Endogenous Nitric Oxide and Myocardial Adaptation to Ischemia
Abstract—Ischemic myocardium does not inevitably undergo necrosis but rather can survive through downregulation of contractile function, ie, “hibernate.” To study the role of endogenous NO in this adaptation, 41 enflurane-anesthetized swine were subjected to 90 minutes of moderate left anterior descending coronary artery hypoperfusion and assigned to placebo (P), to 30 mg/kg NG-nitro-l-arginine (L-NNA) IV to inhibit NO synthase, or to aortic constriction (AO) to match the increased left ventricular pressure observed with L-NNA. During normoperfusion, a regional myocardial external work index (WI, mm Hg · mm, sonomicrometry and micromanometry) was reduced with L-NNA (from 326±27 [SEM] to 250±19, P<0.05) but increased with AO (from 321±16 to 363±19, P<0.05 versus L-NNA). At 10 minutes of ischemia, WI was lower with L-NNA (109±10, P<0.05) than P (180±22) and AO (170±11) and did not change further at 85 minutes of ischemia. Relationships between WI and transmural myocardial blood flow and oxygen consumption were shifted rightward by L-NNA versus P and AO at both 10 and 85 minutes of ischemia. The maximal increment in calcium-activated external work was not different during normoperfusion among groups but was decreased during ischemia with L-NNA. L-NNA transiently increased myocardial contractile calcium sensitivity along with systemic pressure but reduced it during ongoing ischemia. The free-energy change of ATP hydrolysis after an early ischemic decrease recovered toward baseline values in all groups, and necrosis was absent after 2 (triphenyltetrazolium chloride staining) or 8 (histology) hours of reperfusion. Thus, endogenous NO contributes to hibernation by reducing oxygen consumption and preserving calcium sensitivity and contractile function without an energy cost during ischemia. (Circ Res. 2000;87:146-152.)
Ischemic myocardium does not inevitably undergo necrosis but rather can adapt to reduced blood flow through a regulatory reduction in contractile function.1 2 Therefore, loss of contractile function in patients with coronary artery disease frequently does not indicate infarction but rather a downregulated state of myocardial “hibernation.”1 A close relationship between reduced myocardial blood flow and contractile function, ie, perfusion-contraction matching, is a key feature of such hibernation in the experimental setting.3 Further characteristics of myocardial hibernation are recovery of energy metabolism during ongoing ischemia, persistence of an inotropic reserve, recovery of contractile function on reperfusion, and, almost by definition, absence of necrosis.2 No mechanism underlying myocardial hibernation other than reduced calcium responsiveness4 has thus far been identified.
Nitric oxide (NO), among its other beneficial cardiovascular effects,5 is an attractive candidate to be involved in such adaptation to ischemia, and it has been documented to be crucial in the endogenous cardioprotection by delayed preconditioning.6 The activity of the constitutive cardiovascular isoform of NO synthase (NOS III)7 and the production of NO are increased during ischemia.8 The impact of endogenous NO on myocardial function in vivo remains controversial. Inhibition of NO synthesis either has no effect on8 9 or decreases10 regional contractile function during normoperfusion and decreases it during ischemia, potentially by further decreasing ischemic blood flow.8 Apart from its ambiguous effect on regional function, NO rapidly and reversibly inhibits mitochondrial respiration,11 12 and stimulation of NO synthesis decreases oxygen consumption of isolated cardiac and skeletal muscle.13 14 During exercise, myocardial oxygen consumption for a given myocardial work is increased with inhibition of NO synthesis in conscious dogs,15 indicating improvement of the economy of oxygen use for contractile function by endogenous NO. Inhibition of NO synthesis shifts substrate metabolism from fatty acids to glucose and lactate utilization, possibly in compensation for the reduced economy of oxygen use.16
Data on the effects of endogenous NO on the interaction of myocardial contractile function, oxygen consumption, blood flow, and energetics during ischemia on the regional level, ie, the level on which coronary artery disease occurs, are lacking. We therefore investigated the consequences of systemic inhibition of NO synthesis on myocardial adaptation to ongoing ischemia by measuring regional myocardial blood flow, function, and oxygen consumption as well as contractile calcium responsiveness and myocardial energetics.
Materials and Methods
Forty-one anesthetized Göttinger miniswine were instrumented for the measurement of left ventricular pressure and wall thickness.4 A silicon tube was passed around the descending thoracic aorta to permit its constriction. The left anterior descending coronary artery (LAD) and vein were cannulated, and the artery was perfused by an extracorporeal circuit. Heart rate was held constant by left atrial pacing.
