TRPA1 and Sympathetic Activation Contribute to Increased Risk of Triggered Cardiac Arrhythmias in Hypertensive Rats Exposed to Diesel Exhaust

Background: Diesel exhaust (DE), which is emitted from on- and off-road sources, is a complex mixture of toxic gaseous and particulate components that leads to triggered adverse cardiovascular effects such as arrhythmias. Objective: We hypothesized that increased risk of triggered arrhythmias 1 day after DE exposure is mediated by airway sensory nerves bearing transient receptor potential (TRP) channels [e.g., transient receptor potential cation channel, member A1 (TRPA1)] that, when activated by noxious chemicals, can cause a centrally mediated autonomic imbalance and heightened risk of arrhythmia. Methods: Spontaneously hypertensive rats implanted with radiotelemeters were whole-body exposed to either 500 μg/m3 (high) or 150 μg/m3 (low) whole DE (wDE) or filtered DE (fDE), or to filtered air (controls), for 4 hr. Arrhythmogenesis was assessed 24 hr later by continuous intravenous infusion of aconitine, an arrhythmogenic drug, while heart rate (HR) and electrocardiogram (ECG) were monitored. Results: Rats exposed to wDE or fDE had slightly higher HRs and increased low-frequency:high-frequency ratios (sympathetic modulation) than did controls; ECG showed prolonged ventricular depolarization and shortened repolarization periods. Rats exposed to wDE developed arrhythmia at lower doses of aconitine than did controls; the dose was even lower in rats exposed to fDE. Pretreatment of low wDE–exposed rats with a TRPA1 antagonist or sympathetic blockade prevented the heightened sensitivity to arrhythmia. Conclusions: These findings suggest that a single exposure to DE increases the sensitivity of the heart to triggered arrhythmias. The gaseous components appear to play an important role in the proarrhythmic response, which may be mediated by activation of TRPA1, and subsequent sympathetic modulation. As such, toxic inhalants may partly exhibit their toxicity by lowering the threshold for secondary triggers, complicating assessment of their risk.


I. Materials and methods:
A. Implantation of radiotelemeters, and electrocardiogram acquisition and analysis.
Radiotelemeters were implanted in all animals as previously described (Hazari et al. 2009); this methodology was used to track changes in cardiovascular function by monitoring ECG and HR.
Briefly, animals were weighed and anesthetized with ketamine hydrochloride/xylazine hydrochloride solution (1ml/kg, ip Sigma-Aldrich, St. Louis, MO). Using aseptic technique, each animal was implanted with a radiotelemetry transmitter (Model TA11CTA-F40, Data Sciences International, St. Paul, MN) in the abdominal cavity through a small incision. The electrode leads were guided through the abdominal musculature via separate stab wounds and tunneled subcutaneously across the lateral ventral thorax; the distal portions of the leads were secured in positions that approximated those of the lead II of a standard electrocardiogram (ECG). Body heat was maintained both during and immediately following the surgery. All animals were allowed 7-10 days to recover from the surgery and reestablish circadian rhythms. Using a remote receiver (DataART2.1: Data Sciences International, Inc., St. Paul, MN), ECG waveforms were continuously acquired and saved during the 5-min baseline period and aconitine challenge, which did not last longer than 40mins. HR was obtained from the ECG.
ECGAuto software (EMKA technologies USA, Falls Church, VA) was used to visualize individual ECG signals, analyze and quantify ECG segment durations and identify cardiac arrhythmias. Using ECGAuto, P wave, QRS complex, and T wave were identified for individual ECG waveforms and compiled into a library and used for analysis of all experimental ECG traces. The following parameters were determined for each ECG waveform: PR interval, QRS duration, QT corrected for HR (QTc) using Bazett's formula, and ST interval. The Lambeth conventions (Walker et al. 1988) were used as guidelines for the identification of cardiac arrhythmic events in rats. Arrhythmias were identified as occurring sequentially during aconitine challenge as ventricular premature beats (VPBs), ventricular tachycardia (VT) and ventricular fibrillation (VF).
Heart rate variability (HRV) was also calculated as the mean of the differences between sequential HRs for the complete set of ECG signals. For each 1-min stream of ECG waveforms, mean time between successive QRS complex peaks (RR interval), mean HR, and mean HRVanalysis-generated time-domain measures were acquired. The time-domain measures included standard deviation of the time between normal-to-normal beats (SDNN), and root mean squared successive differences (RMSSD). HRV analysis was also conducted in the frequency domain using a fast-Fourier transform. In this study, the spectrum was divided into low-frequency (LF) and high-frequency (HF) regions. The ratio of these two frequency domains (LF/HF) was calculated as an estimate of the relative balance between sympathetic (LF) and vagal (HF) activity.
B. Diesel exhaust generation and exposure. wDE for exposure experiments was generated using Particle size distributions were characterized during each exposure using an engine exhaust particle sizer (EEEPS,TSI Inc.,model 3090,St. Paul,MN). Chamber temperatures, relative humidity, and noise were also monitored, and maintained within acceptable ranges.

II. Tables
A. Supplemental Material, Table 1 (Hazari et al. 2009), for the current study, VF was marked as a random (variable amplitude and frequency) ECG waveform with no clear P-wave or QRS complex, and a wandering baseline.

B. Supplemental Material, Figure 2.
Supplemental Material, Figure 2. Diesel exhaust mediated increase in heart rate is only prevented by blocking TRPA1. One day following exposure, SH rats exposed to high wDE or fDE (A.), or low wDE or fDE (B.) had slightly higher HR when compared to controls. Pre-treatment with the TRPA1 antagonist prevented the increase in HR due to low wDE, but the TRP antagonist and TRPV1 antagonist caused HR to become further increased (C.). Guanethidine treatment of low wDE-exposed rats caused a slight increase, and vagotomy or atropine caused a significant increase in HR (D.). Values are mean ± SEM; * significantly different from controls; p < 0.05, n = 5-6.
Supplemental Material, Figure 3. Exposure to diesel exhaust causes a prolongation of ventricular depolarization and shortening of repolarization in the ECG. High DE and Low DE -SH rats exposed to high wDE or fDE, or low wDE or fDE, had an increase in QRS duration and decrease in ST segment duration when compared to air-exposed controls. DE and TRP -Treatment of rats with the TRPA1, TRP or TRPV1 antagonist before low wDE prevented the increase in QRS duration, and decrease in ST segment duration. DE and Autonomics -Vagotomy and atropine only prevented the increase in QRS duration caused by low wDE. Guanethidine treatment only partially reversