Disruption of inhibitory G-proteins mediates a reduction in atrial beta-adrenergic signaling by enhancing eNOS expression.

OBJECTIVE: Cardiac parasympathetic nerve activity is reduced in most cardiovascular disease states, and this may contribute to enhanced cardiac sympathetic responsiveness. Disruption of inhibitory G-proteins (Gi) ablates the cholinergic pathway and increases cardiac endothelial nitric oxide (NO) synthase (eNOS) expression, suggesting that NO may offset the impaired attenuation of beta-adrenergic regulation of supraventricular excitability. To test this, we investigated the role of endogenous NO production on beta-adrenergic regulation of rate (HR), contraction (CR) and calcium (Ca2+) handling in atria following blockade of Gi-coupled muscarinic receptors. METHODS: Mice were administered pertussis toxin (PTx, n=105) or saline (C, n=100) intraperitoneally. After 3 days, we measured CR, HR, and NOS protein levels in isolated atria. Intracellular calcium (Ca2+) transients and Ca2+ current density (I(Ca)) were also measured in atrial myocytes. RESULTS: PTx treatment increased atrial myocyte eNOS protein levels compared to C (P<0.05). This did not affect basal atrial function but was associated with a significant reduction in the CR and HR response to isoprenaline (ISO) compared with C. NOS inhibition normalized responses in PTx atria with respect to responses in C atria (P<0.05), which were unaffected. Furthermore, PTx did not affect ISO-stimulated HR and CR in eNOS gene knockout mice (n=40). In agreement with these findings, the ISO-mediated increase in Ca2+ transient was suppressed in PTx-treated myocytes (P<0.05), whereas I(Ca) did not differ between groups. CONCLUSION: eNOS-derived NO inhibits beta-adrenergic responses following disruption of Gi signaling. This suggests that increased eNOS expression may be a compensatory mechanism which reduces beta-adrenergic regulation of heart rate when cardiac parasympathetic control is impaired.

Nitric oxide does not modulate the hyperpolarization-activated current, I(f), in ventricular myocytes from spontaneously hypertensive rats.

OBJECTIVE: : In sinoatrial (SA) node cells, nitric oxide (NO) exerts a dual effect on the hyperpolarization-activated current, I(f), i.e. in basal conditions NO enhances I(f) whereas in the presence of beta-adrenergic stimulation it decreases it. Recent studies have shown that I(f) is present in ventricular myocytes from hypertrophied or failing hearts where it may promote abnormal automaticity. Since these pathological conditions are associated with increased sympathetic tone and upregulation of myocardial NO production, we set out to investigate whether I(f) is similarly modulated by NO in hypertrophied ventricular myocytes. METHODS: Left ventricular myocytes were isolated from 18-20-month-old spontaneously hypertensive rats (SHRs). Membrane current was measured under whole-cell or amphotericin-perforated patch-clamp conditions, at 35 degrees C. RESULTS: Application of diethylamine-NO (DEA-NO, 1-100 microM) did not alter the amplitude or voltage dependence of activation of I(f) under basal conditions (half-activation voltage, V(h): control -82.9+/-2.6, DEA-NO -84.0+/-2.6 mV). Similarly, I(f) was not affected by the inhibition of endogenous NO production (L-NMMA, 500 microM) or guanylate cyclase (ODQ, 10 microM). Forskolin (10 microM) or isoprenaline (100 nM) elicited a positive shift in V(h) but subsequent application of DEA-NO did not further affect the properties of I(f). CONCLUSIONS: Our results show that, unlike in SA node cells, in SHR ventricular myocytes basal and adrenergically stimulated I(f) is not modulated by exogenous NO or by constitutive NO or cGMP production.

Ventricular activation during sympathetic imbalance and its computational reconstruction.

We characterized the epicardial activation sequence during a norepinephrine (NE)-induced ventricular arrhythmia in anesthetized pigs and studied factors that modulated it. Subepicardial NE infusion caused the QRS complex to invert within a single beat (n = 35 animals, 101 observations), and the earliest epicardial activation consistently shifted to the randomly located infusion site (n = 14). This preceded right atrial activation, whereas the total ventricular epicardial activation time increased from 20 +/- 4 to 50 +/- 9 ms (P < 0.01). These events were accompanied by a ventricular tachycardia and a drop in left ventricular pressure, which were fully reversed after the infusion was stopped. Epicardial pacing at the infusion site mimicked all electrical and hemodynamic changes induced by NE. The arrhythmia was prevented by propranolol and abolished by cardiac sympathetic or vagal nerve stimulation. Focal automaticity was computationally reconstructed using a two-dimensional sheet of 256 x 256 resistively coupled ventricular cells, where calcium handling was abnormally high in the central region. We conclude that adrenergic stimulation to a small region of the ventricle elicits triggered automaticity and that computational reconstruction implicates calcium overload. Interventions that reduce spatial inhomogeneities of intracellular calcium may prevent this type of arrhythmia.

