Thomas H. Hintze

Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression.

Recently, we have shown that chronic exercise increases endothelium-derived relaxing factor (EDRF)/nitric oxide (NO)-mediated epicardial coronary artery dilation in response to brief occlusion and acetylcholine. This finding suggests that exercise can provide a stimulus for the enhanced production of EDRF/NO, thus possibly contributing to the beneficial effects of exercise on the cardiovascular system. Therefore, the purpose of the present study was to examine whether chronic exercise could influence the production of NO (measured as the stable degradation product, nitrite) and endothelial cell NO synthase (ECNOS) gene expression in vessels from dogs after chronic exercise. To this end, dogs were exercised by running on a treadmill (9.5 km/h for 1 hour, twice daily) for 10 days, and nitrite production in large coronary vessels and microvessels and ECNOS gene expression in aortic endothelial extracts were assessed. Acetylcholine (10(-7) to 10(-5) mol/L) dose-dependently increased the release of nitrite (inhibited by nitro-L-arginine) from coronary arteries and microvessels in control and exercised dogs. Moreover, acetylcholine-stimulated nitrite production was markedly enhanced in large coronary arteries and microvessels prepared from hearts of dogs after chronic exercise compared with hearts from control dogs. One potential mechanism that may contribute to the enhanced production of nitrite in vessels from exercised dogs may be the induction of the calcium-dependent ECNOS gene. Steady-state mRNA levels for ECNOS were significantly higher than mRNA levels for von Willebrands factor (vWF, a specific endothelial cell marker) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a constitutively expressed gene) in exercised dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Stretch-induced programmed myocyte cell death.

To determine the effects of loading on active and passive tensions, programmed cell death, superoxide anion formation, the expression of Fas on myocytes, and side-to-side slippage of myocytes, papillary muscles were exposed to 7-8 and 50 mN/mm2 and these parameters were measured over a 3-h period. Overstretching produced a 21- and a 2.4-fold increase in apoptotic myocyte and nonmyocyte cell death, respectively. Concurrently, the generation of reactive oxygen species increased 2.4-fold and the number of myocytes labeled by Fas protein 21-fold. Moreover, a 15% decrease in the number of myocytes included in the thickness of the papillary muscle was found in combination with a 7% decrease in sarcomere length and the inability of muscles to maintain stable levels of passive and active tensions. The addition of the NO-releasing drug, C87-3754, prevented superoxide anion formation, programmed cell death, and the alterations in active and passive tensions with time of overloaded papillary muscles. In conclusion, overstretching appears to be coupled with oxidant stress, expression of Fas, programmed cell death, architectural rearrangement of myocytes, and impairment in force development of the myocardium.

Role of nitric oxide in the regulation of oxygen consumption in conscious dogs.

The role of nitric oxide (NO) in the regulation of O2 consumption was studied in chronically instrumented conscious dogs. A specific NO synthesis inhibitor, nitro-L-arginine (NLA, 30 mg/kg i.v.), significantly increased mean arterial pressure from 100 +/- 4 to 134 +/- 5 mm Hg (mean +/- SEM) and total peripheral resistance by 157 +/- 16% and reduced cardiac output by 47 +/- 3% and heart rate by 34 +/- 6% after 120 minutes. Changes in arterial blood gases were not observed. There were significant changes in PO2 (-14 +/- 2 mm Hg), O2 saturation (-21 +/- 2%), the percentage of hemoglobin as oxyhemoglobin (-21 +/- 2%), and O2 content (-3.0 +/- 0.9 vol%) and a significant increase in percent reduced hemoglobin (21 +/- 1%) in mixed venous blood, associated with an increase in O2 extraction (5.1 +/- 0.2 vol%) (all P < .01). O2 consumption was increased from 124 +/- 6 to 155 +/- 9 mL/min (P < .05). Methoxamine, titrated to have hemodynamic effects similar to those of NLA (eg, mean arterial pressure increased from 97 +/- 4 to 131 +/- 5 mm Hg), had much smaller effects on venous blood gases, hemoglobin, and O2 extraction (2.3 +/- 0.7 vol%) and no significant effect on O2 consumption. NLA also caused an increase in O2 consumption of 37 +/- 8% (P < .01) in quietly resting conscious dogs that had undergone pretreatment with hexamethonium and atropine, but no significant change in O2 consumption in dogs anesthetized with barbiturate.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced Nitric Oxide Production and Altered Myocardial Metabolism During the Decompensation of Pacing-Induced Heart Failure in the Conscious Dog

