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Cardiovascular Research 1999 44(3):527-535; doi:10.1016/S0008-6363(99)00226-6
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Hemodynamic effects of nitric oxide synthase inhibition at steady state and following tumor necrosis factor-{alpha}-induced myodepression

David R. Murray*, Sumanth D. Prabhu and Gregory L. Freeman

The Department of Medicine, University of Texas Health Science Center at San Antonio and the Audie L. Murphy Memorial Veterans Hospital, San Antonio, TX 78284, USA

* Corresponding author. Tel.: +1-210-567-4602; fax: +1-210-567-6960

Received 26 January 1999; accepted 16 July 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Nitric oxide (NO) has been proposed as a common mediator of tumor necrosis factor-{alpha} (TNF{alpha})-induced vasodilation and myocardial dysfunction. Accordingly, we performed an extensive assessment of the influence of NO synthase inhibition on left ventricle (LV) and circulatory performance in conscious dogs at steady state and after establishment of TNF{alpha} mediated myodepression. Methods: Autonomically blocked, chronically instrumented dogs were studied at steady state and 6 h after initiation of a 1-h rhTNF{alpha} infusion (40 µg/kg). Ventricular performance was evaluated using the pressure–volume framework. Dogs were then treated with either NG-nitro-L-arginine methylester (L-NAME, 40 mg/kg bolus) or angiotensin II (250–500 ng/kg). Results: L-NAME, under control conditions or following recombinant human (rh)TNF{alpha}-induced ventricular dysfunction, produced marked increases in afterload with attendant increases in LV pressure, volume, and prolonged isovolumic relaxation without adversely influencing coronary blood flow. Regardless of whether the dogs received rhTNF{alpha}, L-NAME did not affect the slopes of the end-systolic pressure–volume and stroke-work (SW)-end-diastolic volume (EDV) relations (force-based measure of contractility), whereas the slope of the dP/dtmax–EDV relation, a velocity dependent parameter of LV systolic function, declined. Overall ventricular performance, as seen by the circulation, was reduced by L-NAME in control as well as rhTNF{alpha}-treated dogs, evidenced by rightward shifts of the SW–EDV and dP/dtmax–EDV relations. Similar findings were observed in the separate cohorts of dogs, at steady state and 6 h after rhTNF{alpha}, following angiotensin II at matched systolic pressure. Conclusions: Systemic NO synthase inhibition with L-NAME does not acutely reverse rhTNF{alpha}-induced myocardial dysfunction. The detrimental influence of L-NAME on LV size, relaxation, and velocity-based measures of contractility is likely attributable to its effects on increasing afterload.

KEYWORDS Cytokines; Hemodynamics; Nitric oxide; Ventricular function; Angiotensin


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The role of nitric oxide (NO) as a vasodilator is well established. Analogues of L-arginine which serve as competitive inhibitors of NO synthase (NOS) raise arterial pressure and systemic vascular resistance under basal conditions as well as in the setting of endotoxic shock [1,2]. In fact, NO has been implicated as a primary mediator of lipopolysaccharide (LPS), otherwise known as endotoxin, and tumor necrosis factor-{alpha} (TNF{alpha})-induced hypotension, peripheral vasodilation and vascular hyporesponsiveness to adrenergic agonists [3–5].

In addition to its effects on vasculature, NO appears to regulate basal myocardial function, particularly relaxation and diastolic tone [6,7]. Agents which stimulate NO release from the endothelium (substance P, bradykinin) [8] and exogenous NO donors [9] promote earlier onset and acceleration of left ventricular (LV) pressure decay, along with a fall in peak LV systolic pressure, without affecting the maximal value of the first derivative of LV pressure (dP/dtmax), LV ejection fraction or stroke volume. Moreover, while not affecting steady state contractility, NOS inhibition augments contractile responsiveness to exogenous β-adrenergic stimulation [10] and reverses the negative force frequency relationship in hamster papillary muscle [11]. Taken together, these studies suggest that NO facilitates relaxation while blunting the inotropic response to catecholamines and depressing contractility at higher pacing frequencies.

The impact of NO on myocardial contractility may be dose dependent; high concentration of NO may induce a more profound negative inotropic effect [12]. Endotoxin and proinflammatory cytokines such as Interleukin-1β, and TNF{alpha} induce the expression of inducible NOS (iNOS), leading to an abundant production of NO within the myocardium [13,14]. We and others have shown that TNF{alpha} promotes contractile impairment in vitro and in vivo [15–17]. Whether NO mediates TNF{alpha}-induced myocardial dysfunction has yet to be resolved. While NOS inhibition has been shown to prevent the acute negative inotropic effect of TNF{alpha} in a papillary muscle preparation [16], NO did not promote TNF{alpha}-induced contractile impairment in isolated feline cardiomyocytes [15]. Furthermore, treatment with NOS inhibitors in in vivo models of endotoxic shock or following TNF{alpha} has led invariably to decreases in cardiac output, stroke volume, and oxygen delivery [18,19]. Whether this fall in cardiac output is secondary to a rise in afterload, reduced coronary blood flow, or to a direct effect of NOS inhibitors on myocardial contractility has yet to be established.

