Cardiac Vagal Nerve Activity Increases During Exercise to Enhance Coronary Blood Flow
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Abstract
BACKGROUND:
The phrase complete vagal withdrawal is often used when discussing autonomic control of the heart during exercise. However, more recent studies have challenged this assumption. We hypothesized that cardiac vagal activity increases during exercise and maintains cardiac function via transmitters other than acetylcholine.
METHODS:
Chronic direct recordings of cardiac vagal nerve activity, cardiac output, coronary artery blood flow, and heart rate were recorded in conscious adult sheep during whole-body treadmill exercise. Cardiac innervation of the left cardiac vagal branch was confirmed with lipophilic tracer dyes (DiO). Sheep were exercised with pharmacological blockers of acetylcholine (atropine, 250 mg), VIP (vasoactive intestinal peptide; [4Cl-D-Phe6,Leu17]VIP 25 µg), or saline control, randomized on different days. In a subset of sheep, the left cardiac vagal branch was denervated.
RESULTS:
Neural innervation from the cardiac vagal branch is seen at major cardiac ganglionic plexi, and within the fat pads associated with the coronary arteries. Directly recorded cardiac vagal nerve activity increased during exercise. Left cardiac vagal branch denervation attenuated the maximum changes in coronary artery blood flow (maximum exercise, control: 63.5±5.9 mL/min, n=8; cardiac vagal denervated: 32.7±5.6 mL/min, n=6, P=2.5×10−7), cardiac output, and heart rate during exercise. Atropine did not affect any cardiac parameters during exercise, but VIP antagonism significantly reduced coronary artery blood flow during exercise to a similar level to vagal denervation.
CONCLUSIONS:
Our study demonstrates that cardiac vagal nerve activity actually increases and is crucial for maintaining cardiac function during exercise. Furthermore, our findings show the dynamic modulation of coronary artery blood flow during exercise is mediated by VIP.
Novelty and Significance
What Is Known?
Cardiac function acutely adapts to meet increased metabolic demand during exercise
High vagal tone is correlated with increased exercise capacity
Inhibition of central vagal drive reduces exercise capacity in rodents
What New Information Does This Article Contribute?
Directly recorded cardiac vagal nerve activity increases during exercise
Removal of cardiac vagal innervation reduces cardiac output and coronary artery blood flow during exercise
In animals with an intact cardiac vagal nerve, inhibition of VIP (vasoactive intestinal peptide) during exercise reduces coronary artery blood flow to an equivalent level of animals that have undergone cardiac vagal denervation.
The increase in heart function during exercise was traditionally stated to be predominantly under sympathetic nervous system control, with little to no role for the parasympathetic nervous system during exercise. Recent work suggests this is not the case and that both nervous systems can be active and act complementarily to give an appropriate physiological response. Using a large animal exercise model, our study demonstrates the critical role of the cardiac parasympathetic nervous system during exercise for the first time. We show that cardiac vagal nerve activity increases during exercise and that removing this nerve reduces blood flow through the left main coronary artery during exercise. Vagally mediated coronary artery blood flow control was shown to be mediated by the vagal cotransmitter VIP, not the principal vagal neurotransmitter acetylcholine. Therefore, our findings not only reveal that cardiac vagal nerve activity increases during exercise but that it has an important role in maintaining coronary artery blood flow mediated by the cotransmitter neuropeptide VIP and not acetylcholine.
In This Issue, see p 539
Meet the First Author, see p 540
Exercise elicits a whole-body, integrative physiological response to ensure working muscle receives an adequate supply of oxygen. Cardiovascular fitness and exercise capacity are strongly associated with high resting vagal tone.1–3 However, the role of vagal nerve activity during exercise remains understudied and often controversial.3–7 The simplistic and reductive view that sympathetic and parasympathetic nerve activity act in a binary system to oppose each other has long been questioned.8 However, current reports state that dynamic modulation of the cardiovascular system during exercise is primarily due to actions of the sympathetic nervous system, neurohormonal, and metabolic pathways.4,9,10 Despite the long-standing belief that parasympathetic (vagal) activity in the heart decreases during exercise, recent data have challenged this assumption. Studies in rats,11 cats,12 and humans6 indicate that the simplistic view of vagal withdrawal during exercise needs to be reexamined.
The complexities in studying the vagal control of the heart in human and animal models may have contributed to varying conclusions. The vagus nerve is a mixed nerve bundle containing parasympathetic efferent and sensory afferent nerve fibers.13 Historically, no role for the parasympathetic nervous system during exercise has been concluded due to the assumption that cardiac vagal nerve fibers only innervate the sinoatrial node (SAN) and the absence of changes in cardiac function with cholinergic blockers. We now know that cardiac parasympathetic fibers innervate the whole heart and can modulate contractility,11 as well as, coronary artery blood flow.14 In addition, cardiac vagal efferent nerves are not only limited to its primary neurotransmitter, acetylcholine but also involve cotransmitters VIP (vasoactive intestinal peptide) and neuronal nitric oxide synthase. As such, studies utilizing a cholinergic blocker during exercise can discount the role of the cholinergic system but not the role of the cardiac vagus.
During exercise, cardiac changes are matched with vascular changes to ensure optimal perfusion to the areas of the body that need it most.9,15 This includes the coronary circulation, which acutely adapts to maintain myocardial blood flow during exercise.9,15,16 Although these coronary artery blood flow changes during exercise are a well-studied physiological response,9,16,17 the specific role of the cardiac vagus in modulating coronary blood flow during exercise is not known. It is essential to define the role of cardiac vagal nerve activity (CVNA) during exercise, as many disorders are linked to reduced exercise capacity and reduced vagal tone, including heart failure.18
This study established the first direct cardiac vagal branch nerve recordings during exercise in a large animal model. We tested the hypotheses that cardiac vagal activity is elevated during exercise and that selective denervation of this cardiac vagal branch would attenuate heart function during exercise. We also utilized pharmacological blockade of neurotransmitters released by the cardiac vagus to determine the mechanisms whereby vagal activity altered heart function. Our findings provide a new perspective on how the parasympathetic nervous system modulates heart function during exercise.
METHODS
Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Sheep
Adult (3–6 years old) female Romney sheep (n=45 total) were sourced from the Ngāpouri Liggins research farm, housed in individual crates, and acclimatized to laboratory conditions (18 oC, 50% relative humidity, 12-hour light-dark cycle) and human contact for 1 week before any experiments. Sheep were fed 2 to 2.5 kg/day (Country Harvest pellets) and had access to water ad libitum. All animal experiments and surgical procedures followed relevant guidelines and were approved by the Animal Ethics Committee of the University of Auckland (no. 2268).
Anesthesia for Surgery
General anesthesia was induced with propofol (5 mg/kg IV, AstraZeneca, AUS). Sheep were then intubated and artificially ventilated with a 2% isoflurane-air-O2 mixture to maintain anesthesia. An intercostal nerve block was performed by injecting bupivacaine (0.25%. Aspen, New Zealand) into the second, to sixth intercostal spaces (7.5 µg per injection, ≈0.625 µg/kg total).
Lipophilic Dyes
The left cardiac vagal branch anatomically diverges from the main vagal branch between the pulmonary artery and the azygous vein. A branch can be visualized here, separating from the main vagus, projecting sacral and dorsal behind the left atria. In a subset of animals (n=3), the left cardiac vagal branch was isolated from surrounding tissue. FAST DiO (ThermoFisher: D3898) was applied to the cardiac vagal branch and a 30G needle was used to gently penetrate the nerve fiber bundle and allow the dye to diffuse into the nerve bundle. Silicone (Kwik-Sil, World Precision Instruments, FL) was placed over the nerve to ensure dye crystals remained in contact with the nerve fibers. Sheep were recovered and housed for 21 days. Upon euthanasia, samples were collected from major ganglionic plexi of the heart and coronary artery, placed in 1% paraformaldehyde for 1 hour, and transferred through a sucrose gradient (10%, 20%, and 30%) over 48 hours. Samples were stored at −80 °C before processing.
Imaging of Ganglionic Plexi and Nerve Terminals
Ganglionic plexi were sliced on a cryostat to 12 µm. Samples were mounted onto microscope slides, washed once with PBS, before applying anti-fade VectaShield (Vector Laboratories, CA) and coverslipping. Tissue sections were imaged on an Olympus FV1000 confocal microscope, using a diode 473 nm laser (excitation, 488 nm; emission, 530 nm), 40×/1.00 NA oil immersion objective, using the software FluoView 4.2.
Surgery for Instrumentation of Chronic Flow and Pressure Probes
Following induction and maintenance of anesthesia, a single-tip pressure probe (Millar Inc, TX. Model No. 320-6590) was inserted into the left common carotid artery to get an index of blood pressure (BP). A cannula was also placed just below the location of the carotid body in the common carotid artery for testing chemoreflex responses. Another cannula was inserted into the left jugular vein for drug infusion. A dorsal to ventral incision was made on the left side of the chest, and the fourth rib was removed to access the heart. A Doppler flow probe (size, 28; Transonic, AU) was placed around the ascending aorta to measure cardiac output (CO). A Doppler flow probe (size, 6; Transonic, AU) was placed around the left main coronary artery to measure coronary artery blood flow. The chest was closed, and negative pleural pressure was reestablished. Flow probes were tunneled subcutaneously for connecting to chronic, continuous recording devices after recovery. Sheep were given antibiotic injections (20 mg/kg intramuscular; oxytetracycline, Phoenix, NZ) and analgesia (ketoprofen, 10%, 2 mg/kg intramuscular; Merial, Boehringer Ingelheim, NZ) at the start of surgery, and for the first 3 days post-operatively. Animals were allowed to recover for 7 days post-surgery before beginning the exercise acclimatization protocol. All parameters were recorded from conscious sheep on a desktop computer with a CED Micro-1401 interface and a data acquisition program (Spike2 v8, Cambridge Electronic Design, United Kingdom).
