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Key Points: Vagal reflexes slow heart rate and can change where the heartbeat originates within the sinoatrial node (SAN). The mechanisms responsible for this process - termed leading pacemaker (LP) shift - have not been investigated fully. We used optical mapping to measure the effects of baroreflex, chemoreflex and carbachol on pacemaker entrainment and electrical conduction across the SAN. All methods of stimulation triggered shifts in LP site from the central SAN to one or two caudal pacemaker regions. These shifts were associated with reduced current generation capacity centrally and increased electrical load caudally. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. However, our findings indicate the LP region is defined by both pacemaker rate and capacity to drive activation. Shifts in LP site provide an important homeostatic mechanism for rapid switches in heart rate., Abstract: Reflex vagal activity causes abrupt heart rate slowing with concomitant caudal shifts of the leading pacemaker (LP) site within the sinoatrial node (SAN). However, neither the mechanisms responsible nor their dynamics have been investigated fully. Therefore, the objective of this study was to elucidate the mechanisms driving cholinergic LP shift. Optical maps of right atrial activation were acquired in a rat working heart-brainstem preparation during baroreflex and chemoreflex stimulation or with carbachol. All methods of stimulation triggered shifts in LP site from the central SAN to caudal pacemaker regions, which were positive for HCN4 and received uniform cholinergic innervation. During baroreflex onset, the capacity of the central region to drive activation declined with a decrease in amplitude and gradient of optical action potentials (OAPs) in the surrounding myocardium. Accompanying this decline, there was altered entrainment in the caudal SAN as shown by decreased conduction velocity, OAP amplitude, gradient and activation time. Atropine abolished these responses. Chemoreflex stimulation produced similar effects but central capacity to drive activation was preserved before the LP shift. In contrast, carbachol produced a prolonged period of reduced capacity to drive and altered entrainment. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. Our findings indicate that cholinergic LP shifts are also determined by altered electrical source-to-sink balance in the SAN. We conclude that the LP region is defined by both rate and capacity to drive atrial activation., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αβγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f ) and ryanodine receptor-derived diastolic local subsarcolemmal Ca 2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.

In the present study, channelrhodopsin 2 (ChR2) was specifically introduced into murine cells expressing the Phenylethanolamine n-methyltransferase (Pnmt) gene, which encodes for the enzyme responsible for conversion of noradrenaline to adrenaline. The new murine model enabled the identification of a distinctive class of Pnmt-expressing neuroendocrine cells and their descendants (i.e. Pnmt + cell derived cells) within the heart. Here, we show that Pnmt + cells predominantly localized to the left side of the adult heart. Remarkably, many of the Pnmt + cells in the left atrium and ventricle appeared to be working cardiomyocytes based on their morphological appearance and functional properties. These Pnmt + cell derived cardiomyocytes (PdCMs) are similar to conventional myocytes in morphological, electrical and contractile properties. By stimulating PdCMs selectively with blue light, we were able to control cardiac rhythm in the whole heart, isolated tissue preparations and single cardiomyocytes. Our new murine model effectively demonstrates functional dissection of cardiomyocyte subpopulations using optogenetics, and opens new frontiers of exploration into their physiological roles in normal heart function as well as their potential application for selective cardiac repair and regeneration strategies.

