Nitric oxide and cGMP protein kinase activity in aged ventricular myocytes.

We tested the hypothesis that nitric oxide-induced negative functional effects through cGMP would be reduced in aged cardiac myocytes. Maximum rate of shortening (R(max)) and percent shortening of ventricular myocytes from young (6 mo) and old (3 y) rabbits were studied using a video edge detector. cGMP-dependent phosphorylation was examined by electrophoresis and autoradiography. Myocytes received a nitric oxide donor S-nitroso-N-acetyl-penicillamine (SNAP, 10(-7), 10(-6), and 10(-5) M) followed by KT-5823 (10(-6) M), a cGMP protein kinase inhibitor. Baseline function was similar in young and old myocytes (89.1 +/- 4.5 young vs. 86.4 +/- 8.3 microm/s old R(max), 5.6 +/- 0.3 vs. 5.2 +/- 0.7%shortening). SNAP (10(-5) M) decreased R(max) in both young (25%, n = 6) and old myocytes (24%, n = 7). SNAP also reduced percent shortening by 28% in young and 23% in old myocytes. The negative effects of SNAP were partially reversed by KT-5823 only in young myocytes. Multiple proteins were phosphorylated by cGMP, and KT-5823 could reduce this effect. The degree of phosphorylation was significantly less in old myocytes. These results suggest that the functional response of ventricular myocytes to nitric oxide was preserved during aging. However, the importance of cGMP-dependent protein phosphorylation was decreased, indicating a shift to other pathways.

Ethanol-induced reduction in myocardial oxygen consumption can be attenuated by inhibiting guanylyl cyclase.

We tested the hypothesis that low-dose ethanol-induced reductions in myocardial metabolism were related to increased cyclic guanosine monophosphate (GMP). Anesthetized open chest rabbits were divided into four groups: control (Ringers lactate and vehicle), ETOH (250 mg/kg i.v. ethanol and vehicle), ODQ (Ringers lactate and 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, ODQ 10(-4) M ), and ETOH-ODQ (ethanol and ODQ). ODQ, a soluble guanylyl cyclase inhibitor, or vehicle was applied topically to the epicardium for 15 min, while either Ringers lactate or ethanol was administered intravenously. Oxygen consumption (VO2 ) in both the subepicardium (EPI) and subendocardium (ENDO) was determined from coronary blood flow (radioactive microspheres) and O2 extraction (microspectrophotometry). Cyclic GMP was determined by radioimmunoassay. ETOH significantly decreased VO2 in the subepicardium (9.2 +/- 1.0-5.6 +/- 0.7 ml O2 /min/100 g) and subendocardium (9.7 +/- 0.8-7.1 +/- 0.8) and increased cyclic GMP in the subepicardium (10.2 +/- 1.7-13.8 +/- 0.8 pmol/g) and subendocardium (11.0 +/- 0.5-13.7 +/- 0.9). With ODQ, there was no significant change in the subepicardial (9.5 +/- 1.3) or subendocardial (9.0 +/- 0.9) VO2. However, ODQ caused a significant increase in both wall thickening (12.9 +/- 0.9-17.2 +/- 1.2%) and maximal rate of change in wall thickness (10.8 +/- 0.9-16.3 +/- 1.9 mm/s) and decreased subepicardium (8.3 +/- 1.3) and subendocardium (7.8 +/- 1.2) cyclic GMP. The ETOH-ODQ group had cyclic GMP (subepicardium 9.0 +/- 1.8, subendocardium 8.6 +/- 2.4) and VO2 (subepicardium 7.9 +/- 0.5, subendocardium 8.4 +/- 0.4) values similar to control. Thus, the ethanol-induced rise in cyclic GMP was associated with a decrease in myocardial O2 consumption. When this rise was blocked with a soluble guanylyl cyclase inhibitor, the reduction in metabolic demand was also eliminated. This demonstrated that the alcohol-induced reduction in myocardial metabolism was related to increased cyclic GMP and suggests a novel mechanism for the effect of ethanol.

Interaction between cyclic GMP protein kinase and cyclic AMP may be diminished in stunned cardiac myocytes.

We tested the hypothesis that the importance of the negative functional effects of the cyclic GMP protein kinase would be reduced in stunned (simulated ischemia/reperfusion) cardiac myocytes. Ventricular cardiac myocytes were isolated from New Zealand white rabbits (N=7). Myocytes were studied at baseline and after simulated ischemia (15 min of 95% N(2)-5% CO(2) at 37 degrees C) followed by simulated reperfusion (reoxygenation). Cell shortening was studied with a video edge detector; O(2) consumption was measured using O(2) electrodes. Protein phosphorylation was measured autoradiographically after gel electrophoresis. Functional and metabolic data were acquired after: (1) 8-(4-chlorophenylthio)guanosine-3',5'-monophosphate (PCPT, cGMP protein kinase agonist) 10(-7) or 10(-5) M; (2) 8-Br-cAMP 10(-5) M followed by PCPT 10(-7) or 10(-5) M; (3) beta-phenyl-1, N(2)-etheno-8-bromoguanosine-3',5'-monophosphorothioate, SP-isomer (SP, cGMP protein kinase agonist) 10(-7) or 10(-5) M; (2) 8-Br-cAMP 10(-5) M followed by SP 10(-7) or 10(-5) M. At baseline, percent of shortening (Pcs) and maximal rate of shortening (Rs) were significantly lower in the stunned myocytes (Pcs: 5.0+/-0.2% control vs. 3.8+/-0.3 stunned; Rs: 64.8+/-5.9 microm/s control vs. 46.9+/-4.8 stunned). In both groups, PCPT and SP dose-dependently decreased Pcs and Rs. The effects were slightly, but not significantly, less in stunned myocytes. 8-Br-cyclic AMP significantly increased function in control, but not stunned myocytes (Pcs, 4.5+/-0.5 to 6.2+/-0.8 control vs. 3.1+/-0.2 to 3.6+/-0.2 stunned). The negative functional effects of PCPT and SP were diminished after 8-Br-cyclic AMP in control (from -39% to-29%) and diminished significantly more in the stunned myocytes (-19%). PCPT and cyclic AMP phosphorylated similar protein bands. In stunned myocytes, three (22, 31 and 53 kDa) bands were enhanced less by PCPT.