Cardiac Vagal Branch Stimulation
Left cardiac vagal branch stimulation was performed under anesthesia in a separate group of animals (n=5) to confirm the physiological innervation of this branch. To stimulate the nerve, a hook electrode was placed around the isolated left cardiac vagal branch, with and without cardiac pacing.
Conscious Left Cardiac Vagal Branch Recording
In a total of n=22 sheep, the left cardiac vagal branch was isolated from the main vagal branch and surrounding tissue with a fire-polished glass pipette. A custom-built coil electrode was designed to achieve whole cardiac vagal branch recordings in the conscious animal. Two coil wire (0.25 mm) electrodes were wrapped around the cardiac vagal branch and affixed in place with super glue and silicone. Electrodes were tunneled subcutaneously and exited percutaneously on the dorsum of the sheep. A ground electrode was placed subcutaneously at the left side of the chest. At least 4 days after recovery from this surgery, the cardiac vagal branch and ground electrodes were connected to the data acquisition via a preamplifier, and the signal was amplified (×10 000) and filtered (band pass, 0.1–1.5 kHz). To gain a real-time measure of respiration, a QDC-PRO breathing belt (Nox Medical, Reykjavik, Iceland) was placed on the sheep. Of a total of n=22 sheep that underwent implantation of cardiac vagal branch recording electrode, n=12 had reliable nerve activity, which was maintained 3 to 10 days post-surgery. N=6 of those sheep maintained nerve activity with low signal-to-noise during exercise. Bipolar recording electrodes on intact nerves are susceptible to recording artifacts from other nearby inputs, including diaphragmatic electromyogram (dEMG), ECG, and movement during exercise. All care was taken to only include animals with CVNA during exercise that did not include signals from other sources. One of our criteria for cardiac vagal activity was respiratory modulation. To ensure that the CVNA activity measured during exercise was not dEMG, we carefully examined respiratory effort on the treadmill to ensure cardiac vagal activity was not 1:1. In several sheep, we observed movement artifacts on the treadmill; this was apparent at the onset and stopping of movement, these animals were excluded from the analysis. When ECG was present in the signal at rest, these animals were usually susceptible to recording other artifacts during exercise, so no animals with ECG in the cardiac vagal signal were included in the study.
Signal Averaging
Using custom-written scripts for Spike2 (v8, Cambridge Electronic Design, United Kingdom), 5 minutes of baseline recording of rectified integrated CVNA and respiration recorded using a QDC-PRO (Nox Medical) were signal averaged over a time range of 15 seconds, with a time constant of 1 second.
Baroreflex Activation
To test the response of CVNA to baroreflex activation, conscious BP, heart rate (HR), and CVNA were recorded for a 10-minute baseline and in response to increasing doses of intravenous phenylephrine (25, 50, 100, 200, and 400 mg/minute) until a maximum systolic BP of 150 mm Hg was achieved.
Chemoreflex Activation
In the conscious condition, at rest, potassium cyanide (KCN; 30 µg/kg) was administrated intra-arterially, just below the carotid bifurcation, to selectively target the carotid body as previously described.19
Exercise Test
Seven days post-surgery, sheep were acclimatized to the treadmill exercise protocol for 3 days before the experimental exercise test. A previously optimized exercise of 18 minutes to a maximum intensity of 2.5 km/hour and 15% incline was used for all experiments (Figure S1). At the end of the exercise session, the treadmill was switched off, and the animals were allowed to rest on the treadmill for 15 minutes for postexercise baseline measurements. Exercise recovery measures were taken at 10, 20, 30, 60, and 120 seconds post-exercise. Real-time measurements of BP, CO, coronary artery blood flow, and CVNA were recorded throughout the exercise protocol.
Left Cardiac Vagal Branch Denervation
In a subset of animals (n=6), the left cardiac vagal branch was isolated from the main vagal branch and cut with surgical scissors. Animals were recovered as stated above, with no adverse effects observed with unilateral cardiac vagal branch denervation.