Background: Bradycardic agents are of interest for the treatment of ischemic heart disease and heart failure, as heart rate is an important determinant of myocardial oxygen consumption., Objectives: The purpose of this study was to investigate the propensity of hydroxychloroquine (HCQ) to cause bradycardia., Methods: We assessed the effects of HCQ on (1) cardiac beating rate in vitro (mice); (2) the "funny" current (If) in isolated guinea pig sinoatrial node (SAN) myocytes (1, 3, 10 µM); (3) heart rate and blood pressure in vivo by acute bolus injection (rat, dose range 1-30 mg/kg), (4) blood pressure and ventricular function during feeding (mouse, 100 mg/kg/d for 2 wk, tail cuff plethysmography, anesthetized echocardiography)., Results: In mouse atria, spontaneous beating rate was significantly (P < .05) reduced (by 9% ± 3% and 15% ± 2% at 3 and 10 µM HCQ, n = 7). In guinea pig isolated SAN cells, HCQ conferred a significant reduction in spontaneous action potential firing rate (17% ± 6%, 1 μM dose) and a dose-dependent reduction in If (13% ± 3% at 1 µM; 19% ± 2% at 3 µM). Effects were also observed on L-type calcium ion current (ICaL) (12% ± 4% reduction) and rapid delayed rectifier potassium current (IKr) (35% ± 4%) at 3 µM. Intravenous HCQ decreased heart rate in anesthetized rats (14.3% ± 1.1% at 15mg/kg; n = 6) without significantly reducing mean arterial blood pressure. In vivo feeding studies in mice showed no significant change in systolic blood pressure nor left ventricular function., Conclusions: We have shown that HCQ acts as a bradycardic agent in SAN cells, in atrial preparations, and in vivo. HCQ slows the rate of spontaneous action potential firing in the SAN through multichannel inhibition, including that of If., (Copyright © 2015 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Aims: Clinical markers of cardiac autonomic function, such as heart rate and response to exercise, are important predictors of cardiovascular risk. Tetrahydrobiopterin (BH4) is a required cofactor for enzymes with roles in cardiac autonomic function, including tyrosine hydroxylase and nitric oxide synthase. Synthesis of BH4 is regulated by GTP cyclohydrolase I (GTPCH), encoded by GCH1. Recent clinical studies report associations between GCH1 variants and increased heart rate, but the mechanistic importance of GCH1 and BH4 in autonomic function remains unclear. We investigate the effect of BH4 deficiency on the autonomic regulation of heart rate in the hph-1 mouse model of BH4 deficiency., Methods and Results: In the hph-1 mouse, reduced cardiac GCH1 expression, GTPCH enzymatic activity, and BH4 were associated with increased resting heart rate; blood pressure was not different. Exercise training decreased resting heart rate, but hph-1 mice retained a relative tachycardia. Vagal nerve stimulation in vitro induced bradycardia equally in hph-1 and wild-type mice both before and after exercise training. Direct atrial responses to carbamylcholine were equal. In contrast, propranolol treatment normalized the resting tachycardia in vivo. Stellate ganglion stimulation and isoproterenol but not forskolin application in vitro induced a greater tachycardic response in hph-1 mice. β1-adrenoceptor protein was increased as was the cAMP response to isoproterenol stimulation., Conclusion: Reduced GCH1 expression and BH4 deficiency cause tachycardia through enhanced β-adrenergic sensitivity, with no effect on vagal function. GCH1 expression and BH4 are novel determinants of cardiac autonomic regulation that may have important roles in cardiovascular pathophysiology.

Regular physical exercise reduces the risk of cardiovascular disease and improves outcome in patients with cardiovascular diseases. The dynamic changes in blood pressure and heart rate with acute exercise are independently predictive of prognosis. Quantification of the haemodynamic response to exercise training in genetically modified mouse models may provide insight into the molecular mechanisms underlying the beneficial effects of exercise. We describe, for the first time, the use of radiotelemetry to provide continuous blood pressure monitoring in C57BL/6J mice during a programme of voluntary wheel exercise with continuous simultaneous recording and analysis of wheel rotations and beat-by-beat haemodynamic parameters. We define distinct haemodynamic profiles at rest, during normal cage activity and during episodes of voluntary wheel running. We show that whilst cage activity is associated with significant rises both in blood pressure and in heart rate, voluntary wheel running leads to a further substantial rise in heart rate with only a small increment in blood pressure. With 5 weeks of chronic exercise training, resting heart rate progressively falls, but heart rate during episodes of wheel running initially increases. In contrast, there are minimal changes in blood pressure in response to chronic exercise training. Finally, we have quantified the acute changes in heart rate at the onset of and recovery from individual episodes of wheel running, revealing that changes in heart rate are extremely rapid and that the peak rate of change of heart rate increases with chronic exercise training. The results of this study have important implications for the use of genetically modified mouse models to investigate the beneficial haemodynamic effects of chronic exercise on blood pressure and cardiovascular diseases.

Background: The midbrain periaqueductal grey (PAG) area is important for both pain modulation and cardiovascular control via the autonomic nervous system (ANS). While changes in blood pressure dependent upon dorsal or ventral electrode positioning have been described with PAG deep brain stimulation (DBS), little is known mechanistically about the relationships between pain and cardiovascular regulation in humans. Heart rate variability (HRV) is an established measure of cardiovascular regulation, and an index of autonomic function., Methods and Results: 16 patients undergoing DBS of the rostral PAG for chronic neuropathic pain were investigated post-operatively to determine whether PAG stimulation would alter HRV, and the subjects' perception of pain. Mean heart rate together with HRV, time and frequency domain measures, low frequency (LF) and high frequency (HF) power components of heart rate and the ratio of LF to HF were calculated before and during DBS. Ventral but not dorsal PAG DBS significantly decreased the ratio of LF to HF power (p<0.05, n=8) with HF power significantly increased. Changes in LF/HF ratio correlated significantly with subjective reporting of analgesic efficacy using a visual analogue score (VAS; gamma(2)=0.36, p=0.01, n=16). Diffusion tensor imaging and probabilistic tractography of 17 normal controls' seeding voxels from the mean ventral and dorsal PAG stimulation sites of the 16 patient cohort revealed significant differences between rostral tract projections and separate, adjacent projections to ipsilateral dorsolateral medulla., Conclusions: Ventral PAG DBS may increase parasympathetic activity to reduce pain via anatomical connections distinct from dorsal PAG DBS, which may act by sympathetic mechanisms., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Parasympathetic activity during acute coronary artery occlusion (CAO) can protect against ischaemia-induced malignant arrhythmias; nonetheless, the mechanism mediating this protection remains unclear. During CAO, myocardial electrotonic uncoupling is associated with autonomically mediated immediate (i.e. type 1A) arrhythmias and can modulate pro-arrhythmic dispersion of repolarization. Therefore, the effects of acutely enhanced or decreased cardiac parasympathetic activity on early electrotonic coupling during CAO, as measured by myocardial electrical impedance (MEI), were investigated. Anaesthetized dogs were instrumented for MEI measurements, and left circumflex coronary arterial occlusions were performed in intact (CTRL) and vagotomized (VAG) animals. The CAO was followed by either vagotomy (CTRL) or vagal nerve stimulation (VNS, 10 Hz, 10 V) in the VAG dogs. Vagal nerve stimulation was studied in two additional sets of animals. In one set heart rate (HR) was maintained by pacing (220 beats min(-1)), while in the other set bilateral stellectomy preceded CAO. The MEI increased after CAO in all animals. A larger MEI increase was observed in vagotomized animals (+85 +/- 9 Omega, from 611 +/- 24 Omega, n = 16) when compared with intact control dogs (+43 +/- 5 Omega, from 620 +/- 20 Omega, n = 7). Acute vagotomy during ischaemia abruptly increased HR (from 155 +/- 11 to 193 +/- 15 beats min(-1)) and MEI (+12 +/- 1.1 Omega, from 663 +/- 18 Omega). In contrast, VNS during ischaemia (n = 11) abruptly reduced HR (from 206 +/- 6 to 73 +/- 9 beats min(-1)) and MEI (-16 +/- 2 Omega, from 700 +/- 44 Omega). These effects of VNS were eliminated by pacing but not by bilateral stellectomy. Vagal nerve stimulation during CAO also attenuated ECG-derived indices of ischaemia (e.g. ST segment, 0.22 +/- 0.03 versus 0.15 +/- 0.03 mV) and of rate-corrected repolarization dispersion [terminal portion of T wave (TPEc), 84.5 +/- 4.2 versus 65.8 +/- 5.9 ms; QTc, 340 +/- 8 versus 254 +/- 16 ms]. Vagal nerve stimulation during myocardial ischaemia exerts negative chronotropic effects, limiting early ischaemic electrotonic uncoupling and dispersion of repolarization, possibly via a decreased myocardial metabolic demand.

Hypertension is associated with abnormal neurohumoral activation. We tested the hypothesis that beta-adrenergic hyperresponsiveness in the sinoatrial node (SAN) of the spontaneously hypertensive rat occurs at the level of the L-type calcium current because of altered cyclic nucleotide-dependent signaling. Furthermore, we hypothesized that NO, a modulator of cGMP and cAMP, would normalize the beta-adrenergic phenotype in the hypertensive rat. Chronotropic responsiveness to norepinephrine (NE), together with production of cAMP and cGMP, was assessed in isolated atrial preparations from age-matched hypertensive and normotensive rats. Right atrial/SAN pacemaking tissue was injected with adenovirus encoding enhanced green fluorescent protein (control vector) or neuronal NO synthase (nNOS). In addition, L-type calcium current was measured in cells isolated from the SAN of transfected animals. Basal levels of cGMP were lower in hypertensive rat atria. These atria were hyperresponsive to NE at all of the concentrations tested, with elevated production of cAMP. This was accompanied by increased basal and norepinephrine-stimulated L-type calcium current. Using enhanced green fluorescent protein, we observed transgene expression within both tissue sections and isolated pacemaking cells. Adenoviral nNOS increased right atrial nNOS protein expression and cGMP content. NE-stimulated cAMP concentration and L-type calcium current were also attenuated by adenoviral nNOS, along with the chronotropic responsiveness to NE in hypertensive rat atria. Decreased calcium current after cardiac nNOS gene transfer contributes to the normalization of beta-adrenergic hyperresponsiveness in the SAN from hypertensive rats by modulating cyclic nucleotide signaling.

We tested the hypothesis that a single allele deletion of neuronal nitric oxide synthase (nNOS) would impair the neural control of heart rate following physical training, and that this phenotype could be restored following targeted gene transfer of nNOS. Voluntary wheel-running (+EX) in heterozygous nNOS knockout mice (nNOS(+/-), +EX; n= 52; peak performance 9.1 +/- 1.8 km day(-1)) was undertaken and compared to wild-type mice (n= 38; 9.5 +/- 0.8 km day(-1)). In anaesthetized wild-type mice, exercise increased phenylephrine-induced bradycardia by 67% (measured as heart rate change, in beats per minute, divided by the change in arterial blood pressure, in mmHg) or pulse interval response to phenylephrine by 52% (measured as interbeat interval change, in milliseconds, divided by the change in blood pressure). Heart rate changes or interbeat interval changes in response to right vagal nerve stimulation were also enhanced by exercise in wild-type atria (P < 0.05), whereas both in vivo and in vitro responses to exercise were absent in nNOS(+/-) mice. nNOS inhibition attenuated heart rate responses to vagal nerve stimulation in all atria (P < 0.05) and normalized the responses in wild-type, +EX with respect to wild-type with no exercise (-EX) atria. Atrial nNOS mRNA and protein were increased in wild-type, +EX compared to wild-type, -EX (P < 0.05), although exercise failed to have any effect in nNOS(+/-) atria. In vivo nNOS gene transfer using adenoviruses targeted to atrial ganglia enhanced choline acetyltransferase-nNOS co-localization (P < 0.05) and increased phenylephrine-induced bradycardia in vivo and heart rate responses to vagal nerve stimulation in vitro compared to gene transfer of enhanced green fluorescent protein (eGFP, P < 0.01). This difference was abolished by nNOS inhibition (P < 0.05). In conclusion, genomic regulation of NO bioavailability from nNOS in cardiac autonomic ganglia in response to training is dependent on both alleles of the gene. Although basal expression of nNOS is normal, polymorphisms of nNOS may interfere with neural regulation of heart rate following training. Targeted gene transfer of nNOS can restore this impairment.

Despite its highly diffusible nature, the gaseous signalling molecule nitric oxide (NO) can exert specific effects within the CNS and PNS. To date, the specificity of the actions of NO remains an unsolved puzzle. There are several plausible mechanisms that might account for this specificity in the context of autonomic regulation of heart rate. NO acts at distinct levels within the autonomic nervous system to control cardiac rate, with opposing effects at different sites. We discuss factors that might contribute to this diversity of action, and conclude that the isoform of enzyme involved in producing NO, the spatial proximity of the NO source to the target, and differences in the intracellular coupling within the target cell are all crucial for encoding the functional action of NO.

Parasympathetic control of heart rate involves the exocytotic release of acetylcholine and muscarinic receptor regulation of pacemaking currents. Endogenous nitric oxide can potentially regulate all of these processes; however, recent work suggests that the main functional role of nitric oxide lies in the modulation of acetylcholine release.

Electrical stimulation of the hypothalamus, basal ganglia or pedunculopontine nucleus in decorticate animals results in locomotion and a cardiorespiratory response resembling that seen during exercise. This has led to the hypothesis that parallel activation of cardiorespiratory and locomotor systems from the midbrain could form part of the 'central command' mechanism of exercise. However, the degree to which subcortical structures play a role in cardiovascular activation in awake humans has not been established. We studied the effects on heart rate (HR) and mean arterial blood pressure (MAP) of electrically stimulating the thalamus and basal ganglia in awake humans undergoing neurosurgery for movement disorders (n = 13 Parkinson's disease, n = 1 myoclonic dystonia, n = 1 spasmodic torticollis). HR and MAP increased during high frequency (> 90 Hz) electrical stimulation of the thalamus (HR 5 +/- 3 beats min(-1), P = 0.002, MAP 4 +/- 3 mmHg, P = 0.05, n = 9), subthalamic nucleus (HR 5 +/- 3 beats min(-1), P = 0.002, MAP 5 +/- 3 mmHg, P = 0.006, n = 8) or substantia nigra (HR 6 +/- 3 beats min(-1), P = 0.001, MAP 5 +/- 2 mmHg, P = 0.005, n = 8). This was accompanied by the facilitation of movement, but without the movement itself. Stimulation of the internal globus pallidus did not increase cardiovascular variables but did facilitate movement. Low frequency (< 20 Hz) stimulation of any site did not affect cardiovascular variables or movement. Electrical stimulation of the midbrain in awake humans can cause a modest increase in cardiovascular variables that is not dependent on movement feedback from exercising muscles. The relationship between this type of response and that occurring during actual exercise is unclear, but it indicates that subcortical command could be involved in 'parallel activation' of the locomotor and cardiovascular systems and thus contribute to the neurocircuitry of 'central command'.

Objectives: The role of the nitric oxide (NO)-cGMP pathway in the autonomic modulation of cardiac pacemaking is controversial and may involve an interplay between the L-type calcium current, I(CaL), and the hyperpolarisation activated current, I(f). We tested the hypothesis that following adrenergic stimulation, the NO-cGMP pathway stimulates phosphodiesterase 2 (PDE2) to reduce cAMP dependent stimulation of I(f) and heart rate (HR)., Methods: In the presence of norepinephrine (NE, 1 microM), the effects of the NO donor sodium nitroprusside (SNP) were evaluated in sinoatrial node (SAN)/atria preparations and isolated SAN cells from adult guinea pigs., Results: Contrary to our hypothesis, SNP (10 and 100 microM, n=5) or the membrane permeable cGMP analogue, 8Br-cGMP (0.5 mM, n=6) transiently increased HR by 5+/-1, 12+/-1 and 12+/-2 beats/min, respectively. The guanylyl cyclase inhibitor 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ, 10 microM, n=5) abolished the increase in HR to SNP (100 microM) as did the I(f) blockers caesium chloride (2 mM, n=7) and 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium chloride (ZD7288, 1 microM, n=7). Addition of SNP (10 microM) also transiently increased I(f) in SAN cells (n=5). After inhibition of PDE2 with erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA, 10 microM, n=5), the increase in HR to SNP in the presence of NE was significantly augmented and maintained. RT-PCR analysis confirmed the presence of PDE2 in addition to cGMP inhibited PDE3 mRNA in central SAN tissue., Conclusions: These results suggest that during adrenergic stimulation, activation of the NO-cGMP pathway does not decrease HR, but has a transient stimulatory effect that is I(f) dependent, and is limited in magnitude and duration by stimulation of PDE2.

The role of nitric oxide (NO) in the vagal control of heart rate (HR) is controversial. We investigated the cholinergic regulation of HR in isolated atrial preparations with an intact right vagus nerve from wild-type (nNOS+/+, n = 81) and neuronal NO synthase (nNOS) knockout (nNOS-/-, n = 43) mice. nNOS was immunofluorescently colocalized within choline-acetyltransferase-positive neurons in nNOS+/+ atria. The rate of decline in HR during vagal nerve stimulation (VNS, 3 and 5 Hz) was slower in nNOS-/- compared with nNOS+/+ atria in vitro (P < 0.01). There was no difference between the HR responses to carbamylcholine in nNOS+/+ and nNOS-/- atria. Selective nNOS inhibitors, vinyl-L-niohydrochloride or 1-2-trifluoromethylphenyl imidazole, or the guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one significantly (P < 0.05) attenuated the decrease in HR with VNS at 3 Hz in nNOS+/+ atria. NOS inhibition had no effect in nNOS-/- atria during VNS. In all atria, the NO donor sodium nitroprusside significantly enhanced the magnitude of the vagal-induced bradycardia, showing the downstream intracellular pathways activated by NO were intact. These results suggest that neuronal NO facilitates vagally induced bradycardia via a presynaptic modulation of neurotransmission.

Nitric oxide (NO) decreases norepinephrine (NE) release and the heart rate (HR) response to sympathetic nerve stimulation (SNS). We tested the hypothesis that the enhanced HR response to sympathetic activation following chronic intermittent hypoxia (IH) results from a peripheral modulation of pacemaking by NO. Isolated guinea pig double atrial/right stellate ganglion preparations were studied from animals that had been exposed to IH (n = 20) and control animals (n = 22). The HR response to SNS was significantly enhanced in the IH group compared with the controls. However, the increase in HR with cumulative doses (0.1--10 microM) of bath-applied NE was similar in both groups. Western blot analysis showed less neuronal NO synthase in the right atria from the IH group. In IH animals, the NO synthase inhibitor, N(omega)-nitro-L-arginine (L-NNA; 100 microM) did not alter the increased HR response to SNS, whereas in control animals L-NNA significantly increased the HR response to SNS; an effect that was reversed with excess L-arginine. In conclusion, the enhanced HR response to SNS after IH may be related to a decreased inhibitory action of NO on presynaptic NE release.

Intense exercise or myocardial ischaemia can significantly increase both the concentration of extracellular potassium ([K(+)](o)) and cardiac sympathetic nerve activity. Since changes in [K(+)](o) modulate membrane currents involved in sino-atrial node pacemaking, in particular the voltage-sensitive hyperpolarization-activated current (I(f)), we investigated whether raised [K(+)](o) (from 4 mM to 8 or 12 mM) could directly affect the heart rate response to cardiac sympathetic nerve stimulation (SNS). In the isolated guinea-pig atrial-right stellate ganglion preparation, raised [K(+)](o) significantly decreased the maximum diastolic potential, amplitude and maximum rate of rise of the upstroke of sino-atrial node pacemaker action potentials in 8 and 12 mM [K(+)](o) (P < 0.05). At 12 mM [K(+)](o) these effects were associated with significant decreases in baseline heart rate (4 mM [K(+)](o) = 187 +/- 5 beats min(-1) (bpm); 12 mM = 144 +/- 11 bpm; P < 0.05) and the heart rate response to SNS (1, 3 and 5 Hz; P < 0.05). A 10 % increase in the baseline heart rate with sympathetic activation (3 Hz) was associated with a significant enhancement of the slope of the pacemaker diastolic depolarization at 4 mM [K(+)](o) (increased by 16 +/- 6 %; n = 7; P < 0.05), but not with raised [K(+)](o). When the I(f) current was blocked with 2 mM caesium (n = 8), 12 mM [K(+)](o) had no effect on baseline heart rate and the heart rate response to 3 Hz SNS. The heart rate response to bath-applied noradrenaline (0.01-100 microM) was significantly attenuated by 12 mM [K(+)](o) (at 4 mM [K(+)](o,) EC(50) = -6.31 +/- 0.18; at 12 mM [K(+)](o,) EC(50) = -5.80 +/- 0.10; n = 6, ANOVA, P < 0.05). In conclusion, extreme physiological levels of [K(+)](o) attenuate the positive chronotropic response to cardiac sympathetic activation due to decreased activation of the I(f) current. This is consistent with raised [K(+)](o) protecting the myocardium from potentially adverse effects of excessive noradrenaline. Experimental Physiology (2001) 86.1, 19-25.

