20 mmHg rise in EDP. Two experimental groups were examined, (1) wild-type control hearts and (2) eNOS knockout hearts. Determination of coronary effluent lactate dehydrogenase content. To assess degree of cell death, coronary venous effluent was collected from the pulmonary artery for quantitation of lactate dehydrogenase (LDH) release. To determine whether the preconditioning stimuli alone resulted in enzyme release, a sample was collected immediately before the onset of sustained global ischemia. Following 20 min of global ischemia and 30 min of reperfusion, two aliquots of coronary effluent were collected. Samples were stored at ⫺20°C until analysis. An enzymatic assay (Sigma, St. Louis) was optimized for sensitivity of detection and spectrophotometric analysis (Shimadzu) was used for determination of LDH concentration in each aliquot. An index of total LDH release during reperfusion was calculated by multiplying the enzyme concentration in each aliquot by the duration of its collection. Total LDH release was normalized for wet heart weight [20]. Data analysis and statistical comparisons. Baseline statistics refer to myocardial function just before the onset of ischemia, after 30 min normoxic perfusion. peak ischemic contracture refers to the peak diastolic pressure of the asystolic heart. All analyses were performed using two-way ANOVA for repeated measures with Student– Neuman–Keuls post hoc test. Statistical significance was accepted for P ⬍ 0.05.
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TABLE 1
CF (nL/min/g) AoP (mmHg) LVP (mmHg) EDP (mmHg) LVDP (mmHg) HR (BMP) RPP (mmHg/min) Body wt (g)
Wild-type (n ⫽ 8)
ENOS knockout (n ⫽ 10)
P value*
23.3 ⫾ 4.9 85 ⫾ 3 119 ⫾ 7 5⫾1 114 ⫾ 7 349 ⫾ 11 39806 ⫾ 3015 24.99 ⫾ 0.6
22 ⫾ 2 79 ⫾ 3 118 ⫾ 7 4⫾1 115 ⫾ 7 349 ⫾ 8 40038 ⫾ 2491 32.69 ⫾ 1.85
NS NS NS NS NS NS NS P ⬍ 0.003
Note. All means are ⫾ SEM. CF, coronary flow; AoP, aortic pressure; LVP, left ventricular (systolic) pressure; EDP, end diastolic pressure; LVDP, left ventricular developed pressure; HR, (intrinsic) heart rate; RPP, rate pressure product; Body wt, body weight of animal. *Unpaired t test.
RESULTS
Isolated Hearts Baseline parameters. Baseline functional data for wild-type hearts (n ⫽ 8, 24.99 ⫾ 0.6 g) and eNOS knockout hearts (n ⫽ 10, 32.69 ⫾ 1.85 g) are shown in Table 1. No differences in any baseline functional parameters were observed between wild-type and eNOS knockout hearts. Functional effects of ischemia–reperfusion. The effects on aortic pressure and heart rate resulting from deletion of eNOS in the post-ischemic isolated heart are shown in Figs. 1 and 2. At constant flow and unpaced, eNOS deficiency did not alter aortic pressure or heart rate during ischemia or reperfusion. Global normothermic ischemia abolished heart rate and contractile function in all hearts within 5 min and then caused a rapid increase in diastolic tone. Time to ischemic contracture (time to a 20 mmHg rise in dia-
FIG. 1. Aortic pressure during 20 min of global normothermic ischemia and 30 min of reperfusion. Time 30 marks the onset of ischemia. Wild-type control (n ⫽ 8), eNOS knockout (n ⫽ 10). Values are means ⫾ SEM. No significant differences were noted between groups.
FIG. 2. Heart rate during 20 min of global normothermic ischemia and 30 min of reperfusion. Time 30 marks the onset of ischemia. Wild-type control (n ⫽ 8), eNOS knockout (n ⫽ 10). Values are means ⫾ SEM. No significant differences were noted between groups.
stolic pressure) was 9.8 ⫾ 1.2 min in wild-type and 8.1 ⫾ 0.8 min in eNOS knockout hearts. Functional recovery expressed as rate pressure product (heart rate X developed pressure) is shown in Fig. 3. With the onset of global ischemia, all hearts demonstrated an immediate decline in rate pressure product and full arrest within 2 to 3 min. Upon reperfusion, hearts resumed spontaneous contraction within 1 to 2 min. After an initial increased recovery lasting 2 to 3 min, all hearts demonstrated a rapid decline in function followed by a progressive recovery of rate pressure product for the remainder of reperfusion. The eNOS knockout group (Fig. 3, open symbols), demonstrated an improved initial recovery of function compared to the wild-type group within 2 min of reperfusion (24217 ⫾ 2405 vs 17082 ⫾ 786). This recovery
FIG. 3. Rate pressure product (heart rate ⫻ left ventricular developed pressure) during 20 min of global normothermic ischemia and 30 min of reperfusion. Time 30 marks the onset of ischemia. Wild-type control (n ⫽ 8), eNOS knockout (n ⫽ 10). Values are means ⫾ SEM; *Significant differences from the wild-type group, P ⬍ 0.05.