Pharmacological Tests During Exercise
Protocols using pharmacological blockade were randomized on different days to control runs (Figure 2A). Pharmacological blockers of parasympathetic neurotransmitters were given on the treadmill after 10 minutes of baseline and 10 minutes before starting exercise (Figure S1). Atropine (0.8 mg/kg per min atropine sulfate salt monohydrate, Sigma, catalog no. A0257), was administered intravenously as a 5-mL bolus followed by a 30 mL/hour constant infusion throughout the exercise protocol and recovery.19 VIP receptor antagonist [D-p-Cl-Phe,5 Leu16]-VIP (Tocris Bioscience, United Kingdom, catalog no. 3054) was administered intravenously as a bolus of 20 µg 10 minutes before exercise, and 5 µg bolus immediately before exercise (≈0.4 µg/kg for a 60 kg sheep; Please see the Major Resources Table in the Supplemental Material).
Statistical Analysis
Samples sizes were determined using power calculations (significance criterion of P<0.05 and power of 80%). Using our specific criteria for change in variables (see Supplemental Methods), the minimum sample size of n=6 gave us the ability to detect significant changes of ≈20% (CO), 15% (coronary blood flow), 40% (cardiac vagal activity), and 15% (HR). All animal data were acquired and analyzed in Spike2 (Cambridge Electronics). Data were acquired continuously throughout all exercise and experimental protocols. Exact P – values and number of animals in each group (n) are stated in results, and figure legends. P values are presented with scientific notation with 2 significant figures. Data were considered significant if P<0.05. All data are expressed as mean±SEM, except where indicated. Data were tested for normality using the Shapiro-Wilk test when applicable. Once normality was confirmed, all-time course data (CO, HR, coronary artery blood flow, coronary vascular resistance, and stroke volume) were analyzed using repeated measures 1-way ANOVA to assess the effect of exercise, and repeated measures a 2-way ANOVA to asses differences (interaction) between groups during exercise. The factors used in the 2-way ANOVA were time/exercise intensity, condition and the interaction term, that is, time/exercise intensity×condition. Where normality could not be confirmed (n<6) then appropriate paired, Wilcoxon matched-pairs signed rank test or nonpaired Friedman tests were used. Hemodynamic changes within animals before-after pharmacological agents were analyzed using a paired t test if normally distributed. For the recovery of HR data, the individual data were fitted with a first-order polynomial, and the constants were compared before and after the intervention. All statistical analysis was performed in GraphPad Prism version 9.3.1 (GraphPad, Boston, MA), and exercise recovery data were analyzed in SPSS (IBM, NY).
RESULTS:
Anatomic Innervation of the Left Cardiac Vagal Branch
Lipophilic nerve tracer dye (DiO) applied to the isolated left cardiac vagal branch was expressed at the major cardiac ganglia (Figure 1). The left cardiac vagal branch innervates major regulatory ganglionic plexi, including the right atrial ganglionic plexi, and ganglionic plexi that innervate the left ventricle including the posterolateral left atrial ganglionic plexi, and fat pads around the left coronary artery (obtuse marginal plexi; Figure 1A through 1D). Stimulation of this left cardiac vagal branch under anesthesia reduced HR, indicating functional innervation of the SAN. In addition, when the heart was paced to maintain HR, stimulation of this branch increased coronary artery blood flow (Figure S2), confirming a physiological role and anatomic innervation of the left cardiac vagal branch.
Figure 1. Schematic representation of the ventral view of the heart showing the location of the major cardiac ganglionic plexi and the cardiac vagal branch. A, C, and D, DiO painted onto the left cardiac vagal branch can be traced to multiple cardiac ganglia. A, Posterolateral left atrial ganglionic plexi (PLAGP), (C) right atrial ganglionic plexi (RAGP), and (D), left coronary artery fat pad. Of note, the RAGP and PLAGP are positioned on the dorsal side of the heart. B, Photo of the isolated left cardiac vagal branch. E, Example raw data trace of raw cardiac vagal nerve activity (CVNA; green), integrated iCVNA (green), and respiration (purple) measured using a breathing belt, showing respiratory coupled nerve activity. F, Signal averaging of 5-minute baseline recording of iCVNA and respiration. G, iCVNA during intravenous phenylephrine (PE) to a peak blood pressure of 150 mm Hg, individual animals paired, baseline (pink dot) to after PE (blue dot; n=5. *P=0.031, Wilcoxon signed-rank test), and (H) intraarterial potassium cyanide (KCN), individual animals paired, baseline (pink dot) to after KCN (green dot; n=5. *P=0.031. Wilcoxon signed-rank test).