The aim of the present study was to determine whether cardiac nitric oxide (NO) production changes during the progression of pacing-induced heart failure and whether this occurs in association with alterations in myocardial metabolism. Dogs (n=8) were instrumented and the heart paced until left ventricular end-diastolic pressure reached 25 mm Hg and clinical signs of severe failure were evident. Every week, hemodynamic measurements were recorded and blood samples were withdrawn from the aorta and the coronary sinus for measurement of NO metabolites, O2 content, free fatty acids (FFAs), and lactate and glucose concentrations. Cardiac production of NO metabolites or consumption of O2 or utilization of substrates was calculated as coronary sinus-arterial difference times coronary flow. In end-stage failure, occurring at 29+/-1.6 days, left ventricular end-diastolic pressure was 25+/-1 mm Hg, left ventricular systolic pressure was 92+/-3 mm Hg, mean arterial pressure was 75+/-2.5 mm Hg, and dP/dtmax was 1219+/-73 mm Hg/s (all P<0.05). These changes in hemodynamics were associated with a fall of cardiac NO metabolite production from 0.37+/-0.16 to -0.28+/-0.13 nmol/beat (P<0.05). O2 consumption and lactate uptake did not change significantly from control, while FFA uptake decreased from 0.16+/-0.03 to 0.05+/-0.01 microEq/beat and glucose uptake increased from -2.3+/-7.0 to 41+/-10 microgram/beat (P<0.05). The cardiac respiratory quotient also increased significantly by 28%. In 14 normal dogs the same measurements were performed at control and 1 hour after we injected 30 mg/kg of nitro-L-arginine, a competitive inhibitor of NO synthase .O2 consumption increased from 0.05+/-0.002 mL/beat at control to 0.071+/-0.003 mL/beat after nitro-L-arginine, while FFA uptake decreased from 0.1+/-0.01 to 0.06+/-0.01 microEq/beat, lactate uptake increased from 0.15+/-0.04 to 0.31+/-0.03 micromol/beat, glucose uptake increased from 8.2+/-5.0 to 35.4+/-9.5 microgram/beat, and RQ increased by 23% (all P<0.05). Our results indicate that basal cardiac production of NO falls below normal levels during cardiac decompensation and that there are shifts in substrate utilization. This switch in myocardial substrate utilization also occurs after acute pharmacological blockade of NO production in normal dogs.

Nitric Oxide An Important Signaling Mechanism Between Vascular Endothelium and Parenchymal Cells in the Regulation of Oxygen Consumption

BACKGROUND Nitric oxide (NO) is known to be an inhibitor of mitochondrial function. However, the physiological significance of endothelium-derived NO in the control of tissue respiration is not established. METHODS AND RESULTS Tissue O2 consumption by skeletal muscle slices of the triceps brachii of normal dogs was measured with a Clark-type O2 electrode/tissue bath system at 37 degrees C. S-Nitroso-N-acetylpenicillamine (SNAP), carbachol (CCh), or bradykinin (BK) decreased tissue O2 consumption by 12 +/- 3% to 55 +/- 8%, 15 +/- 6% to 36 +/- 11%, or 21 +/- 5% to 42 +/- 4% at doses of 10(-7) to 10(-4) mol/L, respectively. The effects of both CCh and BK but not SNAP were eliminated by nitro-L-arginine (NLA, 10(-4) mol/L), consistent with SNAP decomposing to release NO and both CCh and BK stimulating endogenous NO production from L-arginine. Oxygen consumption was also decreased by 8-bromo-cGMP. The mitochondrial uncoupler dinitrophenol blocked the effects of 8-bromo-cGMP but only slightly altered those of SNAP, indicating that the major site of action of NO is the mitochondria. In normal, chronically instrumented, resting conscious dogs, blockade of NO synthase by NLA increased mean arterial pressure by 28 +/- 2.5 mm Hg and hind limb vascular resistance by 114 +/- 12% and decreased blood flow by 39 +/- 3%. Most important, NLA also increased O2 uptake by 55 +/- 9% in hind limb skeletal muscle (P < .05), associated with decreases in PO2 and O2 saturation and an increase in reduced hemoglobin in hind limb venous blood. CONCLUSIONS Our results indicate that NO release from vascular endothelial cells appears to play an important physiological role in the regulation of tissue mitochondrial respiration in skeletal muscle and perhaps other organ systems.

