RESEARCH ARTICLE Integrative Cardiovascular Physiology and Pathophysiology

Premature ventricular contractions activate vagal afferents and alter autonomictone: implications for premature ventricular contraction-inducedcardiomyopathy

Siamak Salavatian,1,2 Naoko Yamaguchi,1,2 Jonathan Hoang,1,2 Nicole Lin,1,2 Saloni Patel,1,2

Jeffrey L. Ardell,1,2 J. Andrew Armour,1,2 and Marmar Vaseghi1,2

1University of California, Los Angeles Cardiac Arrhythmia Center, Los Angeles, California; and 2University of California,Los Angeles Neurocardiology Research Center of Excellence, Los Angeles, California

Submitted 20 May 2019; accepted in final form 9 July 2019

Salavatian S, Yamaguchi N, Hoang J, Lin N, Patel S, Ardell JL,Armour JA, Vaseghi M. Premature ventricular contractions activatevagal afferents and alter autonomic tone: implications for prematureventricular contraction-induced cardiomyopathy. Am J Physiol HeartCirc Physiol 317: H607–H616, 2019. First published July 19, 2019;doi:10.1152/ajpheart.00286.2019.—Mechanisms behind developmentof premature ventricular contraction (PVC)-induced cardiomyopathyremain unclear. PVCs may adversely modulate the autonomic nervoussystem to promote development of heart failure. Afferent neurons inthe inferior vagal (nodose) ganglia transduce cardiac activity andmodulate parasympathetic output. Effects of PVCs on cardiac para-sympathetic efferent and vagal afferent neurotransmission are un-known. The purpose of this study was to evaluate effects of PVCs onvagal afferent neurotransmission and compare these effects with aknown powerful autonomic modulator, myocardial ischemia. In 16pigs, effects of variably coupled PVCs on heart rate variability (HRV)and vagal afferent neurotransmission were evaluated. Direct nodoseneuronal recordings were obtained in vivo, and cardiac-related affer-ent neurons were identified based on their response to cardiovascularinterventions, including ventricular chemical and mechanical stim-uli, left anterior descending (LAD) coronary artery occlusion, andvariably coupled PVCs. On HRV analysis before versus afterPVCs, parasympathetic tone decreased (normalized high frequen-cy: 83.6 � 2.8 to 72.5 � 5.3; P � 0.05). PVCs had a powerful impacton activity of cardiac-related afferent neurons, altering activity of 51%of neurons versus 31% for LAD occlusion (P � 0.05 vs. LADocclusion and all other cardiac interventions). Both chemosensitiveand mechanosensitive neurons were activated by PVCs, and theiractivity remained elevated even after cessation of PVCs. Cardiacafferent neural responses to PVCs were greater than any other inter-vention, including ischemia of similar duration. These data suggestthat even brief periods of PVCs powerfully modulate vagal afferentneurotransmission, reflexly decreasing parasympathetic efferent tone.

NEW & NOTEWORTHY Premature ventricular contractions(PVCs) are common in many patients and, at an increased burden, areknown to cause heart failure. This study determined that PVCspowerfully modulate cardiac vagal afferent neurotransmission (exert-ing even greater effects than ventricular ischemia) and reduce para-sympathetic efferent outflow to the heart. PVCs activated bothmechano- and chemosensory neurons in the nodose ganglia. Theseperipheral neurons demonstrated adaptation in response to PVCs. Thisstudy provides additional data on the potential role of the autonomicnervous system in PVC-induced cardiomyopathy.

afferent; autonomic; nodose ganglia; parasympathetic; premature ven-tricular contractions; vagus nerve

INTRODUCTION

Frequent premature ventricular contractions (PVCs) areknown to cause heart failure (1, 13, 20, 47), and increasedburden of PVCs is associated with increased risk of cardiomy-opathy (4, 25, 29, 43). Precise mechanisms behind develop-ment of PVC-induced cardiomyopathy are unclear, but severalstudies have suggested changes in ventricular dynamics anddyssynchrony (31, 43), calcium handling, and oxygen con-sumption (27) as potential mechanisms.

The autonomic nervous system modulates every aspect ofcardiac function (21, 38). Sympathetic activation and parasym-pathetic dysfunction often work in concert to increase risk ofheart failure and sudden death (19, 34, 38, 46). It is possiblethat PVCs cause imbalances in the autonomic nervous systemthat contribute to cardiac dysfunction. PVCs can elevate sym-pathetic tone and have been reported to alter stellate ganglionneural activity (10, 26) and increase muscle sympathetic nerveactivity, coronary sinus norepinephrine levels (15, 28, 41, 50),and the low-frequency component of heart rate variability(HRV) (3), all indices of elevated sympathetic tone. It has alsobeen shown that PVCs can alter the activity of intrinsic cardiacganglion neurons in vivo (16). However, the effect of PVCs oncardiac parasympathetic neurotransmission is unclear. Over80% of the vagal trunk consists of sensory afferent fibers, forwhich neurons reside in the inferior vagal (nodose) ganglia.These neurons transduce visceral activity, including beat-to-beat variability of cardiac function, to alter subsequent para-sympathetic efferent tone (21). Reflex withdrawal of parasym-pathetic tone would eliminate critical peripheral restraininginfluences on cardiac adrenergic function.