Validation of Cardiac Vagal Branch Recordings
(1) Conscious CVNA at rest showed burst activity in synchronization with respiration as demonstrated with signal averaging using the peak of the respiration trace (peak inspiration) as a trigger (Figure 1E and 1F), nerve activity (green), respiration (purple); CVNA peaks posts inspiration (Figure 1F). (2) Increasing BP in conscious animals by performing a baroreflex challenge with phenylephrine increased CVNA (Figure 1G and 1I). (3) Stimulation of the chemoreflex with intraarterial potassium cyanide (KCN) administered to the carotid body increased CVNA (Figure 1H and 1I). Taken together, these data indicate that the recordings are vagal in origin.20,21
CVNA Increases During Exercise
Using an established custom sheep treadmill protocol (Figure S1), conscious real-time CVNA, HR, CO, coronary artery blood flow (CoBF), and BP were recorded during exercise and recovery (Figure 2B and 2C). Exercise increased CVNA (Figure 2D through 2G). This increase in CVNA occurred early, during the initiation of exercise. CVNA increases during the lowest exercise intensity and then plateaus as exercise intensity increases. The change in CVNA was calculated as a percentage change from baseline (Figure 2F; n=6; P=1.0×10−7; Friedman test—effect of exercise), and rectified integrated area under the curve of CVNA (µV; Figure 2G, n=6, P=2.2×10−7, Friedman test—effect of exercise). Hemodynamic changes in HR, CO, and CoBF increased in a graded manner as exercise intensity increased in speed and incline (Figure 2H and 2I, n=9; Figure 2J, n=8).
Figure 2. Effect of exercise on cardiovascular variables. A, Experimental timeline. B, Schematic representation of a sheep on the treadmill. C, Representative recording of all variables recorded during exercise. D and E, Representative raw traces of cardiac vagal nerve activity (CVNA), iCVNA, and heart rate (HR) at baseline and the onset of exercise (D), and during exercise and the start of recovery (E). F, Percentage change in iCVNA during exercise compared to baseline (mean±SEM, n=6, P=1.0×10−7, Friedman, effect of exercise). G, Absolute change in iCVNA during exercise compared to baseline (mean±SEM, n=6, P=2.2×10−7, Friedman, effect of exercise). H through J, Hemodynamic changes during baseline, exercise, and recovery, in control sheep (mean±SEM). H, Heart rate (n=9, *P=4.8×10−12, 1-way ANOVA, effect of exercise). I, Cardiac output (n=9, *P=1.3×10−7, 1-way ANOVA, effect of exercise). J, Coronary artery blood flow (n=8. *P=9.3×10−10, 1-way ANOVA, effect of exercise). BP indicates blood pressure; CoBF, coronary artery blood flow; CO, cardiac output; CVNA, cardiac vagal nerve activity; and HR, heart rate.
Cardiac Vagal Denervation Impairs Cardiac Function and Coronary Artery Blood Flow During Exercise
Left cardiac vagal branch denervation did not significantly affect baseline measurements of HR, CO, or CoBF compared to cardiac vagal-intact sheep (Figure 3A through 3C). Cardiac vagal denervation in a baroreflex challenge resulted in a higher HR when systolic BP was at 150 mm Hg compared to cardiac vagal-intact animals (Figure 3D).
Figure 3. Cardiac vagal denervation attenuates the response to exercise. Resting baseline heart rate (A), cardiac output (B), and coronary artery blood flow (C), in control (blue) and cardiac vagal denervated sheep (red). D, Change in heart rate against systolic blood pressure to a baroreflex challenge with phenylephrine (mean±SEM), n=5 both groups, control (blue) *P=3.0×10−14, cardiac vagal branch denervated (red) *P=2.02×10−3. Friedman, effect of exercise. E through J, Hemodynamic changes during baseline, exercise and recovery, control (blue), cardiac vagal branch denervated (red), mean±SEM. E, Heart rate (control, n=9, *P=4.8×10−12; cardiac vagal denervated, n=6, *P=1.4×10−5, 1-way ANOVA, effect of exercise, #P=0.032, 2-way ANOVA, interaction effect. F, Cardiac output (control, n=9, *P=1.3×10−7; cardiac vagal denervated, n=6, *P=3.4×10−8, 1-way ANOVA, effect of exercise, #P=0.022, 2-way ANOVA, interaction effect). G, Mean arterial blood pressure (control, n=9, *P=1.2×10−3; cardiac vagal denervated, n=6, *P=1.8×10−3, 1-way ANOVA, effect of exercise). H, Coronary artery blood flow (control, n=8, *P=9.3×10−10; cardiac vagal denervated, n=6, *P=1.6×10−3, 1-way ANOVA, effect of exercise, #P=2.5×10−7; 2-way ANOVA, interaction effect. I, Stroke volume (control, n=9, *P=1.4×10−4; cardiac vagal denervated, n=6, *P=2.4×10−3, 1-way ANOVA, effect of exercise, #P=0.038, 2-way ANOVA, interaction effect). J, First-order exponential decay of heart rate recovery during exercise (control, n=9, cardiac vagal denervated, n=6, #P=8.0×10−3, 2-way ANOVA, interaction). K, Tissue sections of the fat pad located near the left coronary artery, labeled with (DiO—green), and nuclear stain (DAPI—blue), showing direct innervation by the left cardiac vagal branch.
