We tested whether or not endogenous nitric oxide (NO) attenuates β-adrenergic inotropic responsiveness during normoperfusion or moderate myocardial ischemia. In 13 anesthetized pigs with a cannulated left anterior descending (LAD) coronary artery, the maximal contractile responses to intracoronary dobutamine and calcium were assessed during normoperfusion and at the end of a 90-min period of moderate ischemia (50% reduction in coronary arterial inflow) without (group 1, n = 6) and with (group 2, n = 7) prior inhibition of NO synthesis [30 mg/kg iv N ω-nitro-l-arginine (l-NNA)]. Contractile function was assessed by a regional work index (sonomicrometry, micromanometry, mm · mmHg). Ingroups 1 and 2 during normoperfusion, the maximal increase of the work index was greater with calcium than with dobutamine. At the end of ischemia in group 1, the baseline work index was decreased by ∼50%, and the subsequent maximal increase of the work index with dobutamine, but not with calcium, was reduced compared with normoperfusion. In group 2 during normoperfusion, l-NNA did not alter the maximal increases of the work index with dobutamine or calcium. At the end of ischemia, the baseline work index was reduced by 64%, and the subsequent maximal increases of the work index with both dobutamine and calcium were reduced compared with normoperfusion; however, the response to calcium was still greater than that to dobutamine. We conclude that endogenous NO does not limit β-adrenergic inotropic responsiveness in normoperfused or moderately ischemic porcine myocardium.
- nitric oxide
- myocardial function
nitric oxide (NO) can depress the cardiomyocyte response to β-adrenergic stimulation. In rat ventricular myocytes and atria, inhibition of NO synthesis potentiated the positive inotropic response to isoproterenol (1,23). In anesthetized dogs in vivo, intracoronary administration of an inhibitor of NO synthesis did not change the contractile response to dobutamine and isoproterenol at rest (3, 12), but did so during autonomic blockade (13) or vagal stimulation (6). In nonfailing human hearts, intracoronary infusion of an inhibitor of NO synthesis did not affect the contractile response to dobutamine (5), whereas stimulation of NO synthesis bysubstance P markedly attenuated the positive contractile effect of dobutamine (2). Accordingly, in patients with left ventricular (LV) dysfunction, in which NO release may be elevated due to expression of inducible nitric oxide synthase (NOS) (8), inhibition of NO synthesis increased the contractile (7) and metabolic (22) responses to dobutamine. It thus appears that in vivo elevated levels of endogenous NO have the potential to limit adrenergic inotropic responsiveness.
The release of NO is increased during myocardial ischemia in anesthetized dogs (18), pigs (9), and conscious rabbits (28) and may impact on myocardial β-adrenergic responsiveness. In anesthetized dogs (18), inhibition of NO synthesis withN ω-nitro-l-arginine methyl ester (l-NAME) increased the inotropic response to isoproterenol during acute moderate myocardial ischemia. However, in this study myocardial oxygen consumption was not limited, and increased during hypoperfusion in l-NAME treated compared with placebo animals before and during the isoproterenol infusion. Furthermore, in separate experiments, increases of regional myocardial function and oxygen consumption in response to intracoronary calcium were also potentiated by l-NAME, therefore, allowing no conclusive statement on the specific effects of NO on β-adrenergic responsiveness during ischemia.
We therefore investigated whether or not endogenous NO decreases β-adrenergic responsiveness during myocardial ischemia in an established model of constant low-flow ischemia in pigs (9, 10, 21). To assess whether or not the recruitment of an inotropic response by β-adrenoceptor stimulation is limited, the maximal contractile effect of dobutamine was compared with that of β-adrenoceptor independent inotropic stimulation by calcium. Because repetitive inotropic stimulation of ischemic myocardium is likely to deplete energy stores and thereby to decrease contractile responses, this comparison was performed in two separate groups of animals, with and without inhibition of NO synthesis.