The purpose of this study was to determine the influence of acute NOS inhibition on ventricular performance in vivo under basal conditions and after the establishment of TNF{alpha}-induced myocardial depression. Unlike in vitro preparations which consistently show a negative inotropic response within 5 to 20 min of exposure [15,16], TNF{alpha} has a biphasic effect on myocardial contractility in the conscious dog, consisting of an early modest improvement in ventricular performance followed by sustained myocardial depression beginning approximately 3 h after TNF{alpha} administration [17]. Accordingly, we studied the effects of NG-nitro-L-arginine methylester (L-NAME) in conscious, chronically instrumented dogs either at baseline or 6 h after recombinant human (rh)TNF{alpha} treatment.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Instrumentation
The investigation confirms with the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Adult mongrel dogs (22–25 kg) of either sex were used; gender did not affect responsiveness to TNF{alpha} in a previous study using the same preparation [17]. The surgical preparation has been previously described in detail [17]. In brief, after intravenous sedation with 2.5% sodium pentothal (6–10 ml) and acepromazine (0.25 mg/kg), the animals were intubated and anesthetized with 1–2% isoflurane. Three sets of piezoelectric crystals were implanted in the endocardium of the LV, oriented orthogonally, to allow for continuous assessment of the anterior–posterior, septal–lateral, and long-axis diameters. A micromanometer-tipped catheter (Konigsberg, Pasadena, CA, USA) and a fluid-filled polyvinyl catheter were placed through the LV apex. A second fluid-filled catheter was positioned in the left-atrium for infusion of drugs and saline while a third fluid-filled catheter was inserted in the aortic arch to measure arterial pressure. Pacing electrodes were sutured to the left atrium, and balloon occluder cuffs were positioned around the inferior vena cava to permit alteration of left ventricular loading during the experiments. In addition, dogs underwent placement of a flow probe (Craig J. Hartley, Baylor College of Medicine Cardiovascular Sciences) around the circumflex artery. Post-operatively, the animals received trimethoprim/sulfadiazine 960 mg daily, for 7 days.

2.2 Experimental protocol
2.2.1 Whole animal studies
Post-operatively, the dog's temperature, body weight, and overall condition were monitored daily. Systemic infection was ruled out with blood cultures. The dogs were studied after full recovery from surgery, a period of at least 10 days. Data were collected as previously described [17] with the animals awake and unsedated. Studies were conducted after pre-treatment with propranolol (2 mg/kg) and atropine (2 mg) to permit atrial pacing at 160 bpm without the development of atrioventricular block. Approximately 10 min after infusion of propranolol and atropine, baseline hemodynamic data were acquired. After steady state recordings were made, vena caval occlusions were performed to define LV function over a range of loading conditions. Immediately after the recording period, the caval occlusion was released. Dogs were paced at 160 bpm for the entire course of study.

Following acquisition of baseline data, six dogs received 40 mg/kg L-NAME as a bolus injection. These experiments were designed to determine the influence of NOS inhibition on basal LV performance. This dose of L-NAME was chosen because it has been previously shown to markedly reduce NOS activity in dogs [20]. Ten min later, after stabilization of systemic pressure, hemodynamic recordings at steady state and during caval occlusion were obtained. Another five dogs received angiotensin II (AT II), rather than L-NAME, to isolate the effects of afterload, independent of NOS inhibition, on our hemodynamic measurements. These dogs were given 250–500 ng/kg AT II as a bolus injection, achieving a LV end-systolic pressure (LV-ESP) similar to that observed with the above dose of L-NAME. After stabilization of systemic pressure, hemodynamic recordings at steady state and during caval occlusion were obtained.

Following measures of baseline hemodynamics (post-hexamethonium 200 mg bolus), another group of 16 animals received 40 µg/kg of rhTNF{alpha} (lot EB6011, Knoll, Whippany, NJ, USA), a dose previously shown in our laboratory to induce LV dysfunction in conscious dogs [17]. Specific activity was 1·107 U/mg protein and the LPS content was <0.5 ng/mg protein (manufacturer's data). Individual doses of active rhTNF{alpha} were reconstituted in 20 ml of saline and administered over 1 h. Hemodynamic recordings before and during caval occlusions were obtained 6 h after finishing the rhTNF{alpha} infusion. Dogs received a repeat dose of hexamethonium (200 mg bolus) prior to this 6-h study.

Next, the dogs received either 40 mg/kg L-NAME (n=6), 0.50 mg/kg AT II (n=5), or saline (n=5). Fifteen min later, after stabilization of systemic pressure, hemodynamic recordings at steady state and during caval occlusion were obtained.

At the end of the studies, the animals were killed by lethal injection of pentobarbital sodium and potassium chloride. The hearts were excised and dissected to confirm proper positioning of the instruments and the absence of heartworms.