Left cardiac vagal branch denervation impaired cardiac function during exercise compared to cardiac vagal-intact sheep. Left cardiac vagal denervation reduced the increase in HR (Figure 3E, P=0.032, 2-way ANOVA interaction), CO (Figure 3F, P=0.022, 2-way ANOVA interaction), stroke volume (Figure 3I, P=0.038, 2-way ANOVA interaction), and CoBF (Figure 3H, P=2.5×10−7, 2-way ANOVA interaction) during exercise, and reduced the rate of HR recovery post-exercise (Figure 3J, P=8.0×10−3, 2-way ANOVA interaction). There was no statistically significant change in mean arterial pressure during exercise between left cardiac vagal denervated and cardiac vagal-intact sheep (Figure 3G). Furthermore, the left cardiac vagal branch directly innervates the fat pads around the left coronary artery, visualized when lipophilic nerve tracer DiO is painted on the left cardiac vagal branch (Figure 3K).
Inhibition of Acetylcholine Does Not Affect Cardiac Function During Exercise
While sheep were standing at rest on the treadmill, administration of atropine caused a significant increase in HR from baseline (Figure 4A, n=7, P=6.5×10−3, paired t test), with no significant change in CO and CoBF. We have previously shown that atropine alone does not alter CoBF in sheep where HR has been fixed with cardiac pacing.19 During administration of atropine during exercise, no statistically significant difference was observed in any hemodynamic parameters compared to controls measured during exercise (Figure 4D through 4I).
Figure 4. Role of acetylcholine in mediating the exercise response. A through C, Paired baseline hemodynamics taken on the treadmill, before (pre-blue) and 10 minutes after (postgreen) atropine (0.08 mg/kg per min) infusion, (A) heart rate, (B) cardiac output, (C) coronary artery blood flow. D through H, Hemodynamic changes with exercise during control (saline: blue), or atropine (green) infusion, mean±SEM. D, Heart rate (control, n=7, *P=7.9×10−7; atropine, n=7, *P=3.2×10−7, 1-way ANOVA, effect of exercise). E, Cardiac output (control, n=7, *P=3.9×10−5; atropine, n=7, *P=1.7×10−5, 1-way ANOVA, effect of exercise). F, Mean arterial blood pressure (control, n=7, *P=1.3×10−3; atropine, n=7, *P=1.1×10−2, 1-way ANOVA, effect of exercise). G, Coronary artery blood flow (control, n=7, *P=8.3×10−7; atropine, n=7. *P=6.5×10−3, 1-way ANOVA, effect of exercise; H), stroke volume (control, n=7, *P=8.3×10−4; atropine, n=7, *P=2.9×10−4, 1-way ANOVA, effect of exercise). I, First-order exponential decay of heart rate recovery during exercise (n=7 both groups).
Exercise-Mediated Increase in Coronary Artery Blood Flow Is Mediated by VIP
VIP antagonism with VIP-[4Cl-D-Phe6, Leu17] (25 µg) caused no significant change in HR, CO, or CoBF at rest in conscious sheep (paired results, Figure 5A through 5C). During exercise, VIP antagonism had no statistically significant effect on HR, CO, stroke volume, mean arterial pressure, or HR recovery (Figure 5D through 5F, 5H, 5I) but had a substantial and significant effect on attenuating the increased CoBF previously observed during exercise (Figure 5G, P=4.8×10−3, 2-way ANOVA interaction).
Figure 5. Role of vasoactive intestinal peptide (VIP) in mediating the exercise response. A through C, Paired baseline hemodynamics taken on the treadmill, before (pre-blue) and 10 minutes after (postorange) VIP receptor antagonist (VIPR Ant: [D-p-Cl-Phe,Leu]-VIP, 25 μg) infusion, (A) heart rate, (B) cardiac output, (C) coronary artery blood flow. D through H, Hemodynamic changes with exercise during control (saline: blue), or VIPR Ant (orange) infusion, mean±SEM. D, Heart rate (control, n=7, *P=2.6×10−11; VIPR Ant, n=7, *P=1.1×10−7, 1-way ANOVA, effect of exercise). E, Cardiac output (control, n=7, *P=1.2×10−6; VIPR Ant, n=7, *P=3.5×10−6, 1-way ANOVA, effect of exercise). F, Mean arterial blood pressure (control, n=7, *P=9.7×10−4; VIPR Ant, n=7, *P=9.9×10−4, 1-way ANOVA, effect of exercise). G, Coronary artery blood flow (control, n=6, *P=7.1×10−7; VIPR Ant, n=6, *P=1.2×10−6, 1-way ANOVA, effect of exercise, #P=4.8×10−3, 2-way ANOVA, interaction; H), stroke volume (control, n=7, *P=5.1×10−5. VIPR Ant, n=7, *P=4.7×10−3, 1-way ANOVA, effect of exercise; I), first-order exponential decay of heart rate recovery during exercise (n=7 both groups).