Regional Myocardial Function, Blood Flow, and Metabolism
A regional myocardial external work index was calculated. Regional myocardial blood flow was measured with microspheres, and myocardial oxygen and lactate consumptions were calculated.4 The cardiac respiratory quotient was determined in 4 pigs each with NG-nitro-l-arginine (L-NNA) and aortic constriction.16 The free energy change of ATP hydrolysis17 and the cGMP concentration were determined from biopsies.
Six transverse myocardial slices from each heart were incubated in a triphenyl tetrazolium chloride solution. In 4 hearts, each with L-NNA and aortic constriction, transmural tissue specimens were taken from the ischemic and the control areas and analyzed by histology; the area of necrotic foci was planimetered and expressed as percentage of the analyzed area.18
Group 1 (n=11)
After measurements of systemic hemodynamics and regional myocardial blood flow, function, and metabolism at baseline, LAD inflow was decreased by 45% for 90 minutes. Measurements were repeated at 10 and 85 minutes of ischemia. Thereafter, the myocardium was reperfused for 2 hours.
Group 2 (n=15)
After baseline measurements, 30 mg/kg IV L-NNA was infused over 30 minutes. During L-NNA infusion, LAD inflow was maintained constant. After a further set of measurements, the protocol was identical to that of group 1. In a subset of 4 animals, reperfusion was prolonged to 8 hours. The arterial−coronary venous nitrite difference was measured with a flow-injection/Griess setup in 4 pigs each of the L-NNA and aortic constriction groups,19 and the cumulative arterial−coronary venous nitrite difference during ischemia was calculated.
Group 3 (n=15)
After baseline measurements, the descending aorta was constricted to increase peak left ventricular pressure by 20 to 25 mm Hg, and LAD inflow was adjusted to increase mean coronary arterial pressure proportionately. After a further set of measurements, the protocol was identical to that in group 1. In a subset of 4 animals, reperfusion was prolonged to 8 hours.
In 8 swine of each group, calcium chloride was infused into the perfusion system at increasing doses to determine the contractile calcium responsiveness during normoperfusion and at 85 minutes of ischemia.4
Data Analysis and Statistics
Data are reported as mean±SEM. Statistical analysis comprised 2-way ANOVA for repeated measures and Fisher’s least significant difference tests when significant overall effects were detected. Linear regression analyses between the regional myocardial external work index and transmural blood flow or oxygen consumption, respectively,20 were compared by ANCOVA. The EC50 for calcium, ie, the added calcium concentration for a 50% increase in fractional work, was calculated.4 Cumulative arterial−coronary venous difference of nitrite over 90 minutes of ischemia and cardiac respiratory quotient with L-NNA or aortic constriction were compared by t tests.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
Representative original recordings from 1 animal from each of the 3 groups are shown in Figure 1A⇓ through 1C.
Effectiveness of NO Synthase Blockade
L-NNA increased the cardiac respiratory quotient from 0.78±0.04 to 0.86±0.04 (P<0.05), whereas aortic constriction did not (0.84±0.03 versus 0.83±0.03, P=NS). Also, L-NNA abolished the bradykinin-induced decrease in mean coronary resistance (from 5.1±1.0 [SEM] to 1.4±0.2 mm Hg · mL−1 · min−1, n=5). With aortic constriction, there was a small net nitrite production before ischemia (arterial−coronary venous difference, −6±2 pmol/mL); during ischemia, there was increased nitrite production (cumulative arterial−coronary venous difference, −1089±478 pmol · 90 min−1 · mL−1). With L-NNA, in contrast, there was net nitrite uptake before ischemia (arterial−coronary venous difference, 3±1 pmol/mL) and during ischemia (cumulative arterial−coronary venous difference, 342±284 pmol · 90 min−1 · mL−1, P<0.02 versus aortic constriction).
Left ventricular pressure was increased with L-NNA and aortic constriction during normoperfusion and at 10 minutes of ischemia, and it was increased at 85 minutes of ischemia compared with placebo (Table 1⇓).
Regional Myocardial Function
During normoperfusion, the regional external work index was reduced with L-NNA, also at pressure match. At 10 minutes of ischemia, the regional external work index was again lower with L-NNA than with placebo or pressure match. With prolongation of ischemia to 85 minutes, the external work index did not change further in any group (Table 1⇑). The maximal increment in calcium-activated external work was not different during normoperfusion among groups but was decreased at 85 minutes of ischemia with L-NNA (Table 2⇓). With increased left ventricular peak pressure by L-NNA or aortic constriction, the EC50 for calcium was decreased. At 85 minutes of ischemia, when the left ventricular peak pressure increase had subsided, the EC50 for calcium was increased by L-NNA but unchanged with placebo and aortic constriction (Table 2⇓).