In this study, we hypothesized that PVCs adversely influ-ence autonomic control by altering the activity of cardiacsensory nodose neurons, subsequently decreasing parasympa-thetic efferent neurotransmission to the heart. We also aimed tocompare the effect of PVCs with ventricular ischemia, a knownmodulator of autonomic tone. Finally, we aimed to assesswhether PVCs purely activate cardiac mechanosensitive neu-rons (as might be expected) or also alter activity of chemosen-sitive neurons, amplifying autonomic dysfunction.

Address for reprint requests and other correspondence: M. Vaseghi, UCLACardiac Arrhythmia Center, 100 Medical Plaza, Ste. 660, Los Angeles, CA90095 (e-mail: mvaseghi@mednet.ucla.edu).

Am J Physiol Heart Circ Physiol 317: H607–H616, 2019.First published July 19, 2019; doi:10.1152/ajpheart.00286.2019.

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METHODS

Sixteen Yorkshire pigs (59.2 � 3.2 kg) were used in this study.Animal experiments were performed in accordance with the NationalInstitutes of Health Guide for the Care and Use of LaboratoryAnimals and approved by the University of California, Los AngelesChancellor’s Animal Research Committee.

Animal Preparation

Animals were sedated (4–8 mg/kg im tiletamine-zolazepam) andintubated. Anesthesia for the surgical portion of the experiment wasprovided using isoflurane (1–5% inhaled). Following completion ofsurgical procedures and for neural recordings, anesthesia wasswitched to �-chloralose (50 mg/kg initial bolus, thereafter 20–35mg·kg�1·h�1 iv; Sigma-Aldrich). The level of anesthesia was ad-justed during the procedure by examining the corneal reflex, jaw tone,and hemodynamic indices. End-tidal CO2 and oxygen saturation viapulse oximetry were monitored throughout the experiment. A waterheating pad (T/PUMP; Gaymar Industries, Orchard Park, NY) wasused to maintain temperature. Twelve-lead electrocardiography(ECG) was recorded via CardioLab System (GE Healthcare). Becauseof sternotomy, anterior precordial leads were placed on left dorsalaspect of the animal. Left and right femoral arteries and veins werecannulated to measure blood pressure and administer saline and drugs,respectively. The carotid artery was cannulated to obtain access to theleft ventricle (LV). ECG and arterial blood pressure were digitized(Power1401; Cambridge Electronic Design, Cambridge, England),stored, and analyzed offline by the Spike2 program (CambridgeElectronic Design). Arterial blood gases were assessed every hour,and the tidal volume or respiratory rate was adjusted or sodiumbicarbonate was given as needed to maintain a normal pH. Sternotomywas performed after a fentanyl bolus (20–30 �g/kg). An overdose ofpentobarbital sodium (100 mg/kg iv; Med-Pharmex) followed bysaturated KCl (1–2 mg/kg iv; Sigma-Aldrich) was used for euthanasia.

Histological Confirmation of the Nodose Ganglion

Nodose ganglia were removed after euthanasia and fixed in 4%paraformaldehyde for 24 h at 4°C and then embedded in paraffin.Tissue was sectioned (5 �m) and rehydrated in two toluene washesfollowed by three ethanol washes and water. Slides were blocked for1 h in 3% BSA-TBS-0.2% Triton X-100 with 5% donkey serum andincubated overnight at 4°C with rabbit anti-PGP9.5 (1:200; ab108986;Abcam) and mouse anti-S100 (1:200; ab4066; Abcam) at 4°C fol-lowed by 2-h incubation at room temperature with Alexa Fluor 555donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-mouse IgG(Invitrogen), respectively. Sections were imaged using a Zeiss LSM880 with Airyscan (Zeiss).

HRV Analysis

HRV was analyzed in 10 animals that had suitable/clean ECGrecordings pre- and post-PVCs using the Lomb periodogram nonpara-metric method for spectral analysis (LabChart software; ADInstru-ments, Colorado Springs, CO). HRV during the 1 min before induc-tion of PVCs was compared with 1 min after cessation of PVCs.Given the short duration of the intervention and period of time (1 min)pre- versus post-PVC evaluated, only the high-frequency componentof HRV was analyzed and used to assess parasympathetic function(0.15–0.4 Hz) (36). For HRV time-domain analysis, the followingparameters were calculated: standard deviation of RR interval, coef-ficient of variance of RR intervals, standard deviation of successiveRR interval differences between adjacent RR intervals, root-mean-square successive difference, and number of pairs of adjacent RRintervals differing by �50 ms to all RR intervals.