DISCUSSION
The 3 main findings of this study are:
Directly recorded CVNA increases during exercise.
CVNA modulates coronary artery blood flow during exercise.
Vagal cotransmitter VIP mediates the increase in coronary artery blood flow observed during exercise.
It is widely accepted that cardiac vagal nerves innervate the whole heart and can have roles beyond HR control.11,14,22 In exercise physiology, much of the study on vagal control of the heart has focused on the regulation of HR alone.19,23,24 Historically, it has been thought that since HR increases, vagal tone must be withdrawn.25,26 While this is a simplified assessment, the technical nature of studying cardiac vagal control in humans and conscious animal models, alongside the repeated conflation that cholinergic blockade equals vagal blockade,4,6,26,27 means that, to date, most measures of vagal tone have been indirect.
A scattering of previous studies have suggested an important role for cardiac vagal activity during exercise.3,6,11 Bilateral vagal denervation in dogs resulted in a decreased exercise capacity, but given that the denervation was not cardiac specific, the putative role of the cardiac vagus was not considered.28 Kadowaki showed in the de-cerebrate cat that simulated exercise increased cardiac vagal activity.12 However, the de-cerebrated nature of the preparation meant that this increase in activity was not considered important in the conscious preparation. The most recent and compelling study came from Machhada et al,11 where they showed that inhibiting vagal outflow from the dorsal vagal motor nucleus reduced exercise capacity and recruitment of vagal activity increased exercise capacity in conscious rodents. These data highlight the possibility that cardiac vagal activity is necessary during exercise, but this has never been directly tested. The challenging technical requirements of directly measuring cardiac vagal activity in the exercising animal have thus far prevented conclusive evidence either supporting or refuting the role of the cardiac vagus during exercise. As far as we know, our study is the first to directly record from the cardiac vagal branch during exercise in the conscious animal, and our data unequivocally confirm the presence of CVNA during exercise.
Directly recorded conscious CVNA is a novel technique. Our data show a strong coupling between CVNA and respiration. CVNA respiratory coupling was previously shown in humans,21 cats,7 and rodents.20 In this study, using signal averaging with a breathing waveform as the trigger, we have shown that CVNA bursts occur in the postinspiratory period, which is in line with the majority of previous studies.20,29,30 We have recorded from the whole left cardiac vagal branch. While the anatomic location and nerve tracer studies confirm that this side branch innervates the heart, we cannot confirm whether the nerve fibers recorded are efferent, afferent, or likely a combination of both.
CVNA Increases During Exercise and Has a Role in Regulating Cardiac Function
Here, we provide the first evidence in a conscious large animal model that selective removal of the left cardiac vagal nerve significantly impacted cardiac function in terms of the ability to elicit maximum HR, CO, and CoBF responses during exercise.
We propose a novel hypothesis that, during exercise, there is an increase in both vagal and sympathetic nerve activity, which has a synergistic effect on maintaining optimum cardiac function. Simultaneous coactivation of both autonomic arms is not a new concept, and indeed with the exception of the baroreflex, has been observed in most other reflexes studied.8,31,32 Simultaneous coactivation may lead to more efficient cardiac function than activation of the sympathetic limb alone.8,32 Conversely, to what may be predicted, sheep with left cardiac vagal branch denervation had a reduced maximum HR during exercise compared to control sheep. These data suggest that coactivation of vagal and sympathetic neural outflow to the heart may be synergistic during exercise, resulting in a paradoxical vagally mediated tachycardia.8,33,34 Neither acetylcholine nor VIP antagonism replicated the attenuated maximum HR after cardiac vagal denervation in vagal-intact sheep. Therefore, the mechanisms underlying this are unknown and warrant further investigation.
Acetylcholine Has No Effect on Cardiac Function During Exercise
Administration of the muscarinic acetylcholine blocker atropine during exercise did not affect cardiac function. Our results are in agreement with previous studies. This reinforces the hypothesis that acetylcholine does not modulate cardiac function during exercise beyond HR control (Figure 6). Studies have assumed that cholinergic blockade equals vagal blockade when examining the response to exercise, and given what we know about co-transmission, this may not be true. Taken together with the vagal denervation data, this finding suggests a separate mechanism for the effects of CVNA during exercise beyond acetylcholine-mediated signaling.