2.3 Data analysis
End-diastole was defined as the time of the peak of the QRS complex of the surface ECG. Under control conditions, end-systole was defined as the peak ratio of LV pressure to LV volume; for caval occlusion data, end-systole was determined using an iterative approach. LV volume, stroke-work (SW), dP/dtmax, and tau (the time constant of isovolumic relaxation) were determined by methods described previously [17]. For all of these parameters, the values for 20–25 consecutive beats, spanning two to three respiratory cycles, were averaged to yield a single result.

The slope and volume intercepts of the ESP–end-systolic volume (ESV) (Pes–Ves) relation [21], the SW–end-diastolic volume (SW–EDV) relation [22], and the dP/dtmax EDV (dP/dtmax–EDV) relation [23] were each determined using a linear least-squares algorithm. For the Pes–Ves relation, end-systolic data were fitted to the equation:

Formula
where Ees is the slope of the relation and V0 is its volume-axis intercept. To allow for comparisons between animals without excessive extrapolation in the pressure–volume plane, V100 was determined for each dog. V100 was the ESV calculated at an ESP of 100 mmHg, using the above equation and its derived slope and volume-axis intercept. For the SW–EDV relation the data were fitted to the equation:

Formula
where Mw is the slope of the relation and Vw is its volume-axis intercept. Vw1000 was defined as EDV derived at a SW of 1000 mmHg·ml. For the dP/dtmax–EDV relation, the data were fitted to the equation:

Formula
where dE/dtmax is the slope of the relation and EDV0 is its volume axis intercept. EDV2000 was defined as EDV derived at a dP/dtmax of 2000 mmHg/s. For analysis of these constructs, only caval occlusions that caused a reduction in LV-ESP of at least 20 mmHg were accepted.

The volume at the onset of ejection (Vej, ml) was defined as occurring at the upper right corner of the P–V loop. Stroke volume (ml) was defined as Vej–Ves; effective arterial elastance (EA, mmHg/ml) was defined as Pes/stroke volume during steady-state conditions [24].

2.4 Statistical analysis
All group data are expressed as mean±SEM. Comparisons between mechanical parameters in dogs before and after either L-NAME or AT II administration (or before and after rhTNF{alpha}) were made using the paired two-tailed t-test. For intragroup comparisons involving rhTNF{alpha}-treated animals (baseline, 6-h post-rhTNF{alpha} and either 6-h saline, L-NAME, or AT II), an analysis of variance of repeated measurements was performed; post-hoc comparisons were performed with the Student–Newman–Keuls test with significance set at p<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effects of L-NAME and AT II on hemodynamics and LV performance
At doses used in this study, L-NAME and AT II led to similar changes in steady state hemodynamics in control dogs not exposed to rhTNF{alpha}. These agents led to increases in mean arterial pressure (MAP) (L-NAME: 165.3±4.5 vs. baseline: 107.7±6.0 mmHg, p<0.001; AT II: 161.2±4.8 vs. baseline: 109.0±3.3 mmHg, p<0.001), LV-ESP (L-NAME: 174.4±7.3 vs. baseline: 110.1±6.4 mmHg, p<0.001; AT II: 173.3±6.4 vs. baseline: 114.8±2.4 mmHg, p<0.001), and LV end-diastolic pressure (LV-EDP) (L-NAME: 12.8±3.8 vs. baseline: 6.5±2.4 mmHg, p<0.05; AT II: 14.6± 1.6 vs. baseline: 6.9±1.5 mmHg, p<0.05) with similar increases in EA (p<0.01 for each). The increases in ventricular pressure following either L-NAME or AT II treatment led to similar increases in LV-EDV (L-NAME: 55.7±9.1 vs. baseline: 49.6± 7.6 ml, p<0.05; AT II: 71.6±3.6 vs. baseline: 65.1±2.8 ml, p<0.01) and LV-ESV (L-NAME: 47.6±7.7 vs. baseline: 38.9±6.4 ml, p<0.01; AT II: 62.0±3.4 vs. baseline: 50.6±2.7 ml, p<0.01) along with a prolongation of tau (L-NAME: 62.9±10.3 vs. baseline: 30.3±2.0 ms, p<0.05; AT II: 48.0±2.0 vs. baseline: 27.1±0.5 ms, p<0.001). Coronary blood flow, SW, and dP/dtmax were not affected by either treatment (data not shown).

Fig. 1 illustrates the Pes–Ves, SW–EDV, and dP/dtmax–EDV relations before and after treatment with either L-NAME or AT II. The respective Pes–Ves relations were essentially superimposable following exposure to either of these agents (Ees: L-NAME, 5.0±0.6 vs. baseline, 4.5±0.2 mmHg/ml; AT II, 4.7±0.4 vs. baseline, 4.5±0.4 mmHg/ml) with similar x-intercepts and derived values for V100. On the other hand, L-NAME and AT II shifted the SW–EDV relations to the right, leading to an increase in Vw and Vw1000, without affecting the slope (Mw: L-NAME, 74.2±9.0 vs. baseline, 74.9±2.0 mmHg; AT II, 70.6±10.7 vs. baseline, 67.7±3.2 mmHg). Regardless of whether the dogs were treated with L-NAME or AT II, the dP/dtmax–EDV relations pivoted on similar x-intercepts with tendencies toward a reduction in slope (dE/dtmax: L-NAME, 49.8±7.3 vs. baseline, 57.0±5.4 mmHg/ml/s, p=0.16; AT II, 37.2±2.8 vs. baseline, 43.1±4.2 mmHg/ml/s, p=0.056) and a rightward shift.