Dose response to nitric oxide in adult cardiac surgery patients.

STUDY OBJECTIVE: To determine the dose responsiveness to nitric oxide in adult cardiac surgery patients, especially in those patients with pulmonary hypertension. DESIGN: Prospective, randomized, nonblinded study. SETTING: University teaching hospital. PATIENTS: 62 consecutive cardiac surgery patients demonstrating pulmonary hypertension immediately before induction of anesthesia. INTERVENTIONS: Subjects were assigned by random number allocation to receive one of five doses of inhaled nitric oxide on termination of cardiopulmonary bypass (CBP; i.e., restitution of pulmonary artery flow). Subjects in Group 1 (n = 11) received 10 ppm of inhaled nitric oxide, Group 2 subjects (n = 12) received 20 ppm, Group 3 subjects (n = 12) received 30 ppm, and Group 4 subjects (n = 12) received 40 ppm. The fifth group (n = 15) received no nitric oxide. This fifth group served as a control and was treated with milrinone only. Those patients who were randomized to the milrinone group, had milrinone initiated by bolus administration (50 microg/kg) 15 min before separation from CPB. Milrinone was maintained at 0.5 microg/kg/min in the operating room thereafter. The conduct of anesthesia, surgery, and CBP were controlled. A therapeutic algorithm dictated the use of vasoactive substances for all patients. MEASUREMENTS: Heart rate, mean arterial pressure, pulmonary vascular resistance (PVR), peripheral vascular resistance, cardiac index, and right ventricular ejection fraction were monitored throughout the operative experience. MAIN RESULTS: There were no significant differences found in demographic data, baseline hemodynamic data, surgical treatment, conduct of CBP, or the use of inotropic or vasoactive drugs among the five treatment groups. The percentage decrease in PVR on treatment with nitric oxide as compared to baseline values was not significantly different among the groups (10 ppm = 38%, 20 ppm = 50%, 30 ppm = 44%, 40 ppm = 36%, milrinone = 58%, p = 0.86). CONCLUSIONS: Treatment with nitric oxide was associated with significant reductions in PVR in all groups. Dosages higher than 10 ppm were not associated with greater reductions in pulmonary vascular tone. In view of the fact that nitric oxide-related toxicity is dose-related, doses greater than 10 ppm do not appear to be justified in this patient population.

Interaction between the opposing functional effects of cyclic AMP and cyclic GMP in hypertrophic cardiac myocytes.

We tested the hypothesis that in isolated cardiac myocytes, the negative functional effects of cyclic GMP would be blunted when the level of cyclic AMP was increased and that this interaction would be altered in renal hypertensive (One-Kidney-One-Clip, 1K1C) cardiac hypertrophic rabbits. Using isolated control and 1K1C ventricular myocytes, cyclic AMP and cell shortening (%) data were collected: 1) at baseline, 2) after the addition of 8-Br-cGMP 10(-7), -6, -5 M, and 3) after forskolin (10(-6) M), an adenylate cyclase activator, followed by 8-Br-cGMP 10(-7), -6, -5 M. Basal levels of cyclic AMP were similar in control vs. 1K1C myocytes (10.2 +/- 1.6 vs. 11.3 +/- 2.6 pmol/10(5) myocytes). We found that 8-Br-cGMP decreased the percent shortening in a dose related manner in both control myocytes (5.1 +/- 0.6 to 3.2 +/- 0.4%) and hypertrophic myocytes (5.2 +/- 0.4 to 3.6 +/- 0.5). The level of cyclic AMP significantly increased after the addition of 8-Br-cGMP in control myocytes (14.1 +/- 2.1), but not in 1K1C myocytes. Forskolin increased the percent shortening in the control myocytes (3.8 +/- 0.1 to 4.8 +/- 0.4), but no significant increase was noted in the hypertrophic myocytes (3.6 +/- 0.3 to 3.7 +/- 0.3). The level of cyclic AMP significantly increased after the addition of forskolin in both control (13.9 +/- 2.0), and 1K1C cells (14.6 +/- 3.8). Forskolin attenuated the negative functional effects of 8-Br-cGMP in the control (4.8 +/- 0.4 to 3.2 +/- 0.1) and 1K1C myocytes (3.7 +/- 0.3 to 2.7 +/- 0.3). The addition of 8-Br-cGMP did not affect the level of cyclic AMP after forskolin in either control (13.9 +/- 2.0 to 14.8 +/- 2.5) or 1K1C myocytes (14.6 +/- 3.8 to 13.8 +/- 1.9). These data indicated that in hypertrophic cardiac myocytes the negative functional effects of 8-Br-cGMP were similar to control, but the positive functional effects of cyclic AMP were blunted. There was an increase in cyclic AMP levels after addition of 8-Br-cGMP in control but not 1K1C cells. We conclude that in control and hypertrophic myocytes, the effects of cyclic GMP were blunted after forskolin, but this did not seem to be related to cyclic AMP phosphodiesterase activity.