Coronary Kinin Generation Mediates Nitric Oxide Release After Angiotensin Receptor Stimulation

Our goal was to determine whether angiotensin II (Ang II) and its metabolic fragments release nitric oxide and the mechanisms by which this occurs in blood vessels from the canine heart. We incubated 20 mg of microvessels or large coronary arteries in phosphate-buffered saline for 20 minutes and measured nitrite release. Nitrite release increased from 27 +/- 2 up to 103 +/- 5, 145 +/- 17, 84 +/- 4, 107 +/- 16, and 54 +/- 4 pmol/mg (P < .05) in response to 10(-5) mol/L of Ang I, II, III, IV, and Ang-(1-7), respectively. The effects of all angiotensins were blocked by N omega-nitro-L-arginine methyl ester (100 mumol/L), indicating that nitrite was a product of nitric oxide metabolism, and by Hoe 140 (10 mumol/L), a specific bradykinin B2 receptor antagonist, indicating a potential role for local kinin formation. The protease inhibitors aprotinin (10 mumol/L) and soybean trypsin inhibitor, which block local kinin formation, inhibited nitrite release by all of the angiotensins. Angiotensin nonselective (saralasin), type 1-specific (losartan), and type 2-specific (PD 123319) receptor antagonists abolished the nitrite released in response to all the fragments. Angiotensin type 1 and type 2 and receptors mediate nitrite release after Ang I, II, III, and Ang-(1-7), whereas only type 2 receptors mediate nitrite release after Ang IV. Similar results were obtained in large coronary arteries. In summary, formation of nitrite from coronary microvessels and large arteries in the normal dog heart in response to angiotensin peptides is due to the activation of local kinin production in the coronary vessel wall.

Reduced Gene Expression of Vascular Endothelial NO Synthase and Cyclooxygenase-1 in Heart Failure

Endothelium-dependent responses are depressed in coronary and peripheral blood vessels after the onset of pacing-induced heart failure in dogs and heart failure of various etiologies in humans. The present study was designed to examine whether these responses were due to decreases in the expression of endothelial cell NO synthase (ecNOS) and cyclooxygenase-1 (COX-1). After 1 month of left ventricular pacing, 8 mongrel dogs were monitored for heart failure as defined by clinical signs and left ventricular end diastolic pressures > 25 mm Hg. Total RNA and protein were isolated from endothelial cells scraped from the thoracic aorta and analyzed by Northern and Western blotting, respectively. Blots probed with 32P-labeled cDNAs for ecNOS and COX-1 were quantified densitometrically, and results were normalized against GAPDH or von Willebrand factor (vWF). In arbitrary units, the ratios of ecNOS to GAPDH were 2.66 +/- 0.77 (mean +/- SEM, n = 17) and 1.12 +/- 0.37 (n = 6 and the ratios of COX-1 to GAPDH were 1.52 +/- 0.52 and 0.56 +/- 0.15 before and after heart failure, respectively. These represent 56% to 64% (P < .05) reductions in ecNOS and COX-1 gene expression. There was no change in the ratios of either COX-1 or ecNOS to vWF. There was also a marked reduction in ecNOS protein after heart failure, estimated at 70%. A marked reduction in nitrite production, a measure of enzyme activity, from thoracic aortas in response to stimulation by either acetylcholine or bradykinin also occurred. To determine whether ecNOS and COX-1 could be independently regulated, an orally active NO-releasing agent, CAS 936, was given to 7 normal dogs for 7 days, and aortic ecNOS and COX-1 mRNAs were analyzed. The ratio of ecNOS to GAPDH was depressed by 52% (P < .05) in aortas from these dogs, whereas the ratio of COX-1 to GAPDH was unchanged. Similar results were found when data were normalized to vWF. These results suggest that at least two endothelial vasodilator gene products are reduced in heart failure, as opposed to a selective defect in NO synthase gene expression.

Amlodipine Releases Nitric Oxide From Canine Coronary Microvessels An Unexpected Mechanism of Action of a Calcium Channel–Blocking Agent

BACKGROUND Recent studies suggest that amlodipine may reduce mortality in patients with heart failure, especially those with dilated cardiomyopathy. In general, drugs that release NO, such as organic nitrates and ACE inhibitors, have been shown to be of substantial benefit in the treatment of heart failure. METHODS AND RESULTS We hypothesized that a portion of the beneficial actions of amlodipine may involve the release or action of NO. Coronary microvessels were isolated from the heart of normal dogs and incubated with increasing doses of the calcium channel blockers nifedipine, diltiazem, and amlodipine or the ACE inhibitors enalaprilat and ramiprilat. Neither nifedipine nor diltiazem increased nitrite production at any dose studied. In marked contrast, amlodipine caused a dose-dependent increase in nitrite production from 74+/-5 to 130+/-8 pmol/mg (by 85+/-21%,10(-5) mol/L, P<.05) that was similar in magnitude to that of either of the ACE inhibitors. Amlodipine also increased nitrite production in large coronary arteries and in aorta. N(omega)-Nitro-L-arginine methyl ester, HOE-140, and dichloroisocoumarin essentially abolished the increase in nitrite production, indicating that (1) nitrite production reflected NO formation, (2) nitrite production was dependent on stimulation of the kinin2 receptor, and (3) nitrite production is most likely secondary to the formation of local kinins. CONCLUSIONS Thus, unlike nifedipine and diltiazem, amlodipine releases NO from blood vessels.