Figure 1
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  		Fig. 1 Influence of L-NAME (A, C, E) and AT II (B, D, F) under control conditions upon the end-systolic pressure–volume (A, B), stroke-work end-diastolic volume (C, D), and dP/dtmax end-diastolic volume (E, F) relationships. Points represent derived values for V100 and V0 (A, B), Vw1000 and Vw (C, D), and EDV2000 and EDV0 (E, F)±SEM. * p<0.05; {ddagger} p<0.01 post-L-NAME or post-AT II vs. respective baseline in each group.

 
Taken together, these data confirm that L-NAME and AT II have comparable hemodynamic effects, likely related to changes in afterload, in dogs not exposed to rhTNF{alpha}.

3.2 Effects of L-NAME and AT II on hemodynamics and LV performance following establishment of rhTNF{alpha}-induced myocardial depression
For the entire cohort of dogs (n=16), rhTNF{alpha} led to LV dilation (LV-EDV: 46.9±2.0 vs. 54.6±3.2 ml, post-rhTNF{alpha}, p<0.01; LV-ESV: 38.0±2.0 vs. 45.4±3.1 ml, post-rhTNF{alpha}, p<0.01) without a change in LV-EDP. Though not statistically significant, rhTNF{alpha} induced a drop in MAP (90.7±2.3 vs. 81.8±5.5 mmHg, post-rhTNF{alpha}) and LV-ESP (100.9±2.6 vs. 93.2±4.0 mmHg, post-rhTNF{alpha}). This drop in pressure was more prominent in the AT II group (Table 2), perhaps accounting for a less dramatic increase in LV volume. rhTNF{alpha} treatment significantly impaired dP/dtmax (2243±76 vs. 1674±66 mmHg/s, p<0.0001) with prolongation of tau (25.8±0.7 vs. 29.6±1.3 ms, p<0.01). Coronary blood flow, SW, and EA were not affected by rhTNF{alpha}. Each group of dogs responded to rhTNF{alpha} in a comparable fashion (Tables 1 and 2Go), including those dogs subsequently given saline (data not shown).


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  		Table 2 Effects of rhTNF{alpha} on hemodynamic parameters obtained from steady state runsa

 

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  		Table 1 Effects of rhTNF{alpha}, L-NAME on hemodynamic parameters obtained from steady state runsa

 
L-NAME and AT II treatment, following 6 h exposure to rhTNF{alpha}, led to nearly identical increases in MAP, LV-ESP, and LV-EDP. In addition, both L-NAME and AT II increased EA. The increase in ventricular pressure was accompanied by a similar degree of ventricular enlargement and a marked prolongation of tau. LV dP/dtmax was not affected by subsequent treatment with L-NAME or AT II. Coronary blood flow tended to increase after both L-NAME and AT II, though the change was not statistically significant. Steady state hemodynamic parameters were not altered in the rhTNF{alpha}-treated dogs given saline (data not shown). The influence of L-NAME and AT II 6 h after rhTNF{alpha} administration on steady state hemodynamics was comparable to that observed in our control dogs (not exposed to rhTNF{alpha}) given these agents.

Fig. 2 illustrates the Pes–Ves, SW–EDV, and dP/dtmax–EDV relations at baseline and 6 h following rhTNF{alpha}, before and after either L-NAME or AT II. rhTNF{alpha} led to a comparable reduction in end-systolic elastance in each group (saline group: 7.4±1.0 vs. 4.8±0.5 mmHg/ml, post-rhTNF{alpha} p<0.05; L-NAME group: 8.7±1.0 vs. 4.9±1.0 mmHg/ml, post-rhTNF{alpha} p<0.05; AT II group: 6.0±0.3 vs. 4.5±0.5 mmHg/ml, post-rhTNF{alpha} p<0.05). Each Pes–Ves relation pivoted on a similar x-intercept and was shifted rightward. During rhTNF{alpha}-induced contractile impairment, treatment with saline, L-NAME, or AT II did not alter the Pes–Ves relation (Fig. 2A, B). rhTNF{alpha} adversely affected the SW–EDV relation in each group of dogs, leading to a consistent decline in the slope, Mw (saline group: 58.4±3.0 vs. 50.2±4.8 mmHg, post-rhTNF{alpha} p<0.05; L-NAME group: 68.2±6.7 vs. 53.6±3.4 mmHg, post-rhTNF{alpha} p<0.05; AT II group: 64.3±4.5 vs. 49.4±3.0 mmHg, post-rhTNF{alpha} p<0.05). rhTNF{alpha} treatment shifted each SW–EDV relation to the right in the physiologic range (Vw1000). Six h after rhTNF{alpha}, saline did not change the SW–EDV relation (data not shown). L-NAME (Fig. 2C) and AT II (Fig. 2D) did not affect Mw (L-NAME: 50.4±11.0 vs. rhTNF{alpha}: 53.6±3.4 mmHg, p=nonsignificant (NS); AT II group: 56.5±3.0 vs. rhTNF{alpha}: 49.4±3.0 mmHg, p=NS), but the SW–EDV relations were shifted even further rightward, leading to significant increases in Vw for each group.