Opposing functional effects of cyclic GMP and cyclic AMP may act through protein phosphorylation in rabbit cardiac myocytes.

1. We tested the hypothesis that the negative functional effects of cyclic GMP (cGMP) oppose the positive effects of cyclic AMP (cAMP) in cardiac myocytes through interaction at the level of their respective protein kinases. 2. Cell shortening was studied using a video-edge detector. The O2 consumption of a suspension of rabbit ventricular myocytes was measured using O2 electrodes. Protein phosphorylation was measured autoradiographically following SDS-PAGE. Data were collected with: (1) 8-bromo-cGMP (8-Br-cGMP) 10(-7) or 10(-5) M; (2) 8-bromo-cAMP (8-Br-cAMP) 10(-7) or 10(-5) M; (3) 8-Br-cAMP 10(-5) M followed by 8-Br-cGMP 10(-7) or 10(-5) M; (4) 8-Br-cGMP 10(-5) M followed by 8-Br-cAMP 10(-7) or 10(-5) M; (5) 8-Br-cGMP 10(-7) or 10(-5) M followed by KT 5720 (cAMP-dependent protein kinase inhibitor) or KT 5823 (cGMP-dependent protein kinase inhibitor) 10(-6) M; and (6) 8-Br-cAMP 10(-7) or 10(-5) M followed by KT 5720 or KT 5823 10(-6) M. 3. 8-Br-cGMP 10(-5) M decreased percent shortening (Pcs) from 6.3+/-0.6 to 3.6+/-0.4% and rate of shortening (Rs) from 66.7+/-4.4 to 41.8+/-4.2 microm s(-1). 8-Br-cAMP 10(-5) M increased Pcs (from 3.7+/-0.2 to 4.8+/-0.2) and Rs (from 50.0+/-3.0 to 60.0+/-3.1). With 8-Br-cAMP 10(-5) M, 8-Br-cGMP 10(-5) M decreased Pcs and Rs less. The positive functional effects of 8-Br-cAMP 10(-7) or 10(-5) M were also diminished with 8-Br-cGMP 10(-5) M. Following 8-Br-cGMP 10(-7) or 10(-5) M, KT 5720 10(-6) M further decreased Pcs to 2.5+/-0.3 and Rs to 30.0+/-4.1. KT 5823 10(-6) M returned Pcs to 4.7+/-0.4 and Rs to 61.3+/-5.3. Following 8-Br-cAMP 10(-7) or 10(-5) M, KT 5720 decreased the elevated Pcs and Rs significantly and KT 5823 10(-6) M further increased these parameters. 4. cGMP and cAMP phosphorylated the same five protein bands. With KT 5720 or KT 5823, all of the bands were lighter at the same concentration of 8-Br-cAMP and 8-Br-cGMP. 5. We conclude that, in rabbit ventricular myocytes, the opposing functional effects of cGMP and cAMP are related to the interaction at the level of their respective protein kinases.

Decreasing cyclic GMP exerts similar positive functional effects on cardiac myocytes regardless of initial level.

We tested the hypothesis that lowering the level of cyclic GMP would have positive functional effects on isolated rabbit ventricular myocytes regardless of the basal cyclic GMP level. Cell shortening data were collected with a video detector; O(2) consumption data were obtained with a Clark electrode; intracellular cyclic GMP levels were obtained by radioimmunoassay. Data were obtained: (1) at baseline; (2) after the addition of 1H-[1,2,4]oxadiazolo[4, 3-alpha]quinoxaline-1-one (ODQ) 10(-6) and 10(-4) mol/l, a selective soluble guanylyl cyclase inhibitor, and (3) after zaprinast 10(-6) mol/l, a cyclic GMP phosphodiesterase inhibitor, followed by ODQ 10(-6) and 10(-4) mol/l. We found that ODQ 10(-4) mol/l significantly decreased the cyclic GMP level from 493 +/- 75 to 301 +/- 78 (fmol/100,000 myocytes) and increased percent shortening (Pcs, %; 4.9 +/- 0.3 vs. 5.8 +/- 0.6) and maximum rate of shortening (Rs, microm/s; 58.7 +/- 5.7 vs. 73.6 +/- 4.9). Zaprinast significantly increased the cyclic GMP level from 419 +/- 140 to 599 +/- 241 and decreased Pcs (6.2 +/- 0.5 vs. 4.4 +/- 0.4) and Rs (65.5 +/- 5.3 vs. 49.6 +/- 4.3). After zaprinast, ODQ 10(-4) mol/l decreased the cyclic GMP level to 439 +/- 139 and increased percent shortening and rate of shortening by a similar percentage compared to the non-zaprinast treated myocytes. We conclude that in rabbit ventricular myocytes, a reduction in the level of myocyte cyclic GMP increases myocyte function independent of the initial cyclic GMP level.

Reduction in the level of cardiac cyclic GMP worsens contractile delay in myocardial stunning.