Experimental protocols employed in this study were approved by the bioethical committee of the district of Düsseldorf, Germany, and they adhere to the guiding principles of the American Physiological Society.
Thirteen Göttinger miniswine were instrumented as described previously (9, 10, 21). In brief, swine were anesthetized with enflurane and N2O, and both common carotid arteries were cannulated, one to measure arterial pressure and one to supply blood for an extracorporeal circuit. A micromanometer was implanted into the left ventricle (LV) through the apex for LV pressure recording. Ultrasonic dimension gauges were implanted in the LV myocardium to measure the thickness of the anterior wall. The left anterior descending (LAD) coronary artery and vein were cannulated, and the artery was perfused from an extracorporeal circuit including a roller pump. The extracorporeal circuit included sidearms for the intracoronary injection of microspheres, dobutamine, and calcium. With the use of an occlusive roller pump, a greater fraction of blood flow occurs during systole, thereby possibly elevating mean coronary arterial pressure. To avoid diastolic hypoperfusion of subendocardial layers, we adjusted coronary inflow to keep minimal coronary arterial pressure above 75 mmHg before the onset of ischemia. Mean coronary arterial pressure, therefore, exceeds LV peak pressure under normoperfusion conditions. Coronary venous blood was drained to an unpressurized reservoir and then returned to a jugular vein by use of a second roller pump. Heart rate was held constant by left atrial pacing.
Regional Myocardial Function
End diastole was defined as the point when the first derivative of LV pressure (LV dP/dt) started its rapid upstroke after crossing the 0 line, and regional end systole as the point of maximal wall thickness within 20 ms before peak negative LV dP/dt(26). Regional myocardial work was estimated as a work index (WI) derived from the regional LV pressure-wall thickness relationship (10). Beginning at end diastole, every 5 ms the difference between the actual LV pressure and the minimal LV pressure was multiplied by the change in wall thickness, and the products obtained during systole were added, their sum representing the WI. Because regional inotropic stimulation leads to accelerated wall thickening and late systolic wall thinning, i.e., ventricular asynchrony, the maximum rather than the end-systolic WI value is reported. WI was calculated using the equation where ed = end diastole, n = actual cardiac cycle, m = sampling point within cardiac cyclen at a sampling interval of 5 ms, LVPn,m = instantaneous LV pressure within cardiac cycle n and at sampling point m, LVPmin = minimum LV pressure, and Wth = wall thickness.
Regional Myocardial Blood Flow and Metabolism
Radiolabeled microspheres (15-μm diameter, 141Ce,114In, 103Ru, 95Nb, or46Sc; DuPont NEN, Boston, MA) were injected into the coronary perfusion circuit (1.5–3 × 105suspended in 1 ml of saline) to determine regional myocardial blood flow and its distribution throughout the LAD coronary artery perfusion bed. Regional blood flows at the crystal site are reported. Oxygen content was measured using anaerobically sampled blood drawn simultaneously from the cannulated coronary vein and an artery (ABL 610, Radiometer). Oxygen consumption of the anterior wall was calculated by multiplying the arterial-coronary venous difference by the coronary inflow and normalized to the area at risk, as determined by the microspheres.
Postmortem, each heart was sectioned into 6–7 transverse slices and incubated in 1% triphenyltetrazolium chloride (TTC) to verify the absence of necrotic tissue.
Group 1 (n = 6).