Figure 2
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  		Fig. 2 Effect of rhTNF{alpha} on the end-systolic pressure–volume (A, B), stroke-work end-diastolic volume (C, D), and dP/dtmax end-diastolic volume (E, F) relationships before and after L-NAME (A, C, E) or AT II (B, D, F). Points represent derived values for V100 and V0 (A, B), VW1000 and VW (C, D), and EDV2000 and EDV0 (E, F). * p<0.05 TNF{alpha} vs. pre-TNF{alpha}, {dagger} p<0.05 L-NAME or AT II vs. respective value post-TNF{alpha} in each group. Dogs in the saline control group behaved similarly following rhTNF{alpha} and were not affected by subsequent saline infusion.

 
As shown in Fig. 2(E, F), rhTNF{alpha} decreased dE/dtmax, the slope of the dP/dtmax–EDV relation (saline group: 62.6±12.3 vs. 35.1±8.5 mmHg/ml/s, post-rhTNF{alpha} p<0.05; L-NAME group: 74.7±8.3 vs. 39.7±5.7 mmHg/ml/s, post-rhTNF{alpha} p<0.05; AT II group: 61.8±2.8 vs. 42.0±5.5 mmHg/ml/s, post-rhTNF{alpha} p<0.05). In the physiologic range this was accompanied by a rightward shift in each of the relations. Treatment with saline 6 h after rhTNF{alpha} did not affect the dP/dtmax–EDV relation (data not shown). Both L-NAME and AT II led to further declines in dE/dtmax (post-rhTNF{alpha}: 39.7±5.7 vs. L-NAME: 28.8±4.6 mmHg/ml/s, p<0.05; post-rhTNF{alpha}: 42.0±5.5 vs. AT II: 28.1±2.9 mmHg/ml/s, p<0.05) and additional rightward shifts in the physiologic range.

In summary, as we have previously seen, rhTNF{alpha} led to depression of LV systolic function 6 h after administration. At that time, elevation of blood pressure with either L-NAME or AT II caused LV dilation, increased LV-EDP, and impaired LV relaxation. While effects on the Pes–Ves relation were neutral, these compounds led to rightward shifts of the SW–EDV relation without change in slope, and further reductions in slope and rightward shifts in the dP/dtmax–EDV relation.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Our data show that contractile depression induced by rhTNF{alpha} is not improved by acute inhibition of NOS, and that while NOS inhibition leads to increased arterial pressure, this may have deleterious effects on the heart. The similar response of hearts 6 h after exposure to rhTNF{alpha} to either L-NAME or AT II suggests that inhibition of NOS had no direct effect on myocardial systolic performance, and that changes seen in cardiac function were reflective of responses to altered loading conditions.

While evidence strongly suggests that NO does not play an appreciable role in myocardial systolic function under normal conditions [8,9,16], overproduction of NO within cardiac tissue has been proposed to mediate endotoxin- and cytokine-induced contractile dysfunction. Myocytes, like vascular endothelial and smooth muscle cells [1,25], have the capacity to express the inducible isoform of NOS after treatment with TNF{alpha}, leading to abundant synthesis of NO [13]. Brady et al. have shown that superfusion with NO solution or the NO donor, nitroprusside, at concentrations above the physiologic range attenuates cardiac myocyte contraction, suggesting a direct negative inotropic effect of NO, possibly via cGMP production [26]. Myocytes obtained from guinea pigs exposed to endotoxin, a potent stimulus for TNF{alpha} release, exhibit contractile impairment which was reversed by L-NAME [27]. Studies performed by Finkel et al. in isolated Syrian hamster papillary muscle have provided evidence that NOS inhibition can prevent TNF{alpha}-mediated contractile dysfunction [16]. Thus, a body of evidence supports the hypothesis that NO overproduction is directly responsible for TNF{alpha}-induced myocardial depression.

Our data refute the notion that NO is the final common pathway of TNF{alpha}-induced myodepression in intact dogs. To the contrary, once LV dysfunction is established following exposure to TNF{alpha}, the adverse influence of enhanced afterload associated with NOS inhibition appears to promote further deterioration of LV performance. As was seen in dogs at steady state, L-NAME led to additional ventricular dilation, increases in ESP and EDP, and marked prolongation of isovolumic relaxation. These findings have been observed in other studies following an abrupt increase in afterload [28,29]. Moreover, load-independent force-based measures of LV contractility (Ees, Mw), markedly reduced by TNF{alpha}, were maintained but did not normalize after L-NAME. The SW–EDV relation was consistently shifted to the right following NOS inhibition, suggesting reduced mechanical performance. This shift is reflected by an increase in Vw1000 after L-NAME such that larger EDVs were required to generate similar levels of SW within the physiologic range. Given that AT II treatment produced similar findings after rhTNF{alpha}, it appears that NOS inhibition affects LV systolic performance via altered ventriculovascular coupling rather than via any direct effect of NO suppression at the level of the myocardium. Impairment of LV relaxation after L-NAME is likely due to both increases in afterload and blockade of NO production [7,28].

