The nitric oxide-induced reduction in cardiac energy supply is not due to inhibition of creatine kinase.

OBJECTIVES: While nitric oxide (NO) is a potent vasodilator already in the nM range, a cGMP-independent negative inotropic effect is observed at higher concentrations. Since inhibition of creatine kinase (CK) by NO-induced nitrosylation has been proposed as a possible mechanism of action, we measured the flux through CK in the intact heart. METHODS: In saline perfused, paced guinea pig hearts 31P NMR spectroscopy was employed to directly assess the cardiac energy status, i.e. free energy of ATP hydrolysis (DeltaG(ATP)) and flux through CK using magnetization transfer in absence and presence of NO. RESULTS: NO (50 microM) doubled coronary flow and induced a rapid drop in left ventricular developed pressure (39+/-10 vs. 81+/-10 mmHg) and MVO(2) (1.3+/-0.8 vs. 3.7+/-0.5 micromol/min/g) (n=7). This effect was associated with an immediate decrease in phosphocreatine (PCr) (-69%) and DeltaG(ATP). During the subsequent 35 min of NO infusion cardiac function and MVO(2) remained depressed, while PCr partially recovered. NO had no effect on the unidirectional forward flux through CK (98 +/- 21 vs. 99 +/- 20 micromol/min/g, n=7) which was 5- to 10-fold greater than the rate of ATP turnover. Upon cessation of NO infusion both cardiac function and PCr rapidly returned to baseline values. The NO-induced fall in the myocardial energy status was associated with an increase in mitochondrial NADH (n=7) as assessed by surface fluorescence. The observed change in fluorescence was similar to that observed with short term ischemia. CONCLUSION: The NO-mediated depression of myocardial function, MVO(2) and energy status is not mediated by changes in CK flux. Most likely a partial blockade of mitochondrial oxidative phosphorylation at the level of cytochrome c oxidase is responsible for this effect.

alpha 1-Adrenoceptor stimulation inhibits the isoproterenol-induced effects on myocardial contractility and protein phosphorylation.

In the present study the influence of alpha 1-adrenoceptor stimulation on the beta-adrenoceptor agonist-induced increases in contractile parameters and protein phosphorylation was determined in isolated perfused hearts and isolated cardiac myocytes, respectively. Methoxamine inhibited the isoproterenol-induced increases in left ventricular pressure and heart rate dose dependently up to 90% and 75%, respectively; the EC50 of this antiadrenergic effect was 4.4 microM. The alpha 1-adrenoceptor antagonist, prazosin (1 microM), greatly diminished methoxamine's inhibitory action, confirming the alpha 1-adrenoceptor-mediated mechanism. The inotropic effect of glucagon was inhibited by methoxamine in a similar manner. Radioligand binding assays with [3H]dihydroalprenolol demonstrated that the antiadrenergic action of methoxamine is not due to an unspecific beta-adrenoceptor blocking property. In an additional experimental series the effects of methoxamine and isoproterenol on the protein phosphorylation pattern of isolated cardiac myocytes were investigated. Isoproterenol increased the phosphorylation state of five proteins (6-kDa, phospholamban; 15-kDa; 28-kDa, troponin I; 97-kDa; 140-kDa) while in the experiments with methoxamine the 15-kDa protein was the only phosphorylated substrate. In the presence of methoxamine the isoproterenol-induced phosphorylation of phospholamban, troponin I and the 97-kDa and 140-kDa protein was markedly inhibited while the phosphorylation state of the 15-kDa protein remained unaltered. The present study clearly demonstrated that alpha 1-adrenoceptor stimulation potently inhibits the beta-adrenoceptor-mediated changes in contractile force and phosphorylation of key regulatory proteins, most likely through modulation of cAMP metabolism.

Short-term hibernation in adult cardiomyocytes is PO(2) dependent and Ca(2+) mediated.

The mechanism of myocardial hibernation, the reversible downregulation of contractile activity on reduction of coronary flow with unchanged cardiac energetics, is presently not understood. The oxygen consumption (VO(2)), shortening fraction (DeltaL), energy status [phosphocreatine (PCr), ATP, and adenosine and lactate release], and free intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured in isolated rat cardiomyocytes at precisely controlled ambient PO(2) (Oxystat). When PO(2) was reduced from 25 to 6 mmHg, VO(2) decreased by 50%, while DeltaL was downregulated from 11.2 +/- 4.1 to 7.6 +/- 4.0%, and energy status was unchanged in the steady state (observation time 12 min). Only transiently PCr decreased, and lactate and adenosine release increased. Further reduction of PO(2) (to 3 mmHg) reduced VO(2) by 80%, decreased PCr by 35%, moderately increased adenosine and lactate release, and progressively reduced DeltaL by 50% (to 5.6 +/- 3.3%). All parameters fully recovered during reoxygenation. PO(2)-dependent downregulation of DeltaL was accompanied by a progressive reduction in systolic [Ca(2+)](i) (from 512 +/- 110 to 357 +/- 91 nmol/l at 6 mmHg and to 251 +/- 69 nmol/l at 3 mmHg), whereas diastolic free [Ca(2+)](i) remained unchanged. Therefore, the mechanism of the reversible, PO(2)-dependent downregulation of contractile activity (myocardial hibernation) involves a substantial reduction of systolic calcium.

Phosphorylation potential, adenosine formation, and critical PO2 in stimulated rat cardiomyocytes.