During normoperfusion, microspheres were injected into the extracorporeal circuit, systemic hemodynamic and regional myocardial dimension data were recorded, and arterial and coronary venous blood samples were drawn. Dobutamine hydrochloride (Lilly; Giessen, Germany) was infused intracoronarily at increasing doses until WI did not increase further. The infusion was then stopped, and when hemodynamic and regional myocardial dimension data had returned to baseline values, calcium chloride (CaCl2 · 2 H2O, Sigma; Deisenhofen, Germany) was infused intracoronarily at increasing doses until WI did not increase further. The maximal increases of WI with dobutamine and calcium infusion are referred to as inotropic reserve. During the dobutamine or calcium infusion, coronary inflow was increased to keep mean coronary arterial pressure constant, and blood samples were taken when WI had reached a plateau. When the effects of calcium had subsided, coronary inflow was decreased by 50% for 90 min. At 10 and 75 min of ischemia, further sets of measurements were obtained. Thereafter, increasing doses of dobutamine, representing the dose-response relationship under normoperfusion conditions, were infused. Five minutes after the end of the dobutamine infusion, increasing doses of calcium were infused. The infusion rates of dobutamine and calcium were adapted to the decreased coronary inflow during ischemia. Blood samples were taken at the maximum increase in WI. After 90 min of ischemia, the myocardium was reperfused for 2 h to verify the absence of necrotic tissue by TTC staining.
Group 2 (n = 7).
During normoperfusion, measurements of systemic hemodynamics, regional myocardial blood flow and function were taken, blood samples were drawn, and the infusion of dobutamine and calcium was performed as ingroup 1. Thereafter, 30 mg/kgN ω-nitro-l-arginine (l-NNA) (Sigma) were infused intravenously over 30 min. This dose has previously been shown to abolish the bradykinin-induced decrease in mean coronary resistance and NO production during ischemia (9). After infusion of l-NNA, another set of measurements was obtained and infusion of dobutamine and calcium was repeated. Coronary inflow was then decreased by 50% for 90 min, and the protocol was continued as in group 1.
Data Analysis and Statistics
Systemic hemodynamic and regional myocardial dimension data were digitized and recorded over a 20-s period during each measurement using CORDAT II software (9). Parameters reported are LV end-diastolic and peak pressures, the maximum LV dP/dt (LV dP/dt max), mean coronary arterial pressure, mean coronary inflow, WI of the anterior wall, and regional blood flows.
The EC50 values for dobutamine and calcium were obtained by plotting the percent increase in the regional WI against the logarithm of the drug concentration in the coronary arterial blood. The plots were fitted by a sigmoidal curve with variable slope using GraphPad Prism 3.0 software (San Diego, CA). The quality of fits exceeded a squared r of 0.85 with exception of the response to dobutamine at the end of the ischemic period in group 2 in which, therefore, a EC50 was not calculated.
Data are presented as means ± SE. Statistical analysis was performed using SigmaStat software (Urbana, IL). In each group, changes in systemic hemodynamics, regional myocardial function, blood flow and oxygen consumption, inotropic reserve, and EC50 were evaluated by a one-way ANOVA for repeated measurements. Data on inotropic reserve obtained at late ischemia were compared between groups using a two-way ANOVA. When ANOVA revealed a significant overall effect, the Student-Newman post hoc test was applied.
Systemic Hemodynamics and Regional Myocardial Oxygen Consumption
Heart rate was kept constant by left atrial pacing and averaged 100 ± 2 beats/min in group 1 and 100 ± 1 beats/min in group 2. Baseline values before dobutamine and calcium infusions were not different at a given time point throughout the protocol in both groups.
LV dP/dt max, coronary inflow and regional myocardial oxygen consumption during normoperfusion conditions were increased by dobutamine and calcium in both groups (Tables1 and 2). After l-NNA infusion in group 2, LV peak pressure and mean coronary arterial pressure were increased, whereas baseline coronary inflow, regional blood flow, and oxygen consumption remained unchanged. LV dP/dt max, coronary inflow and oxygen consumption were increased to a similar extent as before l-NNA by dobutamine and calcium infusion. With dobutamine or calcium infusion at late ischemia, dP/dt max increased in both groups, whereas regional myocardial oxygen consumption in the presence of a constantly reduced coronary inflow remained unchanged.
Regional Myocardial Function and Inotropic Reserve in Response to Dobutamine or Calcium
Data on absolute WI of groups 1 and 2 are presented in Tables 1 and 2. During normoperfusion, the inotropic reserve in response to calcium was greater than that to dobutamine in both groups (Figs. 1 and2). Although baseline WI was reduced byl-NNA (group 2, Table 2), the inotropic reserves in response to dobutamine or calcium remained unchanged compared with normoperfusion (Fig. 2).