Regional Myocardial Metabolism
Regional myocardial oxygen consumption tended to increase with aortic constriction but decreased to similar values during ischemia in all groups (Figure 2⇓). Myocardial lactate consumption was reversed to net lactate production during early ischemia but tended to recover, albeit significantly only in the group with aortic constriction, with no differences among groups. The free energy change of ATP hydrolysis (ΔGATP) was unaffected by L-NNA or aortic constriction. At 10 minutes of ischemia, ΔGATP was reduced but recovered to near baseline values at 85 minutes of ischemia in all groups (Table 1⇑). cGMP did not change throughout the protocol in all groups (Table 1⇑).
Relationships Between Regional Myocardial Function and Oxygen Consumption or Blood Flow
L-NNA shifted the relationships of regional myocardial external work versus myocardial oxygen consumption (Figure 2⇑) or transmural myocardial blood flow (Figure 3⇓) rightward, also at pressure match. Regressions with data at 85 rather than 10 minutes of ischemia were no different.
In none of the experiments was necrosis detected by triphenyltetrazolium chloride staining. Microinfarction, as determined by histology, averaged 1.1±1.6% (anterior myocardium) and 2.2±2.9% (posterior myocardium) with L-NNA and 0.6±0.4% (anterior myocardium) and 1.3±1.1% (posterior myocardium) with aortic constriction.
Endogenous NO increases contractile function at baseline and during moderate ischemia for any given blood flow and myocardial oxygen consumption. Obviously, endogenous NO decreases excess oxygen consumption and preserves contractile calcium sensitivity and, consequently, contractile function at no additional energy cost, thus helping the myocardium to adapt to ischemia and contributing to hibernation.
Critique of Methods
The strengths and limitations of the present model have been discussed before.4 LAD inflow was held constant, and constant inflow avoids hypoperfusion in the absence of the tonic vasodilator influence of endogenous NO. It seems unlikely that a reduction in regional myocardial function possibly seen with L-NNA was counteracted in part by increased coronary arterial pressure, because a Gregg or garden hose phenomenon is not operative in this preparation.21 The effects of L-NNA on systemic pressure were matched by a purely mechanical increase in pressure by aortic constriction and thus accounted for. The aortic constriction not only served to match left ventricular afterload, as reflected by left ventricular peak pressure, but was also associated with a preload recruitment comparable to that seen with L-NNA, as reflected by left ventricular end-diastolic pressure and wall thickness (Table 1⇑). The regional external work index used in the present study comprises both wall excursion and the load against which it occurs and is therefore an appropriate mechanical counterpart to compare with myocardial oxygen consumption and energetics. Apart from the match in loading conditions, both the L-NNA and the aortic constriction groups had a greater percent reduction in blood flow during ischemia; therefore, the most rigorous comparison with respect to the role of endogenous NO is done between the L-NNA and the aortic constriction groups.
Because of the 3D geometry of the left ventricle, we may not have detected small changes in loading conditions. However, with voluntary major changes in loading (ie, aortic constriction versus placebo), we found only small shifts in those relationships between function and flow or function and oxygen consumption; therefore, the substantial shift in those relationships with L-NNA versus aortic constriction cannot be attributed to loading.
The dose of L-NNA in the present study decreased nitrite production, increased systemic pressure, and abolished the bradykinin-induced coronary vasodilation and nitrite production; thus, L-NNA obviously reduced the activity of vascular smooth muscle soluble guanylate cyclase. In contrast, cGMP in total myocardial tissue, which reflects mostly cardiomyocytes, was, as previously described,22 not reduced, whereas myocardial oxygen consumption for a given contractile function was increased with L-NNA.
Endogenous NO, Myocardial Function, and Oxygen Consumption
Systemic inhibition of NO synthesis in anesthetized23 and conscious24 dogs decreases cardiac output, and this decrease is more pronounced than with a pressure match with phenylephrine.23 To avoid systemic effects and altered loading conditions, other studies used intracoronary infusion of NO synthesis inhibitors and measured regional myocardial function; regional myocardial function was either unchanged8 9 or decreased.10 In previous studies with regional ischemia in anesthetized dogs, function was decreased along with blood flow.8 In contrast, the present study even demonstrated a decrease in regional contractile function for any given blood flow during both normoperfusion and ischemia.