Premature Ventricular Contractions

PVCs were induced for 1 min using intermittent endocardial rightventricular (RV) pacing via a quadripolar catheter (Abbott, St. Paul,MN) placed from the femoral vein and attached to a cardiac stimulator(EPS320; Micropace, Canterbury, Australia). PVCs were generatedon average every 5 beats (range 4–7) with random/variable couplingintervals (300–600 ms).

Afferent Nodose Neuronal Activity Recordings

To evaluate cardiac afferent neurotransmission, activity of individ-ual nodose ganglia afferent neurons was recorded at baseline andduring cardiac stimuli, including PVCs. Right and left nodose gangliawere exposed via a lateral neck cut down (Fig. 1A and SupplementalFig. S1; all supplemental material is available at https://doi.org/10.6084/m9.figshare.8139512.v1).

Vagus nerve branches were kept intact during recordings. As nodifferences in the transduction capabilities of left versus right nodoseganglion have been previously observed (44), left (n � 3), right (n �4), or bilateral (n � 9) nodose ganglion soma activity was recorded invivo using custom-made 16-channel linear microelectrode arrays(LMA; Microprobes for Life Science, Gaithersburg, MD; Fig. 1B).The LMA (25-�m-diameter platinum/iridium electrodes, 16 elec-trodes/channels, 250-�m interelectrode distance) was connected to a16-channel preamplifier (NeuroNexus, Ann Arbor, MI). Neural sig-nals were sampled at 20 kHz (filtered at 300–10,000 Hz), amplified,and digitized (SmartBox acquisition system; NeuroNexus). If cardiac-related neuronal activity could not be identified, LMA position wasadjusted slightly and neural activity was re-evaluated. At the end ofeach experiment, nodose ganglia were removed and histology wasperformed for confirmation of the location of the nodose as describedabove (Fig. 1A). Before removal of ganglia, ipsilateral vagus nervestimulation was performed (1 Hz, 1 ms, 6 mA) at the end of theexperiment to confirm backfiring (antidromic activation) of nodoseganglion neurons (Supplemental Fig. S2).

Cardiovascular Interventions

Interventions were performed in all animals in a random order.PVCs were induced as described above. To further characterize thenodose ganglion neurons identified, other cardiac interventions wereperformed. These included 1) anterior LV and RV mechanical stim-ulation and 2) chemical stimulation, 3) rapid RV pacing, 4) inferiorvena cava (IVC) and then 5) aortic artery occlusion and 6) 1 min ofleft anterior descending (LAD) ischemia. Depending on the interven-tion, 10–30 min was allowed between stressors for hemodynamicindices to return to baseline.

Ventricular epicardial afferent inputs. To assess the response ofnodose ganglion afferent neurons to epicardial mechanical stimuli,gentle pressure (~10 g) was applied to the RV and LV anterior wallsfor 15 s via a saline-soaked-cotton-tipped applicator. Thereafter,chemical transduction of individual neurons was tested by 1-minapplication of a gauze soaked in adenosine (100 �M), bradykinin (10�M), capsaicin (1 �M), or veratridine (10 �M) to the anterior RV andLV epicardium (same region that mechanical stimuli had been ap-plied). Warmed saline was used to wash off the chemicals after eachapplication.

Rapid ventricular pacing. Rapid RV pacing was performed for 1min at 15% above the baseline heart rate using the same pacingcatheter used for PVCs. Neuronal responses to ventricular pacingwere evaluated.

Great vessel occlusions. Two segments of umbilical tape wereplaced around the IVC and descending aorta. IVC and then aorticocclusions were performed separately for 30 s to evaluate and com-pare neuronal responses to changes in preload and afterload, respec-tively.

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Regional ventricular ischemia. A silk suture was placed around theLAD coronary artery after its first diagonal branch. The LAD wasoccluded for 1 min to evaluate neural responses to coronary arterialocclusion. Ischemia was confirmed by ST elevation or T-wave inver-sion on ECG and decrease in LV systolic pressure (LVSP).

Neural Signal Processing and Analysis

Detailed spike identification of the activity generated by individualneurons was performed by assessing all neural channels. Simultane-ous and similar waveforms present on all channels were identified asartifacts and removed. Neural waveforms with a signal-to-noise ratio�3 were identified (Fig. 1C). Neuronal activity classification wasperformed using principal component, cluster on measurements,and k-means clustering analysis using Spike2 (5, 16, 33). Timeseries of individual neuronal activity for the entire experiment wastransferred to MATLAB (MathWorks, Natick, MA) for post hocneural analysis.

If activity of a neuron changed significantly during at least onecardiovascular stressor (1-min baseline vs. during the intervention),then the neuron was considered to be a cardiac-related neuron (Fig.1D). Neurons that did not respond to any cardiovascular interventions,and may be sensing other visceral organs, were excluded from the

analysis. Chemosensitive cardiac neurons were defined as those thatresponded to at least one ventricular chemical stimulus and not to theepicardial mechanical stimuli. Mechanical neurons were defined asthose that responded only to mechanical stimuli. Multimodal neuronswere identified as those that responded to at least one mechanical andat least one chemical stimulus.

Statistical Analysis

Data are presented as means � SE. Neuronal activity was com-pared in different time windows (1 min before vs. during intervention)by calculating the average neuronal activity. Significant differences inactivity between time windows were compared based on the Skellamdistribution (40). This test has been validated for neuronal activity ofperipheral ganglia (5, 16, 32, 33) and the central nervous systemneurons (40). A 2-test was used to compare neuronal responses withdifferent stressors. Wilcoxon signed�rank test was used to comparepaired data. One-way ANOVA was used to evaluate differencesamong groups. Adjustment for multiple comparisons was performedusing Tukey multiple comparisons test. A P value � 0.05 wasconsidered statistically significant. Statistical analyses were per-formed using Prism (GraphPad Software, La Jolla, CA).

Fig. 1. Nodose ganglia in vivo neuronal recordings. A: anatomic locations of the nodose ganglion and carotid artery are shown. Protein gene product (PGP) 9.5staining of the nodose ganglion was used to confirm location of the ganglion at the end of the experiments. B: customized 16-channel linear microelectrode arraywas used for individual neuronal recordings. C: representative sorted neuronal action potentials from the nodose ganglion of 1 animal. Representative actionpotentials from individual neurons are illustrated in the boxes. D: representative activities generated by 6 nodose neuronal soma in response to prematureventricular contractions (PVCs) in 1 animal. Increase in the activity of these nodose neurons (P � 0.05) can be observed during PVCs in this animal. CH,electrode channel; LVP, left ventricular pressure (mmHg).

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RESULTS

Hemodynamic Responses

Hemodynamic parameters at baseline and during interven-tions are shown in Table 1 and Supplemental Table S1.

Of note, during the PVC period, excluding the PVC beats, heartrate increased by 3.5 � 1.6 beats/min (3.9 � 1.7%; P � 0.05),whereas mean LVSP decreased by 4.1 � 1.5 mmHg (3.8 � 1.4%;P � 0.01). LVSP and dP/dtmax decreased in response to IVCocclusion by 58.5 � 6.6 mmHg (52.5 � 4.7%; P � 0.01) and651.4 � 153.4 mmHg/s (39.1 � 7.3%; P � 0.01). Aortic occlu-sion increased both LVSP and dP/dtmax by 60.0 � 6.7 mmHg(62.1 � 7.5%; P � 0.01) and 182.3 � 74.7 mmHg/s (25.3 �13.1%; P � 0.05), respectively. LAD occlusion also changedhemodynamic indices significantly by increasing dP/dtmin by370.6 � 62.6 mmHg/s (19.5 � 2.1%; P � 0.01) and decreasingLVSP by 9.7 � 1.3 mmHg (9.2 � 1.0%; P � 0.01) and dP/dtmax

by 159.5 � 25.8 mmHg/s (11.2 � 1.7%; P � 0.01).

Heart Rate Variability

HRV was analyzed before and after induction of PVCs.The heart rate in the minute before initiation of PVCs was96.3 � 4.6 beats/min. The heart rate was 97.4 � 4.7 beats/min in the minute after cessation of PVCs. High-frequencycomponent analysis of HRV showed that PVCs significantlydecreased indices of parasympathetic tone [relative power

of high frequency (HF): 78.6 � 2.7 to 57.9 � 6.8%, P �0.05; normalized power of HF: 83.6 � 2.8 to 72.5 � 5.3,P � 0.05], suggesting parasympathetic withdrawal (Table2). All time-domain parameters pre- versus post-PVCsshowed a consistent trend for decrease in variability, con-sistent with a decrease in parasympathetic tone (Table 2),but did not reach statistical significance. This was likely dueto the limited duration of the intervention as well as theshort period of time that could be used for analysis pre- andpostintervention.

Neuronal Responses to Cardiac Stressors

Given evidence for withdrawal of parasympathetic efferenttone during PVCs, we evaluated whether PVCs affect vagalafferent neurotransmission, which can reflexly decrease centralparasympathetic drive. Of 16 animals, cardiac-related neuronswere meticulously identified in the nodose ganglia of 9 ani-mals, as the nodose ganglia contain neurons from all visceralorgans (26). Location of nodose ganglion was confirmed his-tologically in all 16 animals (Fig. 1A). Of the 212 nodoseafferent neurons (13 � 4 per animal) recorded, 89 cardiac-related neurons were identified based on their significant re-sponses (P � 0.05) to cardiac stressors (Fig. 2A). Basal activityof cardiac afferent neurons was relatively low (0.19 � 0.05Hz). Of these 89 neurons, the greatest number (51%) re-sponded to PVCs, whereas only 31% responded to LADocclusion of similar duration (P � 0.05; Fig. 2B). In addition,19 and 18% of these sensory neurons responded to IVC andaortic occlusions, respectively. Changes in percentage of neu-rons affected by other cardiovascular interventions, includingRV pacing, are shown in Fig. 2B.