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FIG. 4. End diastolic pressure at baseline (30 min perfusion), peak contracture (peak diastolic pressure of the asystolic heart), and final point. Values are means ⫾ SEM; *Significant differences from the wild-type group, P ⬍ 0.05.
was followed by a rapid decline in rate pressure product, with the end result of the wild-type group displaying a marked improvement in functional recovery at 10, 20, and 30 min reperfusion (Fig. 3). Baseline, peak, and final diastolic pressure is shown in Fig. 4. Baseline diastolic pressure was similar in both groups. Peak and final diastolic pressure in the eNOS knockout group was 114 ⫾ 5 and 29 ⫾ 7, significantly higher than that of the wild-type group, which was 95 ⫾ 8 and 18 ⫾ 3 mmHg (P ⬍ .05). Determinants of Tissue Viability Lactate dehydrogenase release. There were no significant differences in total efflux of LDH, and thus cell death, between the two groups (Fig. 5). DISCUSSION
In the present study, with the use of an isolated heart preparation that minimized vascular effects, we found that the deletion of eNOS resulted in significantly depressed LVDP and RPP following ischemia– reperfusion. The eNOS knockout model also suffered more pronounced levels of left ventricular end-diastolic pressure (LVEDP) dysfunction. These data provide compelling evidence that deletion of the major NOS isoform responsible for NO production in the heart (eNOS) results in a pronounced increase in myocardial stunning following ischemia–reperfusion. Moreover, this dysfunction occurs without affecting the rate of cardiac necrosis. The role of NO on myocardial contractility is controversial [21–27]. Gross et al. [22] have reported a decline in cardiac contractile reserve following infusion of the NO donor SNAC, possibly through depression of high energy phosphate metabolism. Hattler et al. [26] have
demonstrated increased myocardial stunning with elevations in NO production in the human heart in the postpulmonary bypass interval, suggesting a link between NO production and myocardial depression. Moreover, Brady et al. [21] have shown that the NO donor sodium nitroprusside when perfused over guinea pig myocytes resulted in depressed myocardial contractility. This decrease in contraction amplitude was reversed by methylene blue, an inhibitor of cGMP (a mediator in the NO pathway). In contrast, Pabla et al. [24] previously reported that in the isolated rat model exogenous NO infusion significantly improved both LVDP and RPP, while preventing elevations in LVEDP. Inhibition of NO using L-NAME exacerbated contractile dysfunction. Furthermore, work by Hasebe et al. [23] showed that inhibition of endogenous NO synthesis by use of L-NAME enhances myocardial stunning. In another study, Weyrich et al. [25] investigated the effects of physiological concentrations of exogenous NO on myocardial contractility in isolated rat cardiomyocytes. These investigators found that physiologically relevant concentrations of NO do not induce physiologically significant negative inotropic effects in the cat or rat heart. These findings are in agreement with the present study, that in fact, endogenous NO synthesis in the myocyte does not induce a negative inotropic effect with ischemia–reperfusion and that endogenous eNOS-derived NO may be important for maintaining normal myocardial contractility. A recent study of ischemia–reperfusion in isolated mouse hearts using eNOS knockout mice by Flogel et al. [27] demonstrated increased ischemia–reperfusion injury in wild-type mice and a protective effect of eNOS deletion on ischemia–reperfusion injury in eNOS knockout mice. Significant differences in the protocol used in this study and that reported by Flogel include a significantly longer period of reperfusion (60 versus 30 min) and a slighter shorter period of ischemia (16 versus 30 min). It may be that the effect of eNOS deletion may vary during the time period of reperfusion: during early reperfusion, the protective effect of
FIG. 5. Determinants of tissue viability assessed by total efflux of LDH. Values are means ⫾ SEM. No significant differences were noted between groups.