Figure 6. Schematic representation of the cardiac effect of increased vagal activity during exercise mediated by acetylcholine and vasoactive intestinal peptide (VIP). mAChR indicates muscarinic acetylcholine receptor; SAN, sinoatrial node; VPAC1, vasoactive intestinal peptide receptor 1; and VPAC2, vasoactive intestinal peptide receptor 2.
VIP Regulates Coronary Artery Blood Flow During Exercise
During exercise, myocardial oxygen consumption increases, as does coronary artery blood flow. The mechanisms behind increased coronary artery blood flow during exercise have been linked to many integrative signaling pathways, including circulating metabolic factors,35–37 adenosine,38–40 K+ ATP41,42 channels, nitric oxide,41,43 and beta-adrenergic signaling.5
There is extensive parasympathetic innervation of the coronary vasculature.44 Parasympathetic regulation of coronary artery blood flow has been shown to be mediated by muscarinic signaling at rest.35,45 While initially identified in the gut, VIP is a potent vasodilator46 and has previously been shown to directly affect the coronary vascular and ventricular myocardium14,46 and increases during exercise.47 VIP as a cotransmitter of vagal nerve fibers has been identified in the heart,14,48 coronary vasculature,49 lungs,50,51 and gastric system.50,52 In our study, antagonism to VIP during exercise resulted in reduced coronary artery blood flow compared to control, indicating that maintenance of coronary artery blood flow during exercise depends on VIP signaling on the coronary vasculature (Figure 6). In recent studies, which mapped cardiac ganglionic plexi, nerves containing VIP were not detected in the SAN, right atrial ganglionic plexi nor was VIP detected in the perivascular innervation of the SAN and right atrium.53 In our study, we have seen VIP labeled in association with cardiac vagal nerve fibers in the coronary artery (Figure S3). The finding that there is no labeling of VIP in the SAN supports our data showing no effect of VIP antagonism on HR at baseline or during exercise. It is unclear if the vasodilatory actions of VIP are due to action on the coronary artery vascular smooth muscle or endothelium.54 However, either option identifies the importance of VIP in maintaining coronary artery blood flow during exercise.
Limitations
A limitation of this study is that only 1 vascular bed, the left coronary artery, was recorded. While this study focused on the effect on cardiac function during exercise, it is important to assess the role of other vascular beds within the heart, such as the right coronary and extra-cardiac vascular beds, such as skeletal and renal arteries. Due to the randomization of exercise and drugs on different days, not all animals got both drugs for numerous reasons, including loss of function of probes or blockage of the venous infusion line. All pharmacological agents (except KCN) used in this study were given systemically intravenously; therefore, the off-target effects of any drug cannot be ruled out. In this context, there is the potential for atropine sulfate to cross the blood-brain barrier. However, previous work within our group has seen comparable results with atropine versus glycopyrrolate (which does not cross the blood-brain barrier). The dose of VIP antagonist used was based on previous studies. We did not observe any evidence of cardiac injury or gastrointestinal effects in sheep with VIP antagonism compared to control days. Only female sheep are used in this study. While we anticipate the observations made in this study will be applicable to males, this will need to be tested in future studies. We have also not corrected for multiple tests done for different variables in this study although the measurement of multiple variables in our animal model is a strength of this study.
Conclusions
Our study has shown that CVNA is crucial in maintaining cardiac function during exercise. VIP serves as a critical mediator of vagally mediated increase in coronary artery blood flow, and muscarinic acetylcholine does not play a significant role in regulating cardiac function during exercise beyond HR control.
Reduced resting vagal activity is a negative prognostic indicator in many cardiovascular diseases.55,56 By gaining a deeper understanding of how CVNA and VIP regulate cardiac perfusion during exercise and improve exercise capacity, we can better understand how these pathways are altered in disease and develop new approaches for promoting cardiovascular health.
ARTICLE INFORMATION
Acknowledgments
The authors thank Maree Schollum, Vanessa Hawkins, Renee Singh, and Melanie Hyslop for their excellent technical and animal welfare support.
Sources of Funding
The data reported in this article were supported by the Health Research Council of New Zealand 23/119 (Fellowship. J. Shanks), the New Zealand Heart Foundation (J. Shanks, R. Ramchandra), and the University of Auckland Faculty Research Development Grant.
Supplemental Material
Expanded Materials and Methods
Major Resources Table
Table S1
Figures S1–S3
Reference 57
| BP | blood pressure |
| CO | cardiac output |
| CoBF | coronary artery blood flow |
| CVNA | cardiac vagal nerve activity |
| HR | heart rate |
| SAN | sinoatrial node |
| VIP | vasoactive intestinal peptide |
Disclosures None.
Footnotes
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