This study investigated the relationship between O2 consumption (VO2) and energy status in isolated rat cardiomyocytes using a system in which O2 supply (PO2) was maintained constant. For this purpose, VO2, phosphocreatine, ATP, intracellular pH, and adenosine of quiescent and stimulated cardiomyocytes were measured while the ambient PO2 was clamped between 0.1 and 120 mmHg. In quiescent cardiomyocytes (VO2: 7.9 +/- 1.2 nmol.min-1.mg protein-1), the threshold below which respiration decreased (critical PO2) was 1.4 mmHg. Above this value, energy status remained constant; below 1 mmHg, both free ADP and adenosine increased. Stimulation increased VO2 threefold and shifted the critical PO2 to 10 mmHg. Above this value, free ADP and adenosine remained unchanged; between 10 and 5 mmHg. VO2 was reduced but this did not change free ADP or adenosine. These findings demonstrate that 1) under well-oxygenated conditions (PO2 > 10 mmHg), VO2 is not controlled by ADP; 2) similarly, the adenosine formation is independent of VO2; a PO2 < 5 mmHg is a prerequisite for enhanced adenosine formation; and 3) when O2 supply becomes limiting, ATP consumption is downregulated without measurable changes in energy status (hibernation).

Nitric oxide reduces energy supply by direct action on the respiratory chain in isolated cardiomyocytes.

To investigate the effect of nitric oxide (NO) on cardiac energy metabolism, isolated cardiomyocytes of Wistar rats were incubated in an Oxystat system at a constant ambient PO2 (25 mmHg) and oxygen consumption (VO2); free intracellular Ca(2+) (fura 2), free cytosolic adenosine [S-adenosylhomocysteine (SAH) method], and mitochondrial NADH (autofluorescence) were measured after application of the NO donor morpholinosydnonimine (SIN-1). In Na(+)-free medium (contracting cardiomyocytes), VO2 increased from 7.9 +/- 1.2 to 26.4 +/- 3.1 nmol x min(-1) x mg protein(-1). SIN-1 (100 micromol/l) decreased VO2 in contracting (-21 +/- 3%) and in quiescent cells (-24 +/- 7%) by the same extent. Inhibition of VO2 was dose dependent (EC(50): 10(-7) mol/l). S-nitroso-N-acetyl-penicillamine, another NO donor, also inhibited VO2, whereas SIN-1C (100 micromol/l), the degradation product of SIN-1, displayed no inhibitory effect. Intracellular Ca(2+) remained unchanged, and inhibition of protein kinases G, A, or C did not antagonize the effect of NO. Mitochondrial NADH increased with NO, indicating a reduced flux through the respiratory chain. In quiescent but not in contracting cardiomyocytes, NO significantly increased adenosine, indicating a reduced energy status. These data suggest the following. 1) NO decreases cardiac respiration, most likely via direct inhibition of the respiratory chain. 2) Whereas in quiescent cardiomyocytes the inhibition of aerobic ATP formation by NO causes reduction in energy status, contracting cells are able to compensate for the NO-induced inhibition of oxidative phosphorylation, maintaining energy status constant.

Inotropic response to beta-adrenergic receptor stimulation and anti-adrenergic effect of ACh in endothelial NO synthase-deficient mouse hearts.

1. The functional consequences of a lack of endothelial nitric oxide synthase (eNOS) on left ventricular force development and the anti-adrenergic effect of acetylcholine (ACh) were investigated in isolated hearts and cardiomyocytes from wild type (WT) and eNOS knockout (eNOS-/-) mice. 2.eNOS expression in cardiac myocytes accounted for 20 % of total cardiac eNOS (Western blot analysis). These results were confirmed by RT-PCR analysis. 3. In the unstimulated perfused heart, the left ventricular pressure (LVP) and maximal rate of left ventricular force development (dP/dtmax) of eNOS-/- hearts were not significantly different from those of WT hearts (LVP: 97 +/- 11 mmHg WT vs. 111 +/- 11 mmHg eNOS-/-; dP/dtmax: 3700 +/- 712 mmHg s(-1) WT vs. 4493 +/- 320 mmHg s)-1) eNOS-/-). 4. The dobutamine (10-300 nM)-induced increase in LVP was enhanced in eNOS-/- hearts. In contrast, L-type Ca2+ currents (ICa,L) in isolated cardiomyocytes of WT and eNOS-/- hearts showed no differences after beta-adrenergic stimulation. Dibutyryl-cGMP (50 microM) reduced basal ICa,L in WT cells to 72 +/- 12 % while eNOS-/- ICa,L was insensitive to the drug. The pre-stimulated ICa,L (30 nM isoproterenol) was attenuated by dibutyryl-cGMP in WT and eNOS-/- cells to the same extent. 5. The Ca2+ (1.5-4.5 mM)-induced increase in inotropy was not different between the two experimental groups and beta-adrenergic receptor density was increased by 50% in eNOS-/- hearts. 6. The contractile effects of dobutamine could be inhibited almost completely by ACh or adenosine. The extent of the anti-adrenergic effect of both compounds was identical in WT and eNOS-/- hearts. Measurement of ICa,L in isolated cardiac myocytes yielded similar results. 7. These data demonstrate that in the adult mouse (1) lack of eNOS is associated with increased cardiac contractile force in response to beta-adrenergic stimulation and with elevated -adrenergic receptor density, (2) the unaltered response of ICa,L in eNOS-/- cardiac myocytes to beta-adrenergic stimulation suggests that endothelium-derived NO is important in mediating the whole-organ effects and (3) eNOS is unimportant for the anti-adrenergic effect of ACh and adenosine.