Impaired Myocardial Fatty Acid Oxidation and Reduced Protein Expression of Retinoid X Receptor-α in Pacing-Induced Heart Failure

Background—The nuclear receptors peroxisome proliferator-activated receptor-&agr; (PPAR&agr;) and retinoid X receptor &agr; (RXR&agr;) stimulate the expression of key enzymes of free fatty acid (FFA) oxidation. We tested the hypothesis that the altered metabolic phenotype of the failing heart involves changes in the protein expression of PPAR&agr; and RXR&agr;. Methods and Results—Cardiac substrate uptake and oxidation were measured in 8 conscious, chronically instrumented dogs with decompensated pacing-induced heart failure and in 8 normal dogs by infusing 3 isotopically labeled substrates: 3H-oleate, 14C-glucose, and 13C-lactate. Although myocardial O2 consumption was not different between the 2 groups, the rate of oxidation of FFA was lower (2.8±0.6 versus 4.7±0.3 &mgr;mol · min−1 · 100g−1) and of glucose was higher (4.6±1.0 versus 1.8±0.5 &mgr;mol · min−1 · 100g−1) in failing compared with normal hearts (P <0.05). The rates of lactate uptake and lactate output were not significantly different between the 2 groups. In left ventricular tissue from failing hearts, the activity of 2 key enzymes of FFA oxidation was significantly reduced: carnitine palmitoyl transferase-I (0.54±0.04 versus 0.66±0.04 &mgr;mol · min−1 · g−1) and medium chain acyl-coenzyme A dehydrogenase (MCAD; 1.8±0.1 versus 2.9±0.3 &mgr;mol · min−1 · g−1). Consistently, the protein expression of MCAD and of RXR&agr; were significantly reduced by 38% in failing hearts, but the expression of PPAR&agr; was not different. Moreover, there were significant correlations between the expression of RXR&agr; and the expression and activity of MCAD. Conclusions—Our results provide the first evidence for a link between the reduced expression of RXR&agr; and the switch in metabolic phenotype in severe heart failure.

Role of Endothelium-Derived Nitric Oxide in the Modulation of Canine Myocardial Mitochondrial Respiration In Vitro: Implications for the Development of Heart Failure

The mechanism responsible for the regulation of cardiac function by endogenous nitric oxide (NO) remains unclear. In this investigation, O2 consumption by freshly isolated myocardial muscle segments from the left ventricular free wall of canine hearts was quantified by a Clark-type O2 electrode at 37 degrees C. S-nitroso-N-acetylpenicillamine (SNAP, 9 +/- 3% to 50 +/- 8%), bradykinin (BK, 14 +/- 3% to 30 +/- 5%), or carbachol (CCh, 15 +/- 4% to 29 +/- 4%) significantly attenuated tissue O2 consumption at doses of 10(-7) to 10(-4) mol/L (mean +/- SE, P < .05). The effects of BK and CCh, but not SNAP, were blocked by 10(-4) mol/L NG-nitro-L-arginine, consistent with both BK and CCh stimulating NO biosynthesis and with SNAP decomposing to release NO, respectively. Similar doses of 8-Br-cGMP caused a respiratory inhibition, but to a lesser extent (9 +/- 2% to 14 +/- 6%). A mitochondrial uncoupler, 2,4-dinitrophenol (at 1 mmol/L), blocked the effects of 8-Br-cGMP, but not those of SNAP, BK, or CCh, suggesting that the major site of action of NO is on mitochondrial electron transport. Myocardial muscle from dogs with pacing-induced heart failure had a basal O2 consumption rate of 251 +/- 21 nmol.min-1.g-1, which was 54% higher than the rate seen in muscle from normal healthy canine hearts. The inhibitory effects of BK and CCh on O2 consumption were not observed in failing cardiac tissue, but SNAP showed an unaltered inhibitory effect. Therefore, our results indicate that NO released from microvascular endothelium by BK, stimulation of muscarinic receptors, and perhaps flow velocity may play an important physiological role in the control of cardiac mitochondrial respiration, and the loss of this regulatory function may contribute to the development of heart failure.