Nitric Oxide Attenuates Neutrophil-Mediated Myocardial Contractile Dysfunction After Ischemia and Reperfusion
Abstract With the knowledge of NO as an antiadhesion molecule, we performed studies to investigate the effects of NO on postischemic polymorphonuclear leukocyte (PMN)–mediated myocardial contractile dysfunction. Studies were performed with isolated perfused rat hearts subjected to 20 minutes of global ischemia and 45 minutes of reperfusion. Human PMNs (50 million) were infused over the first 5 minutes of reperfusion, and the recovery of left ventricular function was compared with baseline values. Infusion of PMNs alone (n=10) led to a 61% reduction in left ventricular developed pressure (LVDP) and a 57% reduction in the pressure-rate product (PRP) at 45 minutes of reperfusion. Infusion of an NO donor, CAS-754 (n=9), resulted in 80.2±6.7% recovery of LVDP and 77.0±8.6% recovery of PRP. Treatment with l-arginine (2.5 mmol/L, n=10) resulted in a similar improvement in the postischemic contractile state of the heart. In contrast, NG-nitro-l-arginine methyl ester (L-NAME) treatment (250 μmol/L, n=10) resulted in an exacerbation of contractile dysfunction, as evidenced by a 93% reduction in LVDP at 45 minutes of reperfusion and a 91% reduction in PRP. The deleterious effects of L-NAME were prevented by l-arginine coperfusion. We failed to observe any cardioprotective effects when NO or l-arginine was administered to hearts subjected to 25 minutes of ischemia and 45 minutes of reperfusion in the absence of PMNs. In conclusion, PMN-mediated myocardial contractile dysfunction is attenuated by NO and exacerbated by blockade of NO synthesis.
Neutrophil (PMN)–mediated injury after myocardial ischemia/reperfusion is associated with endothelial damage1 2 as a consequence of release of a variety of enzymes and oxygen-derived free radicals.3 Subsequent PMN accumulation and aggregation leads to a narrowing of capillary lumen diameter as a result of PMN plugging, resulting in a reduction in coronary flow, ie, “no reflow.”4 Moreover, PMN-mediated myocardial injury after ischemia/reperfusion has been associated with myocardial contractile dysfunction5 6 and myocardial necrosis.3
Several studies have clearly demonstrated a reduction in PMN-induced myocardial contractile dysfunction by use of pharmacological intervention,3 by leukocyte filters depleting the blood of leukocytes before the onset of ischemia6 or during reperfusion,7 and by antibodies directed against adhesion molecules on the PMN surface.8 Furthermore, inhibition of adhesion molecules expressed on endothelial cells has also been shown to reduce diastolic myocardial dysfunction and to prevent edema formation and the low-reflow phenomenon associated with PMN adhesion and aggregation.9 Thus, taken together, these studies indicate that prevention of PMN adhesion may be important in the attenuation of cardiac dysfunction after ischemia and reperfusion. On the other hand, several studies have reported the lack of a PMN contribution toward postischemic myocardial contractile dysfunction.10 11 12 13 These differences may be attributed to the duration and severity of ischemia used or to the specific intervention used.
NO has been shown to reduce PMN adhesion and aggregation and to quench free radicals generated by PMNs.14 Since NO possesses antineutrophil properties, it is feasible that NO may reduce any adverse effects of PMNs on the contractile state of the heart. Several groups have shown the importance of NO in myocardial contractility. Recently, Hasebe et al15 demonstrated that the inhibition of NO in conscious dogs by use of L-NAME enhances myocardial stunning with no significant effect on blood flow. In contrast, using isolated papillary muscle preparations, Finkel et al16 have shown that cytokines have a negative inotropic effect on the heart, which is thought to be mediated by NO. Another recent study has demonstrated that NO has no effect on the contractile function of the normal myocardium.17 Thus, experimental data regarding the effects of NO on cardiac function are conflicting and unresolved.
Myocardial ischemia and reperfusion are associated with PMN infiltration and adhesion to the vascular endothelium.18 Since NO has been shown to inhibit PMN function, the aim of the present study was to investigate the effects of exogenous NO and the effects of l-arginine, the natural substrate for NO synthase, on PMN-mediated myocardial contractile dysfunction after 20 minutes of global zero-flow ischemia and 45 minutes of reperfusion in an isolated rat heart model. We also sought to determine the effects of the manipulation of coronary NO levels on the PMN-independent myocardial injury associated with ischemia and reperfusion of isolated rat hearts.