In group 1 at the end of ischemia, the inotropic reserve in response to calcium was preserved, whereas that to dobutamine was reduced (Fig. 1). After l-NNA (group 2) at the end of ischemia, the inotropic reserves in response to both dobutamine and calcium were reduced; however, the response to calcium was still greater than that to dobutamine (Fig. 2). The inotropic reserves between groups 1 and2 at the end of ischemia did not differ significantly.
The calculated EC50 values for dobutamine (60 ± 7 vs. 65 ± 1 ng/ml) and calcium (76 ± 13 vs. 75 ± 15 μg/ml) were unchanged in group 1. In group 2, the EC50 values for dobutamine and calcium were decreased after l-NNA from 77 ± 10 to 41 ± 6 ng/ml (P < 0.05) and from 62 ± 9 to 42 ± 4 μg/ml, respectively. After l-NNA, the EC50value for calcium increased at late ischemia (117 ± 19 μg/ml; P < 0.05 vs. normoperfusion).
In none of the experiments was necrosis detected by TTC staining.
The main findings of the present study are: 1) in normoperfused myocardium the maximum inotropic reserve in response to intracoronary calcium is greater than that in response to intracoronary dobutamine; 2) inhibition of NO synthesis decreases baseline regional myocardial function, but does not alter the inotropic responses to calcium and to dobutamine during normoperfusion; and3) inhibition of NO synthesis does not prevent the decrease of the inotropic reserve in response to dobutamine after 75–90 min moderate ischemia.
Dobutamine versus NO as cardiac inotropes
The strengths and limitations of the experimental model have been discussed in detail previously (10, 21). The regional WI derived from the LV pressure-wall thickness loop correlates well with the stress-strain loop in dogs during exercise and ischemia (16) and, therefore, provides a reliable measure to determine changes in regional external myocardial work.
At the end of ischemia, the infusion of dobutamine is likely to deplete the energy stores of the myocardium (10, 21), and therefore, the subsequent inotropic response to calcium might have been underestimated. However, the inotropic reserve in response to calcium was preserved in group 1, and a reduced inotropic reserve in response to calcium after 75–90 min of moderate ischemia during inhibition of NO synthesis has been described previously (9) in the same model in the absence of prior dobutamine infusion. Because the inotropic response to calcium is preserved, the decreased inotropic response to dobutamine cannot simply result from reduced energy stores; and furthermore, creatine phosphate content at late ischemia is similar to that at baseline in this animal model (9).
In group 2, the regional WI was increased by 101 ± 9 mm · mmHg with dobutamine and by 169 ± 18 mm · mmHg with calcium, whereas it was decreased by 67 ± 14 mm · mmHg after inhibition of NO synthesis byl-NNA (Fig. 2). Thus endogenous NO elicits a significant inotropic effect (∼40% of the maximal response to calcium). We (9) have previously shown that such inotropic effect of endogenous NO is independent of loading conditions and discussed potential mechanisms in detail.
β-Adrenergic Responsiveness in Normoperfused and Moderately Ischemic Myocardium
During normoperfusion, the inotropic reserve with calcium was by 50–70% greater than that with dobutamine, although oxygen consumption during calcium and dobutamine infusion was increased to the same extent. Similar findings were previously obtained in isolated rat hearts (19) and dog hearts (17) in vivo in which, for a given increase in global LV function, myocardial oxygen consumption increased more with isoproterenol than with calcium, possibly secondary to shifts in substrate metabolism. In addition, pronounced activation of the myocardial β-adrenergic signal-transduction pathway is associated with an oxygen cost itself (11). Therefore, the efficiency of contraction appears to be greater with calcium than with dobutamine.