Sulphonylurea-sensitive K(+)channels (K(ATP)) have been implicated in the release of acetylcholine (ACh) from the vagus nerve in the heart. Our aim was to establish the functional significance of this and to test whether this modulation could interact with stimulation of the NO-cGMP pathway that facilitates the decrease in heart rate (HR) in response to vagal nerve stimulation (VNS). We studied the effect of activation (diazoxide, 100 microM) and inhibition (glibenclamide 30 microM or tolbutamide 5 microM) of K(ATP)channels, and activation of the NO-cGMP pathway with the NO donor, sodium nitroprusside (SNP, 20 microM) or the cGMP analogue, 8-Br-cGMP (0.5 m M) on the HR response to VNS in the isolated guinea pig (Cavia porcellus) double atrial/right vagus preparation (n=40). Tolbutamide increased the bradycardia in response to vagal stimulation at 3 and 5 Hz (P<0.05); effects that were reversed by diazoxide. Glibenclamide also significantly increased the HR response to VNS at 1 and 3 Hz (P<0.05). Diazoxide alone significantly attenuated the HR response to VNS at 5 Hz (P<0.05). Neither glibenclamide nor diazoxide affected the HR response to carbamylcholine (CCh, 50-200 n M). In the presence of a maximal dose of tolbutamide, SNP or 8-Br-cGMP further increased the HR response to VNS at 5 Hz (P<0.05). These results are consistent with the hypothesis that inhibition of sulphonylurea-sensitive channels can increase the HR response to VNS by a pre-synaptic mechanism, and that this modulation may be independent of activation of the NO-cGMP pathway., (Copyright 2000 Academic Press.)

Objectives: We tested the hypothesis that the attenuated heart rate (HR) response to sympathetic activation following swim training in the guinea pig (Cavia porcellus) results from a peripheral modulation of pacemaking by nitric oxide (NO)., Methods: Nitric oxide synthase (NOS) inhibition on the increase in heart rate with sympathetic nerve stimulation (SNS) was investigated in the isolated guinea pig double atrial/right stellate ganglion preparation from exercise trained (6-weeks swimming, n=20) and sedentary animals (n=20). Western blot analysis for neuronal nitric oxide synthase (nNOS) was performed on the stellate ganglion from both groups., Results: Relative to the control group, the exercise group demonstrated typical exercise adaptations of increased ventricular weight/body weight ratio, enhanced skeletal muscle citrate synthase activity and higher concentrations of [3H]ouabain binding sites in both skeletal and cardiac tissue (P<0.05). The increase in heart rate (bpm) with SNS significantly decreased in the exercise group (n=16) compared to the sedentary group (n=16) from 30+/-5 to 17+/-3 bpm at 1 Hz; 67+/-7 to 47+/-4 bpm at 3 Hz; 85+/-9 to 63+/-4 bpm at 5 Hz and 101+/-9 to 78+/-5 bpm at 7 Hz stimulation (P<0.05). The increase in heart rate with cumulative doses (0.1-10 microM) or a single dose (0.1 microM) of bath-applied norepinephrine expressed as the effective doses at which the HR response was 50% of the maximum response (EC50) were similar in both exercise (EC50 -6.08+/-0.16 M, n=8) and sedentary groups (EC50 -6.18+/-0.07 M, n=7). Trained animals had significantly more nNOS protein in left stellate ganglion compared to the sedentary group. In the exercise group, the non-isoform selective NOS inhibitor, N-omega nitro-L-arginine (L-NA, 100 microM) caused a small but significant increase in the heart rate response to SNS. However, the positive chronotropic response to sympathetic nerve stimulation remained significantly attenuated in the exercise group compared to the sedentary group during NOS inhibition (P<0.05)., Conclusions: Our results indicate that there is a significant peripheral pre-synaptic component reducing the HR response to sympathetic activation following training, although NO does not play a dominant role in this response.