We tested the hypothesis that a reduction in the level of myocardial cyclic GMP would worsen the contractile delay associated with myocardial stunning. Two groups of 12 anesthetized open-chest New Zealand white rabbits were utilized. Myocardial stunning was produced by two 15-min occlusions of the left anterior descending coronary artery followed by 15 min of reperfusion. Either control vehicle (saline + 1% DMSO) or 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ 10(-4) M, a guanylate cyclase inhibitor) was topically applied to the left ventricular surface of the rabbit hearts. Left ventricular and aortic pressures along with wall thickness parameters were determined. Coronary blood flow (microspheres) and O(2) extraction (microspectrophotometry) were used to determine myocardial O(2) consumption. Myocardial stunning was observed in the control group through an increased delay in onset of wall thickening (46.2 +/- 7.3 vs 76.6 +/- 17.5 ms). There was no significant effect of stunning on the rate of wall thickening (21.8 +/- 9.5 vs 18.1 +/- 3.4 mm/s) or O(2) consumption (stun 4.6 +/- 0.6, control 4.8 +/- 0.4 ml O(2)/min/100 g). After treatment with ODQ 10(-4) M, both delay (43.9 +/- 9.6 vs 134.1 +/- 30.0 ms) and myocardial O(2) consumption (stun 5.9 +/- 0.6, control 5.9 +/- 0. 7) increased significantly compared to control. There was no significant change in the rate of wall thickening. We conclude that decreasing cyclic GMP worsens stunning by increasing delay in onset of wall thickening and increasing local O(2) costs in the stunned region.

Down regulation of myocardial beta1-adrenoceptor signal transduction system in pacing-induced failure in dogs with aortic stenosis-induced left ventricular hypertrophy.

We recently demonstrated that rapid ventricular pacing caused cardiac failure (Failure) in dogs with aortic stenosis-induced left ventricular hypertrophy (Hypertrophy) and isoproterenol caused no significant increases in function, O2 consumption and intracellular cyclic AMP level in the failing hypertrophied hearts. We tested the hypothesis that alterations in the beta1-adrenoceptor-signal transduction pathway would correlate with the reduced functional and metabolic responses to beta-adrenergic stimulation during the transition from the compensated hypertrophy to failure. Pressure overload-induced left ventricular hypertrophy was created using aortic valve plication in 10 dogs over a 6-month period. Five months after aortic valve plication, congestive heart failure was induced in 5 dogs by rapid ventricular pacing at 240 bpm for 4 weeks. The density of myocardial beta1-adrenoceptors (fmoles/mg membrane protein; fmoles/g wet tissue) was significantly reduced in the Failure dogs (176+/-19; 755+/-136) when compared to those of the Control (344+/-51; 1,551+/-203) and the Hypertrophy (298+/-33; 1,721+/-162) dogs. The receptor affinities were not significantly different among all groups. There was a small but significant decrease in the percentage of beta1-adrenoceptors of the failing hypertrophied hearts (62+/-3%) when compared to that of the hypertrophied hearts (77+/-5%). The basal myocardial adenylyl cyclase activity (pmoles/mg protein/min) was significantly lower in the Failure dogs (45+/-4) than in the Control (116+/-14) and Hypertrophy (86+/-6) dogs. The forskolin (0.1 mM)-stimulated adenylyl cyclase activity was also significantly lower in the Failure dogs (158+/-17) than in the Control dogs (296+/-35) and slightly lower than in the Hypertrophy dogs (215+/-10). There were no significant differences in low Km cyclic AMP-phosphodiesterase activities among all groups. We conclude that down regulation of beta1-adrenoceptors and reduced adenylyl cyclase activities contribute to the decreases in myocardial functions and beta-adrenergic responses in the failing hypertrophied hearts induced by rapid ventricular pacing.

Effects of beta-adrenoceptor stimulation on pacing-induced failure of dog hypertrophic hearts.

1. We tested the hypothesis that the transition to pacing-induced failure in hypertrophic hearts would result in reduced functional and metabolic responses to beta-adrenoceptor stimulation. 2. Isoproterenol (ISO; 0.1 microg/kg per min) was infused into a coronary artery in five anaesthetized open-chest control, five aortic stenosis-induced left ventricular hypertrophy (LVH) and five LVH pacing-induced failure dogs. 3. In both control and LVH dogs, but not in failure dogs, ISO significantly increased local regional work (1,923+/-665 vs 2,656+/-715, 1,185+/-286 vs 1,906+/-562 and 835+/-106 vs 849+/-216g.mm/min, respectively), force (11.1+/-1.4 vs 16.9+/-2.6, 8.6+/-1.5 vs 13.7+/-2.3 and 12.2+/-1.1 vs 11.0+/-1.8g, respectively) and myocardial O2 consumption (7.3+/-2.0 vs 10.0+/-1.5, 8.2+/-1.6 vs 11.6+/-2.6 and 4.4+/-1.5 vs 5.5+/-1.8 mL O2/min per 100 g, respectively). 4. Isoproterenol also significantly increased cAMP in control and LVH dogs (474+/-67 vs 600+/-91 and 473+/-34 vs 619+/-53 pmol/g, respectively). In heart failure, cAMP was significantly lower and there was no significant increase in cAMP in response to ISO (245+/-43 vs 314+/-40pmol/g, respectively). 5. We conclude that there were no significant myocardial functional, O2 consumption or cAMP responses to ISO after the transition from hypertrophy to cardiac failure.

Cyclic GMP attenuates cyclic AMP-stimulated inotropy and oxygen consumption in control and hypertrophic hearts.