Previous studies (15) from our laboratory have shown that 90 min sustained moderate ischemia does not change the expression of calcium handling proteins such as sarcoplasmatic reticulum calcium ATPase, phospholamban, calsequestrin, and troponin inhibitor, and accordingly, the inotropic reserve recruitable by calcium infusion in the present study remained, as expected, unchanged in untreated pigs. Therefore, changes in the β-adrenergic signal-transduction pathway rather than changes at the level of the myofilaments per se are responsible for the reduction of the inotropic response to dobutamine during sustained moderate ischemia. Because ischemia does not alter the density and affinity of β-adrenoceptors in this model (21), and the calculated EC50 for dobutamine was unaltered also, alterations in the β-adrenergic signal-transduction pathway must be located downstream of β-adrenoceptors, but upstream of the myofilaments.
A biphasic pattern of adenylate cyclase activity during prolonged ischemia has been described in isolated rat hearts (24) with a sensitation of adenylate cyclase after 15 min but a pronounced desensitation after 30 and 50 min of ischemia. Studies in dogs with 1 h of coronary artery occlusion also revealed a reduced responsiveness of adenylate cyclase (27), associated with a decreased activity of the stimulatory G protein (25). Finally, a decreased activation of adenylate cyclase by β-adrenoceptor stimulation was also described in human myocardium after cardiopulmonary bypass with cardioplegic arrest (20). Therefore, in the present study, uncoupling between β-adrenoceptors and adenylate cyclase could well explain the decreased inotropic reserve in response to dobutamine during sustained moderate ischemia.
Endogenous Nitric Oxide and β-Adrenergic Responsiveness
Inhibition of NO synthesis during normoperfusion decreased baseline cardiac function, but it did not affect the extent of and the difference between the inotropic reserves recruited by dobutamine and calcium (Fig. 2). Therefore, endogenous NO does not limit maximal β-adrenergic inotropic responsiveness during normoperfusion in our model.
In isolated rat ventricular strips, the NO donorS-nitroso-N-penicillamine caused a rightward shift of the concentration-contractile response curve for isoprenaline (14). Similarly, in anesthetized dogs with autonomic blockade, the inotropic response to a given dose of dobutamine was higher with inhibition of NO synthesis (13); however, the maximum response to dobutamine was not assessed. Also, in the present study, the EC50 for dobutamine was decreased byl-NNA during normoperfusion. In endothelial NOS-knockout mice, the dose-response curve of dobutamine versus LV pressure was shifted upward compared with wild-type mice, whereas the response to calcium remained unaltered (4). One potential explanation for this increased response to dobutamine is related to a 50% increase in the β-adrenoceptor density in endothelial NOS-knockout mice (4); in our preparation, the β-adrenoceptor density and affinity remained unaltered (21).
Inhibition of endogenous NO synthesis with l-NNA did not prevent the ischemia-induced attenuation of the inotropic reserve to dobutamine. Thus the response to dobutamine during ischemia was similar with (group 2) and without (group 1) prior inhibition of NO synthesis. This finding is in contrast to results of Node et al. (18), who reported that during acute moderate ischemia in anesthetized dogs, inhibition of NO synthesis increased the contractile response to isoproterenol and calcium. However, in that study, using a constant reduction in perfusion pressure to induce ischemia, myocardial oxygen consumption also increased significantly with inotropic stimulation during ischemia, and it did so to an even greater extent after blockade of endogenous NO synthesis withl-NAME. It therefore remained unclear whether the increased inotropic response after l-NAME depended directly on the inhibition of NO-mediated effects on cardiomyocyte function or on an improved oxygen delivery. The results of the present study suggest that with a constant limitation in coronary inflow and oxygen consumption, endogenous NO per se does not limit β-adrenergic responsiveness during myocardial ischemia.
This study was supported by the German Research Foundation Grant He-1320/8–3.
Address for reprint requests and other correspondence: G. Heusch, Abteilung für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstr. 55, 45122 Essen, Germany (E-mail:).
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- Copyright © 2001 the American Physiological Society