Objectives: Activation of ATP sensitive K+ channels (K(ATP)) and the NO-cGMP pathway have both been implicated in reducing norepinephrine (NE) release from cardiac sympathetic nerves during stimulation. Our aim was to test whether these pathways could interact and modulate cardiac excitability during sympathetic nerve stimulation (SNS)., Methods: The effect of inhibitors and activators of K(ATP) channels and the NO-cGMP pathway on the heart rate (HR) response to cardiac SNS in the isolated guinea pig (Cavia porcellus) double atrial/right stellate ganglion preparation was studied (n=48)., Results: The K(ATP) channel activator, diazoxide (100 microM, n=6) or hypoxia (0% O2/5% CO2, n=6) significantly attenuated the HR response to 3 Hz SNS by -10+/-4% and -27+/-6% respectively; an effect that was reversed by the K(ATP) channel inhibitor, glibenclamide (30 microM). Glibenclamide (n=6) on its own enhanced the HR response to SNS by 20+/-8%. Bath applied NE (0.1-0.7 microM, n=6) did not affect the HR response to diazoxide, although an increased response to glibenclamide was observed at 0.3 and 0.5 microM NE. In the presence of 8-Br-cGMP (0.5 mM, n=7), diazoxide further decreased the HR response SNS (19+/-3%). The NO synthase inhibitor, N-omega-nitro-L-arginine (100 microM) significantly increased the HR response (13+/-3%) to SNS in the presence of diazoxide (100 microM, n=6). This effect was reversed with excess (1 mM) L-arginine. Conversely, the NO donor, sodium nitroprusside (SNP, 20-100 microM) significantly attenuated the HR response to SNS. The addition of glibenclamide (30 microM, n=10) could still enhance the HR response (42+/-15%) to SNS. Similar results were seen with the cyclic GMP analogue, 8-Br-cGMP (0.5 mM, n=12)., Conclusions: Our results indicate that NO and sulphonlyurea-sensitive channels act in a complementary fashion, but appear to be independent of each other in the regulation of HR during cardiac SNS activation.

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).

Unlabelled: We have recently shown that exogenous nitric oxide (NO) elicits a positive chronotropic response by stimulating the hyperpolarization activated current, I(f)., Objective: To examine whether L-arginine (L-Arg) can mimic the chronotropic effect of NO by enhancing its endogenous production., Methods: In spontaneously beating guinea pig atria we evaluated the heart rate (HR) response to increasing concentrations of L-Arg (1 mumol/l to 10 mmol/l), and compared it with that for D-Arg or L-lysine (L-Lys) (all in free base (FB) or hydrochloride (HCl) formulation)., Results: L-ArgFB > 100 mumol/l caused a reversible dose-dependent increase in HR (peak effect +64 +/- 7 bpm at 10 mmol/l, P < 0.05, n = 8). However, a similar HR response occurred with D-ArgFB (n = 7) or L-LysFB (n = 6). All FB formulations increased the perfusate pH (peak [pH]o = 8.61 +/- 0.03). Although alkalinization can stimulate NO release from the endothelium, this is unlikely to have contributed to HR changes in our preparation, since neither NG-methyl-L-arginine, (100-500 mumol/l, which per se reduced HR by 8 +/- 1%, P < 0.05, n = 9) nor NO scavenging (fresh 5% red blood cells, n = 9) caused a rightward shift of the concentration-response curve to L-ArgFB. Furthermore, as opposed to FB formulations, L-ArgHCl, D-ArgHCl or L-LysHCl > 1 mmol/l significantly decreased HR and [pH]o (n = 17). The chronotropic effects of L-ArFB or L-ArgHCl were reproduced by changing [pH]o with NaOH (n = 8) or HCl (n = 7), whereas the HR increase with L-ArgFB was prevented by clamping [pH]o at 7.42 +/- 0.07 (n = 10)., Conclusions: In vitro, L-Arg can markedly affect HR through a pH-mediated, NO-independent mechanism. Our data show that the opposing changes in [pH]o induced by different formulations of L-Arg can importantly confound the assessment of the biological effects of this amino acid.

Administration of nitric oxide (NO) donors in vivo is accompanied by a baroreflex-mediated increase in heart rate (HR). In vitro, however, NO donors can increase HR directly by stimulating a pathway that involves NO, cGMP, and the hyperpolarization-activated current (I(f)). The aim of this study was to assess the functional significance of this pathway in vivo by testing whether NO donors can increase HR in the anesthetized rabbit independent of the autonomic nervous system. New Zealand White rabbits were vagotomized, cardiac sympathectomized, and treated with propranolol (0.3 mg/kg iv). The NO donor molsidomine (0.2 mg/kg iv) caused a progressive increase (Delta) in HR (DeltaHR, 14 +/- 3 beats/min; P < 0.01). This effect was significantly reduced by the I(f) blocker ZD-7288 (0.2 mg/kg iv; DeltaHR, 2 +/- 3 beats/min; P = not significant). Similar results were seen with sodium nitroprusside. The positive chronotropic effect of sodium nitroprusside (50 microM) was confirmed in the isolated working rabbit heart preparation (DeltaHR, 17 +/- 3 beats/min; P < 0.01). In conclusion, NO donors exert a small, but significant, positive chronotropic effect in vivo that is independent of the autonomic nervous system. These results are also consistent with data in sinoatrial node cells that show that NO donors increase HR by stimulating I(f).