Materials and Methods
CAS-754 was obtained from Dr Rainer Henning (Cassella AG, Frankfurt, Germany). Human serum albumin was purchased from Calbiochem, Percoll was purchased from Pharmacia Biotech AB, and all other chemicals and reagents were purchased from Sigma Chemical Co.
Isolated Heart Perfusion
Male Sprague-Dawley rats (350 to 400 g) were heparinized with 1000 U sodium heparin (Elkins-Sinn, Inc) and anesthetized with intraperitoneal sodium pentobarbital (Abbott Laboratories) at a dose of 35 mg/kg. The hearts were rapidly excised, the ascending aorta was cannulated, and retrograde perfusion was initiated. The hearts were perfused with Krebs’ bicarbonate perfusate containing (mmol/L) glucose 17, sodium chloride 120, sodium bicarbonate 25, calcium chloride 2.5, EDTA 0.5, potassium chloride 5.9, and magnesium chloride 1.2 at 37°C and a constant pressure of 80 mm Hg. The perfusate was bubbled with 95% O2/5% CO2. Two side arms in the perfusion line located just proximal to the heart cannula allowed infusion of PMNs and plasma directly into the heart. To assess contractile function, a microtipped catheter transducer (Millar Instruments Inc) was inserted directly into the left ventricular cavity. Left ventricular pressure was recorded on a Gould TA240 two-channel recorder (Gould Inc). Measurements of developed pressure were calculated as the difference between the peak systolic and end-diastolic pressures. Left ventricular pressures, coronary flow, and heart rate were measured periodically every 5 minutes before a 20-minute period of zero-flow global ischemia and after ischemia for a 45-minute period of reperfusion.
Human PMNs were prepared by the method of Bochner et al.19 Human peripheral venous blood (60 mL) was collected into six 50-mL plastic conical tubes (Sarstedt Inc), each containing 25 mL of 0.9% saline and 0.8 mL of 0.1 mol/L EDTA. The blood-saline mixture was carefully layered over 9 mL of Percoll (specific density, 1.079; Pharmacia, Inc) and centrifuged at room temperature for 20 minutes at 1400 rpm in an IEC Centra GP8R centrifuge (International Equipment Co). The plasma and buffy coat layers were aspirated and discarded, leaving a red blood cell layer along with the PMNs. The erythrocytes were then lysed with 18 mL of deionized ice-cold water, and the blood mixture was inverted several times.20 This was followed by the addition of 2 mL of 10× PIPES to each tube of blood and thorough mixing over a 30-second period. The blood mixture was then centrifuged at 4°C for 5 minutes at 1400 rpm. After this time, the supernatant was poured off, and the pellet was lysed and centrifuged as before. The supernatant was again discarded, and the individual PMN pellets were broken up with 2 mL of PAG buffer, which was composed of 1% glucose, 3% human serum albumin, and 10% 10× PIPES in deionized water. The individual pellets were consolidated into one conical tube, and the final volume was made up to 12 mL with PAG buffer. The PMN-PAG mixture was then spun at 4°C for 5 minutes at 1400 rpm. After the supernatant was discarded, 12 mL of PAG buffer was added to the PMN pellet, and the mixture of cells was respun at 4°C for 5 minutes at 1400 rpm. The PMN pellet was then resuspended with 1 mL PAG buffer per 10 mL of whole blood, and PMNs were counted with a hemocytometer.
Whole rat blood was obtained by performing an open-chest intracardiac puncture by use of a 10-mL plastic syringe with a 20-gauge needle (Becton Dickinson and Co) containing 2000 U sodium heparin (Elkins-Sinn Inc). To obtain platelet-poor plasma, the whole blood was immediately spun in an IEC Centra GP8R refrigerated centrifuge at 3000 rpm for 25 minutes. The plasma layer was collected and stored at 4°C until it was used in the isolated perfused heart.