HANNAN ET AL.: EFFECTS OF DELETION OF eNOS IN HEART MODEL
NO may predominate, while in late reperfusion NO may increase reperfusion injury. In our own data, function in the eNOS knockout group was better than control at 2 min reperfusion but decreased compared to control at all later points. Alternatively, it is possible that during pathological conditions where NO might be produced via the inducible NOS pathway, or when NO is administered at supraphysiological levels, high levels of NO might contribute to the cardiac dysfunction associated with myocardial stunning. Xie et al. [28] have speculated that elevated levels of NO may retard mitochondrial respiration, resulting in a decrease in the ATP synthesis needed for force development, resulting in a negative inotropic effect. It is possible that these opposing observations may be attributed to differential effects on the myocyte to NO at low and high concentrations. The worsening of diastolic function we witnessed in the eNOS knockout group is consistent with a number of studies, which correlated NO in the myocyte with improved myocardial relaxation. Grocott-Mason et al. [12] found that exogenous NO exerts direct myocardial relaxant effects in the isolated guinea pig heart, independent of NO’s known vasodilator activity. NO has been shown to raise intracellular cGMP levels and this, in turn, desensitizes the myofilament response to Ca 2⫹ [11]. Thus, the removal of eNOS-derived NO from the myocardium might eliminate an important cGMP counterregulator. If NO is not present to modulate cGMP elevations, CA 2⫹ influx may go unchecked, resulting in the degree of calcium loading that has been associated with myocardial stunning. It is possible that blocking this mechanism as we did in our experiment through the deletion of eNOS-derived NO contributed to enhanced myocardial stunning. NO may also be involved in optimizing O 2 consumption, as suggested by Xie et al. [28]. They found that exogenous NO infusion using SNAP attenuated O 2 consumption in myocardial tissue. In a study with similar findings, Shen et al. [29] demonstrated that NO inhibition using NLA caused marked elevations in O 2 consumption and decreases in cardiac output in conscious dogs. However, possible adverse actions of this mechanism are likely under pathological conditions where elevated and sustained NO levels (such as might be produced by inducible NOS) could impede the O 2dependent production of ATP. Thus, NO’s effect on O 2 consumption at high concentrations might well result in the negative inotropic effect witnessed in some studies. It is our speculation that at the onset of reperfusion and in the period up to approximately 5 min thereafter, the rapid influx of O 2 into the myocyte causes the rapid and excessive production of NO via eNOS. This increase in NO concentration to well above the physiological threshold causes a transient period of myocardial depression, which dissipates after a prolonged
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period of reperfusion. This mechanism would explain the apparent enhanced myocardial contractility of the eNOS knockout group during the brief initial stage of reperfusion. The eNOS knockouts, lacking the NOS isoform capable of producing such rapid NO elevations in the myocardium, would be unable to experience the pathological levels of NO that might be detrimental to post-ischemic cardiac function. Nevertheless, as reperfusion continues the response to sudden influx O 2 (increased rate of NO synthesis) disappears, and physiological levels of NO synthesis are restored, as are more physiological levels of myocardial contractility. In future studies the rate of NO synthesis in the myocardium as a function of reperfusion duration might yield data that would further elucidate NO’s role on contractility. Chief among NO’s effects believed to be harmful to myocardial function is the possible formation of peroxynitrite, a toxic free radical species. In an investigation by Schulz and Wambolt [30], some free radical species, possibly peroxynitrite, was found to contribute to the myocardial dysfunction associated with ischemia–reperfusion. In contrast with these and other reports, some studies have found that NO may be beneficial to certain physiological processes. In fact, Lefer et al. [31] showed that peroxynitrite reduced the degree of neutrophil-induced myocardial dysfunction after ischemia and reperfusion of the myocardium. However, it is important to note that peroxynitrite is the reaction product of NO and superoxide at equimolar concentrations of these reactants, limiting the maximally achievable concentration of ONOO ⫺ to the highest amount of NO or superoxide present in the myocardium. Basal concentrations of NO occur in the 1–20 nM range [32] and are believed to increase to maximal levels in the low micromolar range (2–5 M) in pathological states where inducible NOS is activated [31]. Consequently, the maximal achievable levels of peroxynitrite that can be formed in vivo would also be in the low micromolar range (2–5 M). Yet the concentration of peroxynitrite necessary to produce lipid peroxidation is well above this level (100 M–1 mM) [33]. Along with its short life span (⬇1.9 s) [13] and the supraphysiological levels of peroxynitrite necessary to exert significant toxic effects, it is highly unlikely for endogenous NO-mediated production of peroxynitrite to play a significant role in myocardial stunning. In conclusion, deletion of eNOS caused impaired diastolic function during ischemia and significant depression of myocardial contractility following ischemia–reperfusion in the isolated mouse heart. Furthermore, this occurred without altering cell viability. Previous reports have for the most part suggested a negative inotropic effect from NO. In that case, eliminating NO should result in improved, not worsened, contractility following ischemia–reperfusion. We suggest that high, nonphysiologic levels of NO might elicit
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the negative inotropic effects found in these studies, but that lower, physiologic endogenous levels do not. Indeed, in light of the depressed contractility of the eNOS knockout animals, our data suggest that eNOSderived NO is in fact cardioprotective at endogenous levels. The lack of eNOS-derived NO appears to increase myocardial dysfunction following ischemia– reperfusion in this isolated mouse model.
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