The absolute value of the change in firing rate from baselinefor each intervention is shown in Fig. 2C. PVCs, LAD, andIVC occlusions caused the greatest change in the firing rates(PVCs: 0.29 � 0.06 Hz; LAD: 0.24 � 0.06 Hz; IVC occlusion:0.19 � 0.06 Hz; Fig. 2C). Cardiac sensory neuronal firingactivity in response to PVCs and LAD was significantly higher(P � 0.01) than epicardial mechanical stimuli, aortic occlu-sion, RV pacing, and chemical stimulation.

Mechanosensitive Versus Chemosensitive Neural Responses

To further evaluate the type of neurons activated by PVCs,we focused on sensory neurons receiving input from theregions of the epicardial RV and LV anterior wall, wherechemical interventions could be directly applied (Fig. 3A).Basal firing rate of different types of cardiac afferent neurons(mechanical vs. chemical vs. multimodal) was not significantlydifferent. PVCs not only activated mechanosensitive neurons

Table 1. Hemodynamic response to applied cardiacstressors (n � 14)

Stimulus HR, % LVSP, % dP/dtmax, % dP/dtmin, %

EMS �0.3 � 0.5 �2.5 � 0.6 �1.4 � 1.2 3.7 � 1.3ADENOS 4.8 � 4.7 1.2 � 1.6 �3.1 � 2.3 �2.1 � 1.8BRADY 4.00 � 3.4 �1.3 � 3.0 6.2 � 4.3 5.8 � 4.2CAPS 6.5 � 6.7 �3.4 � 4.0 65.2 � 77.1 1.1 � 4.1VERAT 0.6 � 2.00 �2.8 � 3.1 �11.6 � 4.4 6.2 � 4.2PVC 3.9 � 1.7* �3.8 � 1.4† 3.7 � 6.1 0.5 � 4.5RVP 13.7 � 2.8† �19.5 � 5.7† �17.6 � 5.0† 26.7 � 5.4†IVC 3.8 � 5.8 �52.5 � 4.7† �39.1 � 7.3† 53.7 � 7.2†AO �11.4 � 3.0† 62.1 � 7.5† 25.3 � 13.1* �50.2 � 30.0†LAD 1.3 � 0.3† �9.2 � 1.0† �11.2 � 1.7† 19.5 � 2.1†

Values are means � SE for percentage change from baseline in heart rate(HR), left ventricular systolic (LVSP), as well as the maximum and minimum1st derivatives of LV pressure (dP/dt). ADENOS, epicardial adenosine appli-cation; BRADY, epicardial bradykinin application; CAPS, epicardial capsaicinapplication; AO, aortic occlusion; EMS, epicardial mechanical stimulation;IVC, inferior vena cava occlusion; LAD, left anterior descending coronaryartery occlusion; PVC, premature ventricular contractions; RVP, right ventric-ular pacing; VERAT, epicardial veratridine application. *P � 0.05 vs. base-line. †P � 0.01 vs. baseline. Values in boldface represent statistically signif-icant changes from baseline.

Table 2. Heart rate variability analysis

HF, % nHF SDRR, ms CVRR SDSD, ms RMSSD, ms pRR50, %

Baseline 78.6 � 2.7 83.6 � 2.8 31.6 � 16.8 0.05 � 0.03 43.75 � 24.0 43.5 � 23.9 0.03 � 0.02Post-PVC 57.9 � 6.8 72.5 � 5.3 14.9 � 8.2 0.02 � 0.01 23.96 � 13.0 24.3 � 13.2 0.03 � 0.02P value �0.05 �0.05 0.37 0.33 0.49 0.51 0.97

Values are means � SE. Wilcoxon signed�rank test was used to compare baseline vs. post-PVC periods; n � 10 animals. CVRR, coefficient of variance ofRR intervals; HF, high-frequency component of heart rate variability; nHF, normalized HF; pRR50, proportion of the number of pairs of successive RR intervalsthat differ by �50 ms divided by all of the RR intervals given as a percentage; PVC, premature ventricular contraction; RMSSD, root mean square of thesuccessive RR differences; SDRR, standard deviation of RR interval; SDSD, standard deviation of successive RR interval differences between adjacent RRintervals.