Determination of Myocardial CK Activity
After 45 minutes of reperfusion, hearts were immersed in 0.25 mmol/L sucrose solution containing 1.0 mmol/L EDTA and 0.1 mmol/L mercaptoethanol, and the hearts were then placed on ice. The left ventricle was minced, and 0.5 g of tissue was placed in fresh 0.25 mmol/L sucrose solution (1:10 [wt/vol]) and homogenized with a Polytron homogenizer (Brinkman Instruments) for 20 seconds twice at 7000 rpm. The homogenates were then centrifuged for 10 minutes at 800g and 4°C. The supernatant was collected and centrifuged again at 36 000g for 30 minutes at 4°C. CK activity and protein concentration of the supernatant were determined as described previously.21 22
The experimental protocols used for the isolated heart ischemia/reperfusion studies are depicted in Fig 1⇓. After a 15-minute stabilization period, baseline LVDP, LVEDP, and coronary flow were measured every 5 minutes for 15 minutes to ensure complete equilibration of the hearts. Flow of Krebs’ perfusate was stopped, creating global zero-flow ischemia for 20 minutes. At the onset of reperfusion (Fig 1A⇓), the hearts were perfused for the first 5 minutes with human PMNs (50 million) and 5 mL of rat plasma along with standard Krebs’ buffer with or without CAS-754 (100 μmol/L, n=9) or l-arginine hydrochloride (2.5 mmol/L, n=10). After 5 minutes, perfusion was continued with Krebs’ buffer alone for an additional 40 minutes, during which serial measurements of coronary flow and developed pressure were performed every 5 minutes. In a separate group of hearts (Fig 1B⇓), L-NAME (250 μmol/L, n=10) or L-NAME (250 μmol/L) in combination with l-arginine (2.5 mmol/L, n=10) was infused over 5 minutes, beginning 10 minutes before ischemia, followed by an additional infusion during the first 5 minutes of reperfusion.
An additional set of experiments was conducted to investigate the effects of NO modulation in the setting of ischemia and reperfusion without HNRP (Fig 1C⇑ and 1D⇑). In these experiments, rat hearts were subjected to 25 minutes of zero-flow global ischemia and 45 minutes of reperfusion with Krebs’ buffer alone. Identical treatment protocols with CAS-754, l-arginine, and L-NAME were studied.
In order to establish that the effects on cardiac function were due to PMN activity alone, additional rat hearts were treated with Krebs’ buffer alone (n=10), rat plasma alone (n=9), human PMNs alone (n=6), or HNRP during the first 5 minutes of reperfusion. Furthermore, the effects of rat plasma alone (n=5), human PMNs alone (n=5), or HNRP (n=5) without ischemia were also investigated.
All data are presented as mean±SEM. Comparisons between the groups during preischemic control conditions as well as after ischemia were made by a two-way repeated-measures ANOVA performed with SuperANOVA (version 1.11, Abacus Concepts, Inc) in conjunction with a post hoc t test using the Bonferroni correction. Values of P<.05 were accepted as statistically significant.
Validation of the Experimental Model
Initial experiments were conducted to determine the effects of rat plasma alone, human PMNs alone, or HNRP in hearts not subjected to ischemia and reperfusion. These results are summarized in Table 1⇓. Clearly, the infusion of plasma alone, human PMNs, or HNRP had no effect on baseline left ventricular contractility, LVEDP, or coronary flow. Additional studies were performed to validate the role of PMNs in the myocardial injury observed in this model of ischemia and reperfusion. Hearts were subjected to 20 minutes of global ischemia and then reperfused with Krebs’ buffer alone, rat plasma alone, PMNs alone, or HNRP, and the results are summarized in Table 2⇓. Postischemic infusion of buffer alone or plasma alone resulted in >80% recovery of left ventricular function and coronary flow at 45 minutes of reperfusion. In contrast, the infusion of human PMNs or HNRP resulted in a 60% reduction in left ventricular function (P<.05 versus buffer alone or plasma alone). Since reperfusion with PMNs alone and HNRP resulted in similar and dramatic attenuation of LVDP, these results suggest that the postischemic decline in LVDP observed with HNRP treatment is attributable to PMN activity.
Myocardial CK Activity
Left ventricular tissue homogenates were analyzed for CK activity to determine the extent of irreversible myocardial cell injury after ischemia and reperfusion (Fig 2⇓). Hearts subjected to 20 minutes of global ischemia and 45 minutes of reperfusion in the presence and absence of 50 million human PMNs and rat plasma were compared with sham hearts subjected to 65 minutes of perfusion after the 15-minute equilibration period. Ischemia and reperfusion alone resulted in a significant (P<.05) loss of CK from the hearts at 45 minutes of reperfusion, which was further exacerbated by the infusion of PMNs and plasma.