Interestingly, both L-NAME and AT II treatment, at steady state and after rhTNF{alpha}, promoted a decline in dE/dtmax, despite preservation of Ees and Mw. Tau, another time-dependent parameter, was also adversely affected by these agents. The different effects of L-NAME and AT II on velocity-based and force-based parameters of contractility are probably related to the degree of afterload stress they produce. At much lower LV systolic pressure and EA, other studies, including those from our own laboratory, have shown that AT II has a neutral effect on both the dP/dtmax–EDV and SW–EDV relations [28,29]. We have recently reported that dissociation of velocity-based and force-based parameters can be produced by ryanodine, an inhibitor of sarcoplasmic reticulum Ca2+ release [30]. Taken together, these findings offer the possibility that large increases in afterload can affect sarcoplasmic reticulum Ca2+ handling. Further studies need to be performed to address these issues.

The inability of L-NAME to reverse TNF{alpha}-induced myodepression in conscious dogs is consistent with other studies. In neonatal rat myocytes, LPS, a potent inducer of TNF{alpha} synthesis and release, has been shown to cause a delayed, time-dependent expression of iNOS mRNA and protein with proportional increases in nitrate and intracellular cGMP [14]. NOS inhibition prevented, but could not reverse, LPS-mediated decreases in peak systolic [Ca2+]i and relative amplitude of cell contraction [14]. In contrast, NOS inhibitors have been ineffective in either preventing or reversing LPS-induced adult rat ventricular dysfunction [31,32]. Quezado et al. [18] evaluated the effects of N{omega}-monomethyl-L-arginine (L-NMMA) given alone or in conjunction with TNF{alpha} in conscious dogs instrumented with pulmonary artery catheters. Consistent with our findings, they found L-NMMA, either in the presence or absence of TNF{alpha}, increased MAP at the expense of decreased cardiac output and increased ventricular filling pressures. They also found deterioration of LV performance as measured by radionuclide gated blood pool scans [18]. However, cardiac output and LV ejection fraction are load-sensitive parameters of cardiac function; the studies by Quezado et al. were not controlled for heart rate or the influence of L-NMMA on afterload [18]. Our studies extend these observations to conscious dogs with controlled heart rate using pressure–volume plane analyses and controlling for the influence of afterload with AT II.

Since NOS inhibitors have been proposed as therapeutic agents to counteract the cardiovascular collapse observed in septic shock [2], our results have direct clinical applicability. TNF{alpha}, produced and released in response to LPS [33] has been shown to play a significant role in the pathophysiology of the cardiovascular derangements in sepsis [34]. As with TNF{alpha}, overproduction of NO has been postulated as the mechanism for LPS-induced hypotension, vascular hyporeactivity, and LV dysfunction [2–5,27]. Similar to our study, NOS inhibitors have been effective in preventing and reversing LPS-induced hypotension, but cause reductions in cardiac output and regional blood flow, impaired oxygen delivery and diminished arterial pH and pO2. [18,19]. Moreover, continuous infusion of L-NMMA in patients with septic shock has been shown to similarly improve arterial pressure while increasing pulmonary vascular resistance, pulmonary capillary wedge pressure, central venous pressure and decreasing cardiac output [2]. Our study complements these data as the increase in afterload stress following L-NAME leads to an increased LV-EDP, prolonged isovolumic relaxation, and impaired velocity-based indexes of LV systolic performance (dE/dtmax).

Our study must be interpreted in light of methodological limitations. First, our analysis assumes linearity of the Pes–Ves, SW–EDV, and dP/dtmax–EDV relations. In a similar intact canine model, Little et al. [35] have described a slight but consistent curvilinearity of the Pes–Ves relation independent of inotropic state. While alterations in afterload might affect the curvilinearity of each relation, our linear regression correlation coefficients remained high in each animal. Secondly, we cannot rule out the possibility of myocardial ischemia related to changes in afterload following L-NAME and AT II. None of the dogs developed ischemic changes on the ECG following administration of either AT II or L-NAME, and L-NAME and AT II did not significantly affect coronary blood flow, either at baseline or after TNF{alpha}. The latter finding was not surprising since NOS inhibition with L-NAME has been shown to have little effect on coronary blood flow in conscious dogs [36]. Unless significant intramyocardial redistribution of blood flow and oxygen delivery occurred, afterload mediated ischemia is unlikely. To address this issue further, studies using coronary sinus lactate sampling need to be conducted. Lastly, this study was performed after treatment with agents to limit changes in autonomic tone following caval occlusion. Conceivably, the effects of rhTNF{alpha} and L-NAME may be modified in conscious animals with intact reflexes.