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(42%), but also altered activity of chemosensitive (25%) andmultimodal neurons (33%) innervating the RV and LV anteriorepicardium (Fig. 3, B and C). No difference in basal activity orchange in firing rate of mechanical sensory nodose neuronsinnervating the RV compared with the LV anterior wall wasnoted. Also, no significant difference between the number ofneurons that responded to the various chemical stimuli wasobserved.

Temporal Profile of Nodose Sensory Neuronal Activity inResponse to PVCs

Analysis of the activity of nodose neurons that responded toPVCs demonstrated that after cessation of PVCs, their activityremained elevated and did not return to baseline for �1 minpostcessation of PVCs (Fig. 4A), suggesting existence of neu-ronal memory. The first five PVCs caused the highest impacton cardiac neuronal activity (Fig. 4B). This activity thensomewhat diminished (Fig. 4B), indicating receptor-mediatedadaptation, given somewhat of a decrease in response to aconstant stimulus (49).

Effects of PVC Coupling Interval on Afferent Activity

To assess the effect of the PVC coupling intervals onneuronal activity, the ratio of the PVC coupling interval to thesinus cycle length was used to define early versus late coupledPVCs, as different sinus rates were observed in differentanimals (Fig. 4C). Increasing afferent neuronal activity wasobserved as the PVC coupling interval increased (Fig. 4D).Cardiac afferent neurons showed the highest activity duringPVCs with the longest coupling interval (ratio of PVC couplinginterval to sinus cycle length of 80–90%), suggesting thatlate-coupled PVCs exert the greatest effect on vagal afferentneural transduction.

DISCUSSION

To our knowledge, this is the first study to evaluate effectsof PVCs on cardiac vagal afferent neurotransmission usingdirect recordings of cardiac nodose sensory neurons in vivo.The major findings of this study are 1) PVCs are powerfulmodulators of cardiac vagal afferent neurotransmission, exert-

Fig. 2. Response of neurons to cardiovascular stimuli. A: 89 cardiac afferent neurons (each row is an individual neuron) were identified based on their responseto cardiovascular interventions, including PVCs. B: percentage of cardiac neurons that responded to each stressor is shown. PVCs engaged the highest numberof afferent neurons. *P � 0.05 vs. other interventions (ANOVA). C: average change in firing rate of all nodose cardiac afferent neurons (n � 89). PVCs, leftanterior descending (LAD) occlusion, and IVC occlusion caused the greatest change in firing rates of neurons. *P � 0.01 (ANOVA).

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ing even greater effects than ventricular ischemia of similarduration, significantly increasing sensory neural inputs to thebrain stem, 2) PVCs activate mechanosensory as well aschemosensory neurons in the inferior vagal ganglia, and 3)nodose ganglion sensory neurons are capable of state-depen-dent adaptations and display memory in response to cardiacstimuli. Because of this capacity, any excitation of this popu-lation of cardiac afferent neurons persists following the initialinsult, further exacerbating the pathology of cardiac arrhyth-mias and cardiomyopathy.

PVC Induced Parasympathetic Efferent Withdrawal

Previous studies of patients with PVCs using HRV analysishave shown existence of sympathetic activation based on anincrease in the low-frequency component (3). PVCs have alsobeen shown to increase muscle sympathetic nerve activity andactivate neurons in the stellate ganglia (10, 15, 26, 50). How-ever, the effects of PVCs on the parasympathetic nervoussystem remained unclear. HRV data have shown controversialresults, with some studies demonstrating a decrease and othersshowing no clear changes in HF component of HRV in thepresence of frequent PVCs (3, 11, 18, 39).

As sympathetic efferent activation and parasympathetic ef-ferent dysfunction often act in concert to increase progressionof cardiomyopathy, we hypothesized that PVCs may lead to alow parasympathetic tone due to activation of cardiac sensoryneurons in the nodose ganglia that provide input to the nucleustractus solitarius of the medulla. To confirm that parasympa-thetic tone was indeed decreased, we measured HRV beforeand immediately after cessation of PVCs to avoid the con-founding factor of presence of PVCs on HRV analysis that mayhave complicated results of other studies. We found that,indeed, the HF component decreased, suggesting with para-sympathetic withdrawal. As such, we hypothesized that affer-

ent parasympathetic activation contributes to parasympatheticefferent withdrawal associated with PVCs.

Sensory Transduction of PVCs by the Nodose Ganglia

The nodose ganglia provide sensory innervation from thelungs, gastrointestinal tract, and other visceral organs. Only asmall percentage of these neurons are cardiovascular (26, 32).Therefore, it is not unexpected that in some animals, despiterecording multiple neurons, we could not identify neurons thatresponded to cardiovascular interventions.