We tested the hypothesis that increasing myocardial cyclic GMP would attenuate cyclic AMP induced positive inotropy and O2 consumption, in part, through changes in cyclic AMP and that renal hypertension-induced cardiac hypertrophy (HYP) would alter this relationship. Anesthetized, open chest rabbits (N = 48) were divided into four groups of control (CON) and HYP animals which received vehicle (VEH), isoproterenol 10(-6)M (ISO), 3-morpholinosyndnonimine 10(-4)M, (SIN-1), or a combination of ISO+SIN-1. Coronary blood flow (microspheres) and O2 extraction (microspectrophotometry) were used to determine O2 consumption in both subepicardium (EPI) and subendocardium (ENDO). Left ventricular change in wall thickness (%) was increased significantly by ISO in both CON (16 +/- 4 to 31 +/- 6) and HYP (17 +/- 2 to 24 +/- 3). Percent change in wall thickness was similar in the CON, SIN-1, and ISO+SIN-1 groups. Myocardial O2 consumption (ml O2/min/100 g) was increased by ISO in CON (10.3 +/- 1.0 to 13.6 +/- 2.0 EPI; 10.9 +/- 1.0 17.1 +/- 1.7 ENDO) and HYP (8.2 +/- 1.4 to 12.3 +/- 2.2 EPI; 6.6 +/- 1.4 to 14.8 +/- 1.8 ENDO). Oxygen consumption was unaffected by SIN-1 in CON and HYP animals. ISO+SIN-1 caused attenuated ISO-induced increases in O2 consumption in CON in EPI and ENDO, and in EPI in HYP. Cyclic GMP (pmol/g) was unchanged by ISO in CON and HYP, and increased by SIN-1 in CON (8.1 +/- 1.3 to 19.2 +/- 2.3 EPI) and HYP (9.1 +/- 1.5 to 12.8 +/- 2.0 EPI). Cyclic GMP remained elevated with ISO+SIN-1 in both groups. Cyclic AMP (pmol/g) was increased significantly by ISO in CON (496 +/- 43 to 725 +/- 106 EPI; 534 +/- 44 to 756 +/- 148 ENDO) and insignificantly in HYP (435 +/- 50 to 566 +/- 35 EPI; 497 +/- 51 to 583 +/- 47 ENDO). Cyclic AMP levels were unaffected by SIN-1 in either group. Isoproterenol induced increases in cyclic AMP were blunted by ISO+SIN-1 in CON (496 +/- 43 to 537 +/- 59 EPI) and not affected in HYP. The current study demonstrated attenuation of cyclic AMP mediated increased inotropy and O2 consumption by increasing cyclic GMP, which appeared, in part, related to cyclic GMP-induced reduction in cyclic AMP. This effect of cyclic GMP on cyclic AMP was not observed in myocardial hypertrophy.

Nitroprusside attenuates myocardial stunning through reduced contractile delay and time.

We hypothesized that myocardial stunning would be reversed through increased cyclic GMP caused by nitroprusside, and that this would be accomplished through a decreased proportion of regional work during diastole. Hearts were instrumented to measure left ventricular pressure, and regional myocardial mechanics were recorded using a miniature force transducer and ultrasonic dimension crystals in eight open-chest anesthetized dogs. Following baseline (CON), the left anterior descending coronary artery (LAD) was occluded for 15 min, followed by a 30-min recovery (STUN). Then intracoronary LAD infusion of sodium nitroprusside (NP) (4 microg/kg/ min) was begun. The time delay (msec) to regional shortening increased significantly from 18+/-13 to 73+/-13 following stunning, but was reduced to 49+/-18 by NP. Total regional work (g*mm/min) at baseline (1368+/-401 CON) was unchanged with stunning (1320+/-333 STUN), but reduced (961+/-240) following NP. Time to peak force development (msec) increased significantly with stunning from 284+/-13 (CON) to 333+/-11 (STUN), but was reduced to 269+/-12 following NP. The percentage work during systole was reduced from 96%+/-2% (CON) to 77%+/-7% (STUN), but returned to 98%+/-1% with NP. Regional O2 consumption was unaffected by either treatment. Cyclic GMP was unchanged by stunning (2.9+/-0.3-2.9+/-0.4 pmol/g) but increased significantly with NP (4.6+/-0.6). These data indicated that regional myocardial stunning could be attenuated by nitroprusside, which increased cyclic GMP, decreased contractile delay, increased the proportion of work done during systole, and reduced time of shortening.

Nitroprusside reverses lengthened time of contraction in stunned canine cardiac myocytes.

We tested the hypothesis that stunning reduces the function of isolated canine ventricular myocytes and that nitroprusside (NP) reverses this effect. After stunning (15 min occlusion, 45 min reperfusion), isolated myocytes were prepared from control (circumflex artery) and stunned (left anterior descending) regions of the hearts of seven dogs. The myocytes were examined at baseline and with NP (10(-6,-5,-4) M) for oxygen consumption (MVO2, nl O2/min/10(5) cells), cyclic guanosine monophosphate (cyclic GMP; fmol/10(5) cells), and cell contraction. Basal MVO2 was not significantly different between control and stunned myocytes (888 +/- 108 vs. 716 +/- 94). NP caused a dose dependent decrease in MVo2 (control, 262 +/-51; stunned, 287 +/- 59, NP 10(-4) M). Basal cyclic GMP levels were comparable between control and stunned myocytes (117 +/-28 vs. 124 +/- 18). NP produced a similar dose-dependent increase in cyclic GMP in control and stunned myocytes. Baseline cell shortening (%) was similar in control vs. stunned myocytes (12.1 +/- 1.2 vs. 11.0 +/- 0.9). NP reduced shortening (6.9 +/- 0.3 vs. 7.3 +/- 0.5, NP 10(-4) M). There was no baseline difference in maximal rate of shortening (microm/s) between control and stunned myocytes (164 +/- 14, 157 +/- 20). With NP, a decrease in the maximal rate of shortening was seen in both groups (128 +/- 12, 139 +/- 21, NP 10(-4) M). The time of contraction (s) was significantly longer in stunned (0.20 +/- 0.03) versus control (0.13 +/- 0.01). NP significantly lengthened the time of contraction in controls in a dose-dependent manner (0.33 +/-0.05, NP 10(-4) M). In stunned myocytes, however, low-dose NP (10(-6) M) caused a decrease in the time of contraction (0.15 +/-0.03). High-dose NP (10(-4) M) did not significantly lengthen time of contraction in stunned cells (0.23 +/- 0.02). The time of relaxation followed a similar pattern. We conclude that part of the effect of NP in low doses in stunned myocardium is to reduce the lengthened time of contraction and relaxation characteristic of stunning.