Vagal control of heart rate is modulated by extracellular potassium.

Heart rate (HR) recovery from heavy exercise is associated with a shift in cardiac sympatho-vagal balance and a transient hypokalaemia. Since changes in extracellular potassium ([K+]0) affect membrane currents in the sino-atrial node, in particular the acetylcholine-activated potassium current (I(K,ACh)), the hyperpolarization-activated current (I(f)) and the L-type calcium current (I(Ca,L)), we investigated whether mimicking [K+]0 concentrations seen during and immediately after exercise could directly modulate the HR response to vagal nerve stimulation (VNS) in the isolated guinea-pig atria preparation pre-stimulated with noradrenaline (NA, 1 microM). Lowering [K+]0 from 4 to 3 mM significantly enhanced the HR response to VNS (5 Hz, 5 V, 30 s, deltaHR 84.5 +/- 14.1 bpm and 119.3 +/- 18.2 bpm, respectively). Increasing [K+]0 to 8 or 10 mM significantly decreased the drop in HR with VNS in comparison to the response to 3 mM K+ Tyrode (deltaHR 56.4 +/- 9.1 bpm and 52.1 +/- 8.7 bpm, respectively). These results could be simulated using the OXSOFT heart sino-atrial node computer model by activating I(K,ACh) during changes in [K+]0. However, changing [K+]0 in the model had no significant effect on the decrease in beating frequency brought about by decreasing I(f) or I(Ca,L). We conclude that the magnitude of the decrease in HR with VNS is enhanced in low [K +]0 and reduced in high [K+]0. The increased efficacy of cardiac vagal activation in low [K+]0 might therefore facilitate the drop in HR after heavy exercise where there is a transient hypokalaemia. Modelling suggests this result may be explained by the effects of changes in [K+]0 on the current-voltage relationship for I(K,ACh).

NO-cGMP pathway accentuates the decrease in heart rate caused by cardiac vagal nerve stimulation.

The role of the cardiac muscarinic-receptor-coupled nitric oxide (NO) pathway in the cholinergic control of heart rate (HR) is controversial. We investigated whether adding excessive NO or its intracellular messenger cGMP could significantly modulate the HR response to vagal nerve stimulation (VNS) in the anesthetized rabbit and isolated guinea pig atria. The NO donor molsidomine (0.2 mg/kg iv) significantly enhanced the decrease in HR seen with right VNS (5 Hz, 5 V, 30 s) in vivo. A qualitatively similar effect was seen with the NO donor sodium nitroprusside (SNP; 10 and 100 microM) during VNS in vitro. This effect was still present when the baseline shift in HR caused by SNP was eliminated by using the specific hyperpolarization-activated current antagonist 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium chloride (ZD-7288, 1 microM). The accentuated decrease in HR with SNP during VNS was mimicked by the stable analog of cyclic GMP, 8-bromoguanosine 3',5'-cyclic monophosphate (0.5 mM). This, however, was not seen with bath application of the stable analog of acetylcholine, carbamylcholine chloride (100 nM). We conclude that excessive NO enhances the magnitude of the decrease in HR caused by VNS. This effect appears to involve a presynaptic action via a cGMP-dependent pathway because it was not mimicked by bath-applied carbamylcholine chloride.

Effect of nitric oxide synthase inhibition on the sympatho-vagal contol of heart rate.

The role of nitric oxide (NO) in the sympatho-vagal control of heart rate was investigated in the cardiac sympathectomized and vagotomized anaesthetised rabbit and in the isolated guinea-pig atria with intact vagus nerve. Specific inhibition of neuronal nitric oxide synthase (nNOS) with 1-(2-trimethylphenyl) imidazole (TRIM, 50 mg kg(-1) i.v. in vivo) significantly enhanced the magnitude of the change in heart rate (HR) with sympathetic nerve stimulation (SNS, 31.6+/-4.5 bpm control vs. 49.7+/-6.0 bpm in TRIM, P < 0.05, 10 Hz). This effect was reversed by L-arginine (deltaHR 37.2+/-4.1 bpm, 50 mg kg(-1) i.v.). An enhanced HR response to SNS was also seen with the non-isoform specific inhibitor, N-omega-nitro-L-arginine (L-NA, 50 mg kg(-1) i.v.). Infusing isoprenaline (0.2 microg kg(-1) min(-1)) did not mimic the change in HR response to SNS with TRIM. There was, however, no significant effect of inhibition of NOS with TRIM L-NA or NG-monomethyl-L-arginine (L-NMMA, 20 mg kg(-1) i.v.) on the magnitude of the change in HR with vagal nerve stimulation (5 Hz) in vivo. There was also no significant effect of NOS inhibition on the change in HR with vagal nerve stimulation in vivo in the presence of pre-adrenergic stimulation or in the presence of propranolol (0.5 mg kg(-1) i.v., 2, 5 and 10 Hz stimulation). This result was confirmed in the isolated guinea-pig atria with the specific nNOS inhibitor, 7-nitroindazole (7-NiNa, 100 microM) at 1, 2, 3 or 5 Hz stimulation frequency. Our data suggest that endogenous NO plays an inhibitory role in cardiac sympathetic neurotransmission, but there was no convincing evidence from our results for a major role for endogenous NO in vagal control of heart rate, with or without prior adrenergic stimulation.