In conclusion, our results demonstrate that NOS inhibition does not influence steady state force-based indexes of contractility (Ees, Mw), suggesting that basal NO production is not involved in regulating LV systolic performance under control conditions. Deterioration of velocity-based measures of LV contractility (dE/dtmax), prolongation of tau, and rightward shifts of the SW–EDV and dE/dtmax–EDV relations were mimicked by AT II, suggesting a nonspecific effect of afterload in this model. Similarly, while NOS inhibition is effective in enhancing arterial pressure, it did not normalize established ventricular dysfunction caused by rhTNF{alpha}. To the contrary, NOS inhibition appears to be detrimental to the depressed heart as evidenced by increased LV-EDP and prolonged velocity-based indexes of LV contraction and relaxation. The results of this study imply that either NO is not involved in TNF{alpha}-induced myodepression or that NO exerts its effect via toxic intermediaries, rendering delayed blockade of iNOS ineffective in reversing myocardial injury. As TNF{alpha} is a primary mediator of the cardiovascular derangements which accompany septic shock, treatment of patients with NOS inhibitors in this condition to improve blood pressure could lead to LV dysfunction, pulmonary edema and impaired oxygen delivery.

Time for primary review 37 days.


   Acknowledgements
 
The authors thank Danny Escobedo and Cindy Ramirez for excellent technical assistance, Robin E. Morris for processing the manuscript, Merardo Monzon for software development, and John Schoolfield for statistical consultation. We also wish to thank Knoll Pharmaceuticals for providing rhTNF{alpha}. This work was supported by an American Heart Association Grants-in-Aid (D.R. Murray, S.D. Prabhu) and the Research Service of the Department of Veteran's Affairs (G.L. Freeman, S.D. Prabhu, D.R. Murray).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Moncada S., Palmer R.M.J., Higgs E.A. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev (1991) 43:109–142.[Web of Science][Medline]
  2. Petros A., Lamb G., Leone A., et al. Effects of a nitric oxide synthase inhibitor in humans with septic shock. Cardiovas Res (1994) 28:34–39.[Abstract/Free Full Text]
  3. Julou-Schaeffer G., Gray G.A., Fleming I., et al. Loss of vascular responsiveness induced by endotoxin involves the L-arginine pathway. Am J Physiol (1990) 259:H1038–H1043.[Web of Science][Medline]
  4. Kilbourn R.G., Gross S.S., Jubran A., et al. NG-methyl-L-arginine inhibits tumor necrosis factor-induced hypotension: implications for the involvement of nitric oxide. Proc Natl Acad Sci USA (1990) 87:3629–3632.[Abstract/Free Full Text]
  5. Szabo C., Mitchell J.A., Thiemermann C., Vane J.R. Nitric oxide mediated hyporeactivity to noradrenaline precedes nitric oxide synthase induction in endotoxic shock. Br J Pharmacol (1993) 108:786–792.[Web of Science][Medline]
  6. Kelly R.A., Balligand J.-L., Smith T.W. Nitric oxide and cardiac function. Circ Res (1996) 79:363–380.[Free Full Text]
  7. Shah A.M. Paracrine modulation of heart cell function by endothelial cells. Cardiovasc Res (1996) 31:847–867.[Abstract/Free Full Text]
  8. Paulus W.J., Vantrimpont P.J., Shah A.M. Paracrine coronary endothelial control of left ventricular function in humans. Circulation (1995) 92:2119–2126.[Abstract/Free Full Text]
  9. Paulus W.J., Vantrimpont P.J., Shah A.M. Acute effects of nitric oxide on left ventricular relaxation and diastolic distensibility in humans: assessment by bicoronary sodium nitroprusside infusion. Circulation (1994) 89:2070–2078.[Abstract/Free Full Text]
  10. Balligand J.-L., Kelly R.A., Marsden P.A., et al. Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. Proc Natl Acad Sci USA (1993) 90:347–351.[Abstract/Free Full Text]
  11. Finkel M.S., Oddis C.V., Mayer O.H., Hattler B.G., Simmons R.L. Nitric oxide synthase inhibitor alters papillary muscle force–frequency relationship. J Pharmacol Exp Ther (1995) 272:945–952.[Abstract/Free Full Text]
  12. Mohan P., Brutsaert D.L., Paulus W.J., Sys S.U. Myocardial contractile response to nitric oxide and cGMP. Circulation (1996) 93(6):1223–1229.[Abstract/Free Full Text]
  13. Schulz R., Nava E., Moncada S. Induction and potential biological relevance of a Ca2+-independent nitric oxide synthase in the myocardium. Br J Pharmacol (1992) 105:575–580.[Web of Science][Medline]
  14. Kinugawa K.-I., Kohmoto O., Yao A., Serizawa T., Takahashi T. Cardiac inducible nitric oxide synthase negatively modulates myocardial function in cultured rat myocytes. Am J Physiol (1997) 272(Heart Circ Physiol 41):H35–H47.[Web of Science][Medline]