In isolated cardiomyocytes, the positive inotropic effect of low exogenous NO concentrations was attributed to cGMP-mediated inhibition of phosphodiesterase, with a subsequently diminished breakdown of cAMP,25 or to direct activation of adenylcyclase.26 An increase in cAMP formation, possibly resulting from adrenergic drive, will increase not only contractile function but also myocardial oxygen consumption.27 Therefore, inhibition of NO synthesis is expected to lower contractile function along with myocardial oxygen consumption when its effect is mediated through reduction of cAMP formation. In the present study, however, L-NNA decreased contractile function independently from a decrease in cGMP and myocardial oxygen consumption.
In isolated rat cardiac and skeletal muscle mitochondria, low levels of exogenous NO reversibly inhibit mitochondrial respiration, in part by inhibition of cytochrome c oxidase.11 12 Consistent with this observation, the acetylcholine- and bradykinin-induced NO release also decreases oxygen consumption in isolated canine left ventricular13 and skeletal muscle,14 in left ventricular muscle of nonhuman primates,28 and in failing human hearts.29 Although the above studies agree on reduced oxygen consumption, they did not simultaneously measure contractile function and myocardial energetics. In the present study, myocardial lactate consumption and the free energy change of ATP hydrolysis were not altered by L-NNA either during normoperfusion or during ischemia. Confirming previous observations by Recchia et al,16 there was even a compensatory shift in substrate use from free fatty acids to glucose and lactate with inhibition of NO synthase by L-NNA, as indicated by the increase in the cardiac respiratory quotient. The reduction of myocardial oxygen consumption for any given contractile function by endogenous NO might therefore be even greater if it occurred with the same preferential glycolytic substrate use.
Apparently, endogenous NO reduces excess myocardial oxygen consumption such that myocardial energetics and contractile function are not reduced along with reduced myocardial oxygen consumption. Apart from its effect on mitochondrial respiration, endogenous NO appears to preserve the calcium sensitivity of the contractile machinery during ischemia (Table 2⇑). An initial transient improvement in calcium sensitivity was seen with L-NNA and pressure match and is probably related to increased cardiomyocyte stretch30 31 ; this stretch-related effect subsided along with the increase in pressure during ongoing ischemia, and at 85 minutes of ischemia, the reduction in calcium responsiveness with L-NNA becomes fully apparent, whereas calcium responsiveness with aortic constriction is not altered. Pharmacological calcium sensitization improves contractile function without increasing myocardial oxygen consumption,32 33 and therefore, the preservation of calcium sensitivity by endogenous NO together with the reduction of excess myocardial oxygen consumption might well explain the improved economy of oxygen use for contractile function during ongoing ischemia.
Endogenous NO and Myocardial Hibernation
The concept of hibernation implies a downregulation of contractile function as an adaptation to a reduction in myocardial blood flow that serves to permit recovery of myocardial energetics and to maintain myocardial integrity and viability during persistent ischemia.1 2 From the present study, it appears that endogenous NO is not involved in the immediate downregulation of baseline contractile function and the recovery of myocardial energetics during ischemia, because both perfusion-contraction matching and the recovery of the free energy change of ATP hydrolysis also occurred with inhibition of NO synthesis by L-NNA. At any given blood flow and oxygen consumption, however, contractile function was lower without than with endogenous NO. The parallel rightward shift of both the external work–blood flow and external work–oxygen consumption relationships with L-NNA indicates that the differences between L-NNA and aortic pressure match at baseline are also maintained during ischemia, suggesting that the role of NO during ischemia, even at increased concentration, is the same as that at baseline. Thus, endogenous NO does not appear to mediate the downregulation of baseline contractile function during ischemia but rather to contribute to successful myocardial adaptation to ischemia by reducing oxygen consumption and maintaining contractile function as high as possible without any additional energy costs, probably through preservation of contractile calcium sensitivity.
In conclusion, we have shown, to the best of our knowledge for the first time, that (1) endogenous NO reduces myocardial oxygen consumption and thus improves regional myocardial function for any given level of myocardial blood flow, oxygen consumption, and energetics; (2) endogenous NO preserves contractile calcium sensitivity during myocardial ischemia; and (3) endogenous NO contributes to hibernation, ie, adaptation to myocardial ischemia, by preserving regional contractile function without any effect on myocardial energetics.
This study was supported by the German Research Foundation (He 1320/8-3) and the IFORES program of the University of Essen Medical School.
- Received May 8, 2000.
- Accepted May 23, 2000.
- © 2000 American Heart Association, Inc.
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