A previous study suggested that LAD occlusion is, in gen-eral, a very potent modulator of cardiac afferent neurons (32).In this study, we were surprised to find that variably coupledPVCs engaged even more cardiac-related neurons than LADocclusion, IVC occlusion (which decreased cardiac preload by50–60 mmHg), or aortic occlusion (which increased cardiacafterload by �60 mmHg). Variably coupled PVCs also acti-vated more sensory neurons than rapid RV pacing, consistentwith a previous porcine study demonstrating that chronic PVCsare more likely to cause cardiomyopathy than rapid RV pacingat 140 beats/min (47). Our data suggest that even brief periodsof PVCs are important stressors and modulators of the para-sympathetic nervous system.

Sensory Transduction of Mechanical and Chemical Milieu

A major strength of our study is evaluation and character-ization of individual cardiovascular neuronal types rather thanmeasurement of global vagal nerve activity, which reflects theaverage effects of afferent and efferent neurotransmission.Cardiac afferent neurons in the nodose ganglia have beenshown to be mechanosensitive, chemosensitive, or capable oftransducing both types of stimuli (multimodal neurons) (2, 44).We hypothesized that PVCs, in altering mechanical stretch and

Fig. 3. Type of afferent neurons modulated by premature ventricular contractions (PVCs). A: location of right ventricular (RV) and left ventricular (LV)application of mechanical and chemical stimuli is shown by the shaded area (white square). Mechanical and chemical stimuli were applied to the same receptivefield. B: PVCs activated both mechanosensitive and chemosensitive neurons. C: percentages of mechanical, chemical, and multimodal neurons that respondedto PVCs are shown. ADE, adenosine; BRA, bradykinin; C, chemical neurons; CAP, capsaicin; CHEM, chemical stimulation; EMS, epicardial mechanicalstimulation; LAD, left anterior descending; M, mechanical neurons; MM, multimodal neurons; VER, veratridine.

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synchrony (47), would likely activate mechanosensitive neu-rons. We were surprised to find that PVCs also activatedchemosensitive neurons. These data suggest that PVCs maycause changes in supply/demand that may lead to release ofreactive oxygen species and activate chemosensitive neurons.Arrhythmias, including PVCs, can be associated with releaseof cellular reactive oxygen species by altering both ion channel(calcium channel) and mitochondrial function (22, 27, 48).

Of note, 33 (37%) of cardiac afferent neurons responded toPVCs without responding to any of the other RV and LVepicardial mechanical or chemical stimuli. This might be dueto the fact that 1) the sensory axons of these neurons are in thedorsal/posterior aspect of the heart or on the endocardium,which would not be affected by our local anterior RV and LV

applied mechanical or chemical cardiac stimuli or 2) theseneurons may sense other molecules/chemicals released duringPVCs not tested by our chemical interventions.

Temporal Profile of Neuronal Activity During PVCs

Cardiac sensory nodose neurons were maximally activatedimmediately after the initiation of PVCs and specifically duringthe period encompassing the first five PVCs. After the first fivePVCs, these afferent neurons remained active but at a lowerfiring rate. These data suggest that neurons of the nodoseganglia adapt during an ongoing pathology. It is important tonote, however, that activity of these neurons remained elevatedcompared with baseline for �1 min postcessation of PVCs.

Fig. 4. Response of cardiac afferent neurons activated with premature ventricular contractions (PVCs). A: response of neurons that were activated during PVCsshows elevated firing rates even after cessation of PVCs (response evaluated over the minute following discontinuation of PVCs). B: response of neurons as afunction of the PVC number during the 1-min period is shown. C: representative example of a PVC coupling interval and underlying sinus cycle length. Ratioof the coupling interval to the sinus cycle length was used to assess effect of early vs. late-coupled PVCs on neuronal activation. Representative sinus cycle length(569 ms, as shown) and PVC coupling interval (395 ms, as shown) are shown. D: response of neurons to PVCs as a function of the coupling interval to sinusrhythm ratio. Late-coupled PVCs (ratio of 80–90%) caused the greatest neuronal activity. *P � 0.05 (ANOVA). BL, baseline; CI, coupling interval; CL, cyclelength; LVP, left ventricular pressure.

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These data are consistent with our HRV data indicating thatwithdrawal of cardiac parasympathetic efferent tone persistedafter arrhythmia termination.

Effects of PVC Coupling Interval on Afferent Activity

It has been shown that PVCs with the longer couplinginterval cause more pronounced LV dyssynchrony, and thismay be a more important variable than even location of PVCsin causing heart failure (31). In this study, PVCs with thelongest coupling interval caused a greater activation ofcardiac sensory neurons than shorter coupling intervalPVCs. This could potentially be due to the more pronouncedLV dyssynchrony observed in previous studies with longercoupled PVCs (31).