Cyclic GMP and cyclic AMP induced changes in control and hypertrophic cardiac myocyte function interact through cyclic GMP affected cyclic-AMP phosphodiesterases.

We tested the hypothesis that the negative functional effects of cyclic GMP (cGMP) would be greater after increasing cyclic AMP (cAMP), because of the action of cGMP-affected cAMP phosphodiesterases in cardiac myocytes and that this effect would be altered in left ventricular hypertrophy (LVH) produced by aortic valve plication. Myocyte shortening data were collected using a video edge detector, and O2 consumption was measured by O2 electrodes during stimulation (5 ms, 1 Hz, in 2 mM Ca2+) from control (n = 7) and LVH (n = 7) dog ventricular myocytes. cAMP and cGMP were determined by a competitive binding assay. cAMP was increased by forskolin and milrinone (10(-6) M). cGMP was increased with zaprinast and decreased by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxilin-1-one (ODQ) both at 10(-6) and 10(-4) M, with and without forskolin or forskolin + milrinone. Zaprinast significantly decreased percent shortening in control (9 +/- 1 to 7 +/- 1%) and LVH (10 +/- 1 to 7 +/- 1%) myocytes. It increased cGMP in control (36 +/- 5 to 52 +/- 7 fmol/10(5) myocytes) and from the significantly higher baseline value in LVH (71 +/- 12 to 104 +/- 18 fmol/10(5) myocytes). ODQ increased myocyte function and decreased cGMP levels in control and LVH myocytes. Forskolin + milrinone increased cAMP levels in control (6 +/- 1 to 15 +/- 2 pmol/10(5) myocytes) and LVH (8 +/- 1 to 18 +/- 2 pmol/10(5) myocytes) myocytes, as did forskolin alone. They also significantly increased percent shortening. There were significant negative functional effects of zaprinast after forskolin + milrinone in control (15 +/- 2 to 9 +/- 1%), which were greater than zaprinast alone, and LVH (12 +/- 1 to 9 +/- 1%). This was associated with an increase in cGMP and a reduction in the increased cAMP induced by forskolin or milrinone. ODQ did not further increase function after forskolin or milrinone in control myocytes, despite lowering cGMP. However, it prevented the forskolin and milrinone induced increase in cAMP. In hypertrophy, ODQ lowered cGMP and increased function after forskolin. ODQ did not affect cAMP after forskolin and milrinone in LVH. Thus, the level of cGMP was inversely correlated with myocyte function. When cAMP levels were elevated, cGMP was still inversely correlated with myocyte function. This was, in part, related to alterations in cAMP. The interaction between cGMP and cAMP was altered in LVH myocytes.

Pacing-induced cardiac failure of hypertrophic hearts: effects of cyclic GMP reduction.

BACKGROUND: We tested the hypothesis that pacing-induced cardiac failure of hypertrophic hearts would reduce the functional and metabolic responses of these hearts to guanylate cyclase inhibition and this was associated with alterations in cyclic GMP. MATERIALS AND METHODS: Methylene blue (MB, 2 mg/kg/min, guanylate cyclase inhibitor) was infused into the left anterior descending coronary artery in 5 control, 5 left ventricular hypertrophy (LVH), and 5 LVH pacing-induced failure dogs. Regional myocardial work was calculated as the integrated product of force and segment shortening and regional myocardial O(2) consumption (VO(2)) from coronary blood flow and O(2) extraction measurements. Cyclic GMP was determined by radioimmunoassay. RESULTS: MB increased regional work (635 +/- 169 vs 1649 +/- 500, 781 +/- 184 vs 1569 +/- 203 g * mm/min) and VO(2) (8.3 +/- 1.4 vs 10.9 +/- 1.4, 7.3 +/- 0.7 vs 9.1 +/- 0.7 ml O(2)/min/100 g) in both control and LVH dogs but not in failure dogs (536 +/- 234 vs 623 +/- 193, 3.6 +/- 1.1 vs 4.7 +/- 1.9). MB also decreased cyclic GMP in control dogs (1170 +/- 142 vs 812 +/- 105 pmol/g). LVH dogs had elevated baseline cyclic GMP (5875 +/- 949) compared to control dogs but also demonstrated decreased cyclic GMP in response to MB (2820 +/- 372). In failure dogs, basal cyclic GMP was also elevated (4650 +/- 613) compared to control dogs but there was a lack of response to MB (3670 +/- 640). CONCLUSIONS: We conclude that the myocardial function, VO(2) and cyclic GMP responses to methylene blue are diminished in the transition from hypertrophy to cardiac failure.

Cyclic GMP protein kinase mediates negative metabolic and functional effects of cyclic GMP in control and hypertrophied rabbit cardiac myocytes.