Effects of NO on PMN-Mediated Postischemic Myocardial Injury
Preischemic baseline values of LVDP did not differ significantly between the groups studied (data not shown). After 20 minutes of global zero-flow ischemia, LVDP in HNRP-alone hearts at 45 minutes of reperfusion was reduced by 61% compared with baseline. Treatment with CAS-754 for the first 5 minutes of reperfusion resulted in a pronounced recovery of LVDP at 45 minutes of reperfusion (80.2±6.7% versus 38.8±7.8% in HNRP-alone hearts, P<.01). Moreover, the recovery in LVDP was rapid in onset and improved steadily throughout reperfusion (Fig 3⇓). Hearts treated with l-arginine showed a similar profile of LVDP recovery that was indistinguishable from CAS-754 at 45 minutes of reperfusion (Fig 3⇓). In contrast, recovery of hearts subjected to L-NAME infusion was consistently <10% of baseline throughout reperfusion, with a 93% reduction in LVDP at 45 minutes of reperfusion (P<.05 versus HNRP). This profound reduction in LVDP was reversed by l-arginine coperfusion, resulting in 92.0±8.8% recovery of LVDP at 45 minutes of reperfusion compared with 6.7±2.5% recovery in hearts perfused with L-NAME alone. Perfusion with L-NAME or L-NAME in combination with l-arginine in hearts that were not subjected to ischemia had no significant effect on any of the variables measured (data not shown).
Left ventricular PRP was used as an additional index of cardiac function. Hearts reperfused with HNRP demonstrated a 57% reduction in PRP at 45 minutes of reperfusion compared with baseline (Fig 4⇓). This effect was significantly attenuated by both CAS-754 and l-arginine (both P<.01 versus HNRP) but profoundly exacerbated by L-NAME (P<.05 versus HNRP). Once again, the effects of L-NAME were overcome by l-arginine coperfusion (80.4±11.7% versus 9.3±1.6% recovery in the L-NAME group, P<.01).
Baseline LVEDP was very similar in all groups studied (Fig 5⇓). After ischemia and reperfusion with HNRP for 5 minutes, LVEDP was elevated significantly to 58.2±7.7 mm Hg compared with 40.6±9.3 mm Hg in the CAS-754–treated group and 25.9±6.7 mm Hg in the l-arginine–treated group (P<.01 versus HNRP) at the same time point. LVEDP remained significantly elevated (>45 mm Hg) in the control group throughout the 45-minute reperfusion period. In contrast, LVEDP steadily declined during reperfusion in hearts treated with CAS-754 or l-arginine (Fig 5⇓). At 45 minutes of reperfusion, the LVEDP was 46.6±6.6 mm Hg in the control hearts compared with 21.0±5.5 mm Hg in the hearts receiving CAS-754 and 15.8±5.2 mm Hg in the hearts treated with l-arginine (both P<.01 versus HNRP). However, LVEDP was significantly augmented to 64.7±6.0 mm Hg in the L-NAME–treated group at 45 minutes of reperfusion. Again, l-arginine coperfusion reversed the detrimental effects of L-NAME and reduced LVEDP to values similar to those observed with l-arginine alone (22.9±9.4 mm Hg, P<.01 versus L-NAME).
Under baseline conditions, coronary flow was similar (P=NS) in all groups (data not shown). After reperfusion, in hearts receiving HNRP alone coronary flow remained >70% of baseline values (Fig 6⇓). Similarly, coronary flow remained at values very similar to baseline throughout reperfusion in the hearts treated with CAS-754 and l-arginine. In contrast, coronary flow was significantly depressed by 50% at 15 minutes of reperfusion in hearts receiving L-NAME, with a further decline to 45% of baseline by 45 minutes of reperfusion (P<.01 versus HNRP alone). The coronary flow reduction by L-NAME was attenuated by l-arginine coperfusion (55.0±6.2% recovery versus 44.7±4.2% recovery in the L-NAME–treated hearts) at 45 minutes of reperfusion, although this difference did not reach statistical significance.
Histological Analysis of PMN Accumulation
Histological analysis of heart biopsies was performed to determine whether treatment with CAS-754 or l-arginine altered the accumulation of PMNs within the postischemic heart. Clearly, the hearts treated with CAS-754 and l-arginine exhibited a marked reduction in PMN accumulation compared with the HNRP hearts (Fig 7⇓, top left, top right, and bottom left panels). Conversely, L-NAME administration significantly enhanced PMN accumulation in the ischemic/reperfused heart (Fig 7⇓, bottom right).