  15. Yokoyama T., Vaca L., Rossen R.D., et al. Cellular basis for the negative inotropic effects of tumor necrosis factor-{alpha} in the adult mammalian heart. J Clin Invest (1993) 92:2303–2312.[Web of Science][Medline]
  16. Finkel M.S., Oddis C.V., Jacob T.D., et al. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science (1992) 257:387–389.[Abstract/Free Full Text]
  17. Murray D.R., Freeman G.L. Tumor necrosis factor-{alpha} induces a biphasic effect on myocardial contractility in conscious dogs. Circ Res (1996) 78:154–160.[Abstract/Free Full Text]
  18. Quezado Z.M.N., Karzai W., Danner R.L., et al. Effects of L-NMMA and fluid loading on TNF-induced cardiovascular dysfunction in dogs. Am J Respir Crit Care Med (1998) 157:1397–1405.[Web of Science][Medline]
  19. Mitaka C., Hirata Y., Ichikawa K., et al. Effects of nitric oxide synthase inhibitor on hemodynamic change and O2 delivery in septic dogs. Am J Physiol (1995) 268(Heart Circ Physiol 37):H2017–H2023.[Web of Science][Medline]
  20. Breslow M.J., Tobin J.R., Bredt D.S., et al. Nitric oxide as a regulator of adrenal blood flow. Am J Physiol (1993) 264(Heart Circ Physiol 33):H464–H469.[Web of Science][Medline]
  21. Kass D.A., Maughan W.L. From "EMAX" to pressure–volume relations: a broader view. Circulation (1988) 77:1203–1212.[Free Full Text]
  22. Glower D.D., Spratt J.A., Snow N.D., et al. Linearity of the Frank–Starling relationship in the intact heart: the concept of preload recruitable stroke work. Circulation (1985) 71:994–1009.[Abstract/Free Full Text]
  23. Little W.C. The left ventricular dP/dtmax–end-diastolic volume relation in closed chest dogs. Circ Res (1985) 56:808–815.[Abstract/Free Full Text]
  24. Sunagawa K., Maughan W.L., Burkhoff D., Sagawa K. Left ventricular interaction with arterial load studied in isolated canine ventricle. Am J Physiol (1983) 245:H773–H780.[Web of Science][Medline]
  25. Shibano T., Vanhoutte P.M. Induction of NO production by TNF and lipopolysaccharide in porcine coronary arteries without endothelium. Am J Physiol (1993) 264:H403–H407.[Web of Science][Medline]
  26. Brady A.J.B., Warren J.B., Poole-Wilson P.A., Williams T.J., Harding S.E. Nitric oxide attenuates cardiac myocyte contraction. Am J Physiol (1993) 265(Heart Circ Physiol 34):H176–H182.[Web of Science][Medline]
  27. Brady A.J.B., Poole-Wilson P.A., Harding S.E., Warren J.B. Nitric oxide production within cardiac myocytes reduces their contractility in endotoxemia. Am J Physiol (1992) 263:H1963–H1966.[Web of Science][Medline]
  28. Freeman G.L., Little W.C., O’Rourke R.A. The effect of vasoactive agents on the left ventricular end-systolic pressure–volume relation in closed-chest dogs. Circulation (1986) 74:1107–1113.[Abstract/Free Full Text]
  29. Cheng C.-P., Suzuki M., Ohte N., et al. Altered ventricular and myocyte response to angiotensin II in pacing-induced heart failure. Circ Res (1996) 78:880–892.[Abstract/Free Full Text]
  30. Prabhu S.D., Rozek M.M., Murray D.R., Freeman G.L. Ryanodine and left ventricular function in intact dogs: dissociation of force-based and velocity-based indexes. Am J Physiol (1997) 273(Heart Circ Physiol 42):H1561–H1568.[Web of Science][Medline]
  31. Klabunde R.E., Coston A.F. Nitric oxide synthase inhibition does not prevent cardiac depression in endotoxic shock. Shock (1995) 3:73–78.[Web of Science][Medline]
  32. Meng X.-L., Brown J.M., Fullerton D.A., Banerjee A., Harken A.H. Nitric oxide synthase is not involved in cardiac contractile dysfunction in a rat model of endotoxemia without shock. Shock (1997) 7:111–118.[Web of Science][Medline]
  33. Michie H.R., Manogue K.R., Spriggs D.R., et al. Detection of circulating tumor necrosis factor after endotoxin administration. New Engl J Med (1988) 318:1481–1486.[Abstract]
  34. Tracey K.J., Fong Y., Hesse D.G., et al. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteremia. Nature (Lond) (1987) 330:662–664.[CrossRef][Medline]
  35. Little W.C., Cheng C.P., Peterson T., Vinten-Johansen J. Response of the left ventricular end-systolic pressure–volume relation in conscious dogs to a wide range of contractile states. Circulation (1988) 78:736–745.[Abstract/Free Full Text]
  36. Smith T.P. Jr., Canty J.M. Modulation of coronary autoregulatory responses by nitric oxide: evidence for flow-dependent resistance adjustments in conscious dogs. Circ Res (1993) 73:232–240.[Abstract/Free Full Text]

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