Clinical Implications

PVCs are routinely observed in many patients with anestimated prevalence of 1–4% (23). However, several studieshave shown that a high burden of PVCs is associated withdevelopment of left ventricular dysfunction and heart failureover time (4, 12, 29). The extent of LV dysfunction alsocorrelates with frequency of PVCs. Currently, there are noclear cut-off points that delineate the PVC burden at whichcardiomyopathy may develop (4, 6, 17). However, severalstudies have suggested that a frequency �10%, and especially�24%, is associated with development of cardiomyopathy (4,17, 37). In line with these studies, we chose to deliver PVCs atan average burden of every 5 beats (~20%). Of note, mostpatients with PVC-induced heart failure do not have scar oncardiac imaging, suggesting an important role for the auto-nomic nervous system in development of this disease. Treat-ment of PVCs, including with medications that reduce burdenof PVCs or catheter ablation of PVCs, results in improvementof LV function and heart failure (4, 14, 17, 24, 35, 42). Byevaluating both the afferent and efferent limbs of the cardiacparasympathetic nervous system, neural data from this studyprovide supportive evidence for powerful modulation of vagalneurotransmission by even brief periods of PVCs. Effects ofPVCs were greater than LAD ischemia or burst RV pacing ofsimilar duration. As these effects persist following the cessa-tion of PVCs, it appears that this neural transduction involvesmemory. These data have implications for autonomic imbal-ances that can be caused by PVCs and can contribute todevelopment of cardiomyopathy. Furthermore, given the im-portant role of the autonomic nervous system in developmentof PVC-induced cardiomyopathy, it is possible that in patientsin whom ablation or medication fail to treat PVCs, autonomicmodulation can provide an additional avenue for therapy.Consistent with this, bilateral stellate ganglion blockade andcardiac sympathetic denervation have been reportedly used incase series of patients to treat refractory PVCs and ventriculararrhythmias (9, 45).

Limitations

The basal activity of nodose ganglion neurons measured inthis study was relatively low, which may reflect effects ofanesthesia. To minimize effects of isoflurane, neural recordingswere obtained after anesthesia was switched to �-chloralose.Because inhaled isoflurane can suppress neuronal responses tocardiovascular interventions, the results of this study may

represent a conservative estimate of the effects of PVCs onparasympathetic function. Given the acute nature of thesestudies, the effects of PVCs were evaluated over short dura-tions. It is possible that long-term autonomic effects of PVCsin development of cardiomyopathy may not parallel those seenin this study. However, given the powerful modulatory effectof PVCs on neural activity observed in this study and thepersistence of these effects beyond cessation of PVCs, it wouldbe expected that chronic frequent PVCs would continue tomodulate afferent neuronal neurotransmission and reflexly de-crease cardiac parasympathetic efferent tone. As HRV is non-invasive, it has been frequently used to assess autonomic tonein clinical studies and has been associated with increased riskof death in setting of heart failure (30); however, HRV havecan have significant limitations, especially in assessing situa-tions where heart rate changes. A decrease in HRV can bedriven by an increase in heart rate (7, 8). In this study, therewas a small increase in heart rate from 96 to 97 beats/min whencomparing the pre-PVC with the post-PVC period, and it ispossible that a small portion of the HRV changes are driven bythis mild increase in heart rate. Parameters in this study werestatistically significant in the frequency domain but not thetime domain. This may be due to the short duration of theintervention and period for pre- and post-PVC time analysis aswell as lack of large numbers of animals. Finally, as theprimary goal of this study was to compare the effect of PVCson afferent neurotransmission and parasympathetic efferentoutflow compared primarily with LAD ischemia, effects ofpremature atrial contractions and various locations of PVCswere not assessed. However, evaluation of these comparisonsremains an important part of future studies in our laboratory.

Conclusions

PVCs cause vagal afferent activation and decrease parasym-pathetic efferent tone to the heart. As such, even brief periodsof PVCs act as significant modulators of cardiac control,continuing to exert a powerful influence on neurotransmissioneven after the event and more than ventricular ischemia.

ACKNOWLEDGMENTS

We thank Dr. Amer Swid and Janki Mistry for technical assistance and Drs.Kalyanam Shivkumar, Pradeep Rajendran, and Peter Hanna for review of themanuscript.

GRANTS

This work was supported by National Institutes of Health Grants DP2-HL-132356 and SPARC OT2OD023848 to M. Vaseghi.

DISCLOSURES

M. Vaseghi and J. L. Ardell have founder shares in Neurcures, Inc.University of California, Los Angeles, has patents developed by M. Vaseghiand J. L. Ardell relating to cardiac neural diagnostics.

AUTHOR CONTRIBUTIONS

S.S., J.L.A., J.A.A., and M.V. conceived and designed research; S.S., N.Y.,J.H., S.P., and M.V. performed experiments; S.S., N.L., S.P., and M.V.analyzed data; S.S., J.L.A., J.A.A., and M.V. interpreted results of experi-ments; S.S. and M.V. prepared figures; S.S. and M.V. drafted manuscript; S.S.,N.Y., J.H., N.L., S.P., J.L.A., J.A.A., and M.V. edited, revised, and approvedfinal version of manuscript.

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