Effects of NO on PMN-Independent Postischemic Myocardial Injury
The results of experiments investigating the effects of administration of the NO donor CAS-754, l-arginine, and L-NAME in hearts subjected to 25 minutes of ischemia and 45 minutes of reperfusion in the absence of PMNs are shown in Table 3⇓. Hearts subjected to 25 minutes of global ischemia exhibited profound myocardial contractile dysfunction after 45 minutes of reperfusion. LVDP and coronary flow recovered only ≈40% and 70%, respectively. In addition, LVEDP was significantly elevated to 72±11 mm Hg. The addition of exogenous NO with CAS-754 (100 μmol/L) failed to attenuate the left ventricular dysfunction or the reduction in coronary flow. Furthermore, treatment with l-arginine (2.5 mmol/L) at the time of reperfusion also failed to provide any substantial cardioprotective effects. Inhibition of coronary NO synthesis with L-NAME (250 μmol/L) did not have any significant effects except for a further reduction in coronary flow, which was significantly (P<.05) less than for the CAS-754 and l-arginine groups.
Administration of l-arginine has previously been shown to attenuate endothelial and myocardial injury in feline and canine models of ischemia and reperfusion.23 24 Furthermore, work by Siegfried et al25 and Lefer et al26 demonstrated significant cardioprotective effects of an NO donor, SPM 5185, administered 10 minutes before reperfusion, resulting in attenuated neutrophil accumulation and myocardial necrosis. However, experimental studies investigating whether an NO donor or l-arginine has any protective effect against PMN-induced myocardial contractile dysfunction have not been performed.
In the present study, we demonstrated that exogenous NO and l-arginine profoundly attenuate PMN-mediated cardiac dysfunction after 20 minutes of global zero-flow ischemia in the isolated rat heart. Treatment with the NO donor and l-arginine significantly enhanced both LVDP and PRP. Furthermore, both treatment strategies resulted in an attenuation of the increase in LVEDP observed in untreated hearts, while having no overall effect on coronary flow. Moreover, inhibition of NO synthesis, using the NO synthase inhibitor L-NAME, caused a worsening of the contractile state of the heart, diminishing LVDP and PRP while exacerbating the increase in LVEDP and markedly reducing coronary flow. Interestingly, the effects observed in L-NAME–treated hearts were reversed by l-arginine coperfusion.
A notable aspect of the present study is the evaluation of the effects of NO modulation in isolated rat hearts that underwent global ischemia and reperfusion without infusion of PMNs during reperfusion. In contrast to our experiments with PMN infusion, we failed to observe any protective effects of exogenous NO or l-arginine in hearts subjected to a more severe ischemic insult and then reperfused with physiological buffer alone. This is an important finding, which suggests that the predominant cardioprotective effect of NO in the setting of myocardial reperfusion injury is the inhibition of PMN-mediated coronary vascular and myocardial cell injury.
The effects of NO on myocardial contractility are controversial at present.16 17 27 28 Finkel et al16 have reported that the negative inotropic effects of cytokines (tumor necrosis factor-α, interleukin-6, and interleukin-2) on the hamster heart are mediated by NO, since the effects of these cytokines are inhibited by the NO synthase inhibitor L-NMMA and reversed with l-arginine. Moreover, Brady et al27 have used cultured guinea pig cardiac myocytes to demonstrate attenuation of myocyte contraction by NO. The nitrovasodilator sodium nitroprusside was shown to decrease myocyte contraction amplitude, whereas the guanylate cyclase inhibitor methylene blue reversed the reduction in myocyte shortening caused by sodium nitroprusside. In contrast, Amrani et al28 have shown that L-NMMA in isolated rat myocytes has neither a positive nor a negative effect on contraction. Weyrich et al17 previously reported that administration of l-arginine or NO does not alter the inotropic state of the normal myocardium in the cat or rat. The results of the present study clearly indicate that NO is not a negative inotrope in the postischemic rat myocardium perfused with PMNs.