We tested the hypothesis that in isolated cardiac myocytes, the negative metabolic and functional effects of cyclic guanosine monophosphate (GMP) are mediated by cyclic GMP protein kinase activity, and that these effects are altered in renal hypertensive (one-kidney, one-clip, 1K1C) cardiac hypertrophic rabbits. By using isolated cardiac myocytes from control and 1K1C rabbits, oxygen consumption (Mvo2; O2 nl/ min/10(5) cells), cyclic GMP (fmol/10(5) cells), and cell shortening (percentage) data were collected (a) at baseline; (b) with cyclic GMP protein kinase inhibitors KT5823 (10(-6) M) or Rp8-pCPT-cGMP (5 x 10(-6) M); (c) with the cyclic GMP phosphodiesterase inhibitor zaprinast (10(-6), 10(-4) M); and (d) with zaprinast (10(-6), 10(-4) M) and protein kinase inhibitors. Basal levels of cyclic GMP were similar in control versus 1K1C myocytes (62 +/- 10 vs. 66 +/- 17 pmol/10(5) myocytes). Zaprinast produced a dose-dependent increase in cyclic GMP in both control and 1K1C myocytes. The addition of KT5823 did not significantly affect cyclic GMP levels. Zaprinast significantly and dose dependently decreased Mvo2, and KT5823 partially restored it in control and 1K1C. Zaprinast also significantly decreased percentage shortening, and KT5823 partially restored it in control. Similar results were obtained with Rp-8pCPT-cGMP, although neither inhibitor was effective without zaprinast. The hypertrophied myocytes demonstrated comparable responses to all agents. These data suggest that the cyclic GMP protein kinase activity was not significant under basal conditions; however, the importance of cyclic GMP protein kinase in control and 1K1C myocytes was significant under conditions of increased intracellular cyclic GMP.

Altered effects of acetylcholine on cyclic AMP and GMP induced changes in O2 consumption of hypertrophic dog cardiac myocytes.

1. We hypothesized that acetylcholine would attenuate the metabolic effect of increasing cAMP and decreasing cGMP on cardiac myocyte O2 consumption (VO2) in dog, and this effect would be altered in left ventricular hypertrophy (LVH) produced by aortic valve placation. 2. Steady-state VO2 of a suspension of ventricular myocytes from control (n = 7) and LVH (n = 6) dogs was measured by Clark O2 electrodes during electrical stimulation (5 ms, 1 Hz, in 2 mm Ca2+). Cyclic AMP and cyclic GMP were determined by radioimmunoassay. Cellular cAMP was increased by forskolin (adenylate cyclase stimulator) and cGMP was decreased by LY83583 (guanylate cyclase inhibitor) both at 10(-7,-6,-5,-4) M with and without 10(-6) M acetylcholine. 3. Baseline cGMP level in LVH (62 +/- 10 fmol 10(-5) myocytes) was significantly greater than that in control (20 +/- 3), although the myocyte VO2 (356 +/- 39 nL O2 min(-1) 10(-5) myocytes) and cAMP levels (3.9 +/- 0.6 nmol 10(5-1) myocytes) were similar to control (312 +/- 23 and 6.9 +/- 3.1). 4. Forskolin increased myocyte cAMP in both control and LVH myocytes and increased VO2 by 51 +/- 13 in control and 91 +/- 65 in LVH myocytes. LY83583 decreased myocyte cGMP levels in control and LVH myocytes and increased VO2 by 128 +/- 57 in control and 43 +/- 26 in LVH myocytes. 5. Acetylcholine altered the cAMP, cGMP, and VO2 levels in control to 2.4 +/- 0.4, 30 +/- 3 and 213 +/- 27 and LVH to 2.5 +/- 0.3, 85 +/- 9 and 261 +/- 32. Acetylcholine attenuated the maximal effects of forskolin on VO2 to 32 +/- 27 in control and 66 +/- 56 in LVH myocytes. Acetylcholine also decreased the maximal effects of LY83583 to 82 +/- 50 in control and 19 +/- 19 in LVH myocytes. 6. The positive metabolic effects of both increases in myocyte cAMP and decreases in cGMP were blunted by acetylcholine. There was a significant increase in myocyte cGMP with forskolin in LVH myocytes. Acetylcholine decreased the increased myocyte VO2 caused by elevated cAMP or decreased cGMP in both control and LVH myocytes, although the absolute decrease in cAMP was reduced and the absolute values of cGMP were higher in LVH myocytes.

Cyclic GMP reduces ventricular myocyte stunning after simulated ischemia-reperfusion.

We tested the hypothesis that the second messenger activated by nitric oxide, cyclic GMP, would reduce the effects of myocyte stunning following simulated ischemia-reperfusion and that this was related to cyclic GMP protein kinase. Ventricular cardiac myocytes were isolated from New Zealand White rabbits (n = 8). Cell shortening was measured by a video edge detector and protein phosphorylation was determined autoradiographically after SDS gel electrophoresis. Cell shortening data were acquired at: (i) baseline followed by 8-Bromo-cGMP 10(-6) M (8-Br-cGMP) and then KT 5823 10(-6) M (cyclic GMP protein kinase inhibitor) and (ii) simulated ischemia (20 min of 95% N(2)-5% CO(2) at 37 degrees C) followed by simulated reperfusion (reoxygenation) with addition of 8-Br-cGMP 10(-6) M followed by KT 5823 10(-6) M, (iii) addition of 8-Br-cGMP prior to ischemia followed by the addition of KT 5823 10(-6) M after 30 min of reoxygenation. In the control group, 8-Br-cGMP 10(-6) M decreased percentage shortening (%short) (5.0 +/- 0.6 vs 3.8 +/- 0. 4) and the maximum velocity (V(max), microm/s) (48.6 +/- 6.9 vs 40.2 +/- 6.4). KT 5823 10(-6) M added after 8-Br-cGMP partially restored %short (4.6 +/- 0.5) and V(max) (46.6 +/- 8.0). After stunning, baseline myocytes had decreased %short (3.4 +/- 0.2) and V(max) (36. 0 +/- 4.2). After the addition of 8-Br-cGMP, the %short (2.7 +/- 0. 2) and V(max) (27.6 +/- 2.5) decreased further. The addition of KT 5823 did not change either the %short or the V(max). The myocytes with 8-Br-cGMP during ischemia had increased %short (4.2 +/- 0.2) and V(max) (37.2 +/- 3.4) when compared to the stunned group. The addition of KT 5823 did not significantly alter %short (3.3 +/- 0.4) or V(max) (29.2 +/- 5.0) in the myocytes pretreated with 8-Br-cGMP. Protein phosphorylation was increased by 8-Br-cGMP in control and stunned myocytes. KT 5823 blocked this effect in control but not stunned myocytes, suggesting some change in the cyclic GMP protein kinase. Ischemia-reperfusion produced myocyte stunning that was reduced when 8-Br-cGMP was added prior to but not after ischemia.