Inhibition of nitric oxide synthase slows heart rate recovery from cholinergic activation.

The role of nitric oxide (NO) in the cholinergic regulation of heart rate (HR) recovery from an aspect of simulated exercise was investigated in atria isolated from guinea pig to test the hypothesis that NO may be involved in the cholinergic antagonism of the positive chronotropic response to adrenergic stimulation. Inhibition of NO synthesis with NG-monomethyl-L-arginine (L-NMMA, 100 micro M) significantly slowed the time course of the reduction in HR without affecting the magnitude of the response elicited by bath-applied ACh (100 nM) or vagal nerve stimulation (2 Hz). The half-times (t1/2) of responses were 3.99 +/- 0.41 s in control vs. 7. 49 +/- 0.68 s in L-NMMA (P < 0.05). This was dependent on prior adrenergic stimulation (norepinephrine, 1 micro M). The effect of L-NMMA was reversed by L-arginine (1 mM; t1/2 4.62 +/- 0.39 s). The calcium-channel antagonist nifedipine (0.2 micro M) also slowed the kinetics of the reduction in HR caused by vagal nerve stimulation. However, the t1/2 for the reduction in HR with antagonists (2 mM Cs+ and 1 micro M ZD-7288) of the hyperpolarization-activated current were significantly faster compared with control. There was no additional effect of L-NMMA or L-NMMA+L-arginine on vagal stimulation in groups treated with nifedipine, Cs+, or ZD-7288. We conclude that NO contributes to the cholinergic antagonism of the positive cardiac chronotropic effects of adrenergic stimulation by accelerating the HR response to vagal stimulation. This may involve an interplay between two pacemaking currents (L-type calcium channel current and hyperpolarization-activated current). Whether NO modulates the vagal control of HR recovery from actual exercise remains to be determined.

Interactive effects of K+, acid, norepinephrine, and ischemia on the heart: implications for exercise.

We tested the hypothesis that cardiac ischemia uncouples the beneficial interaction among hyperkalemia, acidosis, and raised plasma catecholamines when these chemicals are changed to mimic their exercise levels. Potassium chloride, lactic acid, and norepinephrine (NE) were infused intravenously for 2 min into anesthetized, artificially ventilated, thoracotomized rabbits during either occlusion of the left circumflex artery (3 min; n = 10) or after a period of prolonged ischemia (20 min; n = 7) that led to a small infarction. NE (1 microg x kg(-1) x min(-1) iv) offset the negative cardiac effects of hyperkalemia (up to 8.7 +/- 0.7 mM) and acidosis (arterial pH 7.09 +/- 0.03) in normal hearts. Cardiac performance was not significantly depressed by either acute or chronic ischemia before any infusions. However, the protective effect of NE during acute ischemia or after prolonged ischemia with hyperkalemia and acidosis was substantially reduced. These results show that cardiac ischemia attenuates the protective action of NE and increases the depressive effects of hyperkalemia and acidosis. Whether myocardial ischemia amplifies the cardiotoxic effects of hyperkalemia and acidosis during vigorous exercise by attenuating the beneficial effect of catecholamines remains to be determined.

Effects of high potassium and the bradycardic agents ZD7288 and cesium on heart rate of rabbits and guinea pigs.

The effects of 2 mM cesium (Cs+) and a novel selective bradycardic agent ZD7288 (0.64 microM) on sinoatrial Node (SAN) pacing rate were investigated in an isolated guinea pig SAN/atrial preparation, rabbit SAN preparation, and isolated working rabbit heart preparation. The effect of Cs+ and ZD7288 on the response of the preparations to increased extracellular potassium concentration ([K+]o) was also studied. Cs+ reduced beating frequency by 24% in isolated rabbit SAN (n = 16, p < 0.01) and by 21% in isolated working rabbit heart (n = 9, p < 0.01). ZD7288 decreased beating rate by 53% in guinea pig SAN (n = 7, p < 0.01) and by 38% in isolated working rabbit heart (n = 6, p < 0.01). In all three preparations, increased [K+]o significantly decreased the rate (p < 0.01) in normal Tyrode's solution but had no effect in the presence of Cs+ and caused tachycardia (p < 0.01) in the presence of ZD7288. We conclude that Cs+ and ZD7288 decrease pacing rate in rabbits and guinea pigs, possibly through modulation of the hyperpolarization-activated current (I(f)). ZD7288 is a more effective bradycardic agent than Cs+.