There has been much interest in the effects of PMNs on left ventricular contractility after ischemia and reperfusion. To directly investigate the role of PMNs in myocardial function, several groups have used the approach of depleting whole animals of PMNs6 7 10 12 or perfusing isolated hearts with PMNs.29 However, evidence from O’Neill et al10 has shown that dogs made neutropenic by using specific antiserum showed no functional improvement after 15 minutes of ischemia, even though ≈90% of the leukocyte population was depleted. Furthermore, use of leukocyte filters has resulted in a similar lack of effect, ie, no improvement in cardiac function after a 10-minute ischemic period despite severe neutropenia.12 Thus, it would appear that the results of leukocyte depletion in whole animals by use of antiserum or filters are conflicting.
More recently, experimental studies of postischemic myocardial contractile function have focused on the inhibition of the PMN CD11/CD18 adhesion complex by using specific monoclonal antibodies. Kraemer et al30 demonstrated that inhibition of neutrophil-endothelial interactions by use of an anti-CD18 antibody was associated with a reduction in myocardial injury and an attenuation of PMN-mediated contractile dysfunction. Furthermore, inhibition of CD18 in an isolated rat heart model has been shown to preserve coronary flow, reduce reperfusion injury, and maintain cardiac function, which was shown to be associated with a significant reduction in myeloperoxidase activity.8 However, Schott et al11 failed to demonstrate any attenuation in myocardial contractile dysfunction after the inhibition of PMN adhesion in an open-chest canine model. Thus, whether inhibition of PMN adhesion is effective in preventing postischemic myocardial contractile dysfunction remains uncertain.
NO is an antiadhesion molecule that has been shown to inhibit PMN adhesion to the endothelium; however, its effects on PMN-induced cardiac dysfunction after coronary ischemia and reperfusion have not been investigated. It has been shown that inhibition of NO synthesis results in increased PMN adherence to postcapillary venules that can be prevented by both exogenous NO31 and l-arginine reversal.32 Furthermore, an impairment of NO production in a feline model of ischemia and reperfusion has been correlated to an increase in PMN adherence to the coronary endothelium, an effect subsequently diminished by NO.18 Recently, it has been shown that NO administration in a splanchnic model of ischemia/reperfusion reduces P-selectin–mediated PMN rolling along the endothelium,33 thereby reducing PMN adhesion and attenuating PMN-induced injury. Additionally, Lefer et al34 have shown that augmentation of NO levels with NO donors or l-arginine reduces the expression of intercellular adhesion molecule-1 on human aortic endothelial cells in culture.
Our observations complement those of Hasebe et al.15 They demonstrated in a conscious dog model that inhibition of NO synthesis using L-NAME enhanced myocardial stunning transmurally, independent of any effect on blood flow. In the present study, although we also demonstrated a similar enhancement of myocardial dysfunction, perhaps as a consequence of enhanced PMN adhesion35 and free-radical production, we also noted a marked reduction in coronary flow. However, the effects of NO inhibition on both function and flow were reversed by l-arginine cotreatment.
In conclusion, our findings are the first to demonstrate the effects of exogenous NO and l-arginine on PMN-mediated contractile injury in an isolated rat heart model after global ischemia. NO treatment significantly improved both LVDP and PRP, while attenuating the enhancement in LVEDP observed in control hearts, resulting in >80% recovery of function compared with <7% recovery of myocardial contractility in L-NAME–treated hearts. Moreover, the detrimental effects of NO inhibition by L-NAME were reversed by l-arginine coperfusion, reaffirming the involvement of the NO pathway. Thus, NO plays a vital role in the preservation of myocardial contractile function after 20 minutes of global zero-flow ischemia. Furthermore, the cardioprotective effects of NO are not observed in hearts subjected to severe global ischemia and reperfusion in the absence of PMNs. Hence, the next step toward a fuller understanding of the role of NO in the setting of myocardial ischemia/reperfusion would be to establish the precise cellular mechanism of action of NO in attenuating PMN-mediated myocardial dysfunction.
Selected Abbreviations and Acronyms
|HNRP||=||human neutrophils+rat plasma|
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|LVDP||=||left ventricular developed pressure|
|LVEDP||=||left ventricular end-diastolic pressure|
Dr Pabla is a recipient of a Wellcome Prize Studentship from the Wellcome Trust. The authors are very grateful to Dana B. Salzberg, Khoa D. Vo, and Paul R. Jeffords for their expert technical assistance during the course of these studies. We gratefully acknowledge the support and generous supply of CAS-754 received from Dr Rainer Henning, PhD, of Cassella AG, Frankfurt, Germany.
- Received August 17, 1995.
- Accepted September 29, 1995.
- © 1996 American Heart Association, Inc.
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