Relationship between decreased function and O2 consumption caused by cyclic GMP in cardiac myocytes and L-type calcium channels.

We tested the hypothesis that part of the decreased function and metabolism caused by cyclic guanosine monophosphate (GMP) in beating cardiac myocytes is related to inhibition of L-type calcium channels. The steady state oxygen consumption (VO2) of a suspension of ventricular myocytes isolated from hearts of New Zealand white rabbits was measured using oxygen electrodes. Cellular cyclic GMP levels were determined by radioimmunoassay. Cell shortening was measured with a video edge detector. The VO2 was obtained after: (1) adding sodium nitroprusside (NP 10(-8),(-6),(-4) M), (2) pretreatment by BAY K8644 10(-5) M (BAY, L-type calcium channel activator), nifedipine 10(-4) M (NF, L-type calcium channel blocker) or forskolin 10(-7) M (FK, adenylate cyclase activator), then adding NP 10(-8),(-6),(-4) M, (3) pretreatment with both FK 10(-7) M and NF 10(-4) M and subsequently adding NP 10(-8),(-6),(-4) M. NP 10(-4) M decreased VO2 from 707 +/- 34 to 410 +/- 13 (nl O2/min per 10(5) myocytes), decreased the percentage of shortening (Pcs) from 5.7 +/- 0.6 to 3.7 +/- 0.5 and the rate of shortening (Rs) from 65.5 +/- 4.5 (microns/s) to 46.2 +/- 5.5. NP 10(-4) M also increased cyclic GMP from 264 +/- 70 (fmol/10(5) myocytes) to 760 +/- 283. Both BAY and FK increased VO2, Pcs and Rs without changing cyclic GMP. NF decreased Pcs, Rs and VO2. Similar metabolic and functional effects of NP were observed with pretreatment with these agents separately, compared to NP alone, and the elevation of cyclic GMP level was not different from the control group. With FK alone, NP 10(-4) M decreased VO2 by 51%, Pcs by 44% and Rs by 39%. In the presence of both FK and NF, the negative effects of NP were diminished significantly. NP 10(-4) M decreased VO2 by 37%, Pcs by 25% and Rs 20%. Thus, in beating cardiac myocytes, the negative metabolic and functional effects of cyclic GMP were related to inhibition on L-type calcium channels only when adenylate cyclase was stimulated.

Negative metabolic effects of cyclic GMP in quiescent cardiomyocytes are not related to L-type calcium channel activity.

We tested the hypothesis that the negative metabolic effects of elevating cyclic GMP act through inhibition of L-type calcium channels in quiescent cardiac myocytes. The steady state O2 consumption (VO2) of ventricular myocytes, isolated from hearts of New Zealand white rabbits, was measured in a glass chamber using Clark-type oxygen electrodes. The cellular cyclic GMP levels were determined by radioimmunoassay at baseline with either 0.5 mM or 2.0 mM of Ca2+, sodium nitroprusside at increasing concentration (10(-8),(-6),(-4) M) with and without pretreatment by BAY K8644 10(-5) M (L-type Ca2+ channel activator) in 0.5 mM Ca2+, or nitroprusside with and without pretreatment with nifedipine 10(-4) M (L-type Ca2+ channel blocker) in 2.0 mM Ca2+. In the 0.5 mM Ca2+ medium, basal VO2 was 459 +/- 104 (nl O2/min per 10(5) myocytes) with a corresponding cyclic GMP level of 112 +/- 23 (fmol/10(5) myocytes). With nitroprusside 10(-4) M, VO2 was decreased to 285 +/- 39 and cyclic GMP level was significantly elevated to 425 +/- 128. In the same medium, VO2 was slightly increased by BAY K8644 10(-5) M while the cyclic GMP level did not change. With BAY K8644 10(-5) M, nitroprusside 10(-4) M decreased VO2 and increased cyclic GMP to a level which was similar to cells treated with nitroprusside alone. In the 2.0 mM Ca2+ medium, the basal VO2 and cyclic GMP were 518 +/- 121 and 137 +/- 24. In the presence of nitroprusside 10(-4) M, VO2 was decreased to 295 +/- 49 and cyclic GMP was increased to 454 +/- 116. In the same medium, nifedipine 10(-4) M significantly decreased VO2, while the cyclic GMP level was comparable to the baseline. After nifedipine 10(-4) M, nitroprusside 10(-4) M decreased VO2 and increased cyclic GMP to levels which were similar to control. Therefore, in quiescent cardiac myocytes, the negative metabolic effects associated with cyclic GMP were not primarily mediated through inhibition of L-type Ca2+ channels.