This study was designed to determine whether nitric oxide (NO) modulates the positive chronotropic and inotropic (in paced atria) responses to cardiac sympathetic nerve stimulation (SNS) in the isolated guinea-pig double atrial/right stellate ganglion preparation. The ganglion was stimulated at 1, 2, 3 and 5 Hz at constant voltage and the changes in heart rate or force of contraction were measured. The selective neuronal NO synthase (nNOS) inhibitors TRIM (1-(2-trifluoromethylphenyl) imidazole; 100 microM) and 7-NiNa (Na+ salt of 7-nitroindazole; 100 microM) significantly enhanced the positive chronotropic and inotropic responses to SNS. Similar results for heart rate were seen with the non-isoform-selective NOS inhibitor N(omega)nitro-L-arginine (L-NA; 100 microM). All effects were reversed with L-arginine (1 mM). The NO donor sodium nitroprusside (SNP; 100 microM) increased baseline heart rate and force of contraction, and attenuated the positive chronotropic and inotropic responses to SNS. SNP also decreased the positive chronotropic response to bath-applied noradrenaline (NA; 1 microM). In contrast, 7-NiNa did not alter the increase in heart rate with bath-applied NA (0.1 or 1 microM). The guanylyl cyclase inhibitor ODQ (10 microM) enhanced (mimicking nNOS inhibition) and the cyclic GMP (guanosine 3':5'-cyclic monophosphate) analogue 8-Br-cGMP (8-bromoguanosine 3':5'-cyclic monophosphate; 1 mM) attenuated (mimicking exogenous NO) the positive inotropic response to SNS. Taken together, these results are consistent with endogenous NO, synthesized from nNOS, inhibiting the positive chronotropic and inotropic responses evoked by cardiac SNS via a cyclic GMP-dependent pathway.

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.

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.

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.

We investigated the chronotropic effect of increasing concentrations of sodium nitroprusside (SNP, n = 8) or 3-morpholinosydnonimine (SIN-1, n = 6) in isolated guinea pig spontaneously beating sinoatrial node/atrial preparations. Low concentrations of NO donors (nanomolar to micromolar) gradually increased the beating rate, whereas high (millimolar) concentrations decreased it. The increase in rate was (1) enhanced by superoxide dismutase (50 to 100 U/mL, n = 6), (2) prevented by the guanylyl cyclase inhibitors 6-anilino-5,8-quinolinedione (5 mumol/L, n = 6) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (10 mumol/L, n = 6), and (3) mimicked by 8-bromo-cGMP (n = 6) with no additional positive chronotropic effect of SIN-1 (n = 5). The response to 10 mumol/L SNP (n = 28) or 50 mumol/L SIN-1 (n = 16) was unaffected by IcaL antagonism with nifedipine (0.2 mumol/L) but was abolished after blockade of the hyperpolarization-activated inward current (I(f)) by Cs+ (2 mmol/L) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride (1 mumol/L). The effect on I(f) was further evaluated in rabbit isolated patch-clamped sinoatrial node cells (n = 21), where we found that 5 mumol/L SNP or SIN-1 caused a reversible Cs(+)-sensitive increase in this current (+130% at -70 mV and +250% at -100 mV). In conclusion, NO donors can affect pacemaker activity in a concentration-dependent biphasic fashion. Our results indicate that the increase in beating rate is due to stimulation of I(f) via the NO-cGMP pathway. This may contribute to the sinus tachycardia in pathological conditions associated with an increase in myocardial production of NO.

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+.

Three male and four female subjects were acutely exposed to normoxic and hypoxic gas mixtures (FIO2 = 17.39%, 14.40%, 11.81%) in a single-blind randomized fashion during four treadmill runs to volitional exhaustion. Maximal scores for oxygen consumption (VO2), carbon dioxide production and heart rate decreased linearly (p less than 0.01) with increasing hypoxia. Conversely, maximal scores for ventilation, ventilatory equivalent (VE/VO2) and R increased linearly (p less than 0.01) with decreasing FIO2. During hypoxia, no significant differences in work time or respiratory compensation threshold were evident. However, female subjects had significantly higher (p less than 0.05) VE/VO2 scores and showed a relative decrease in VO2max that was significantly less (p less than 0.01) than male subjects. It was concluded that young highly active females, when compared to males of similar age and relative condition, have a stronger adaptive response to acute hypoxia during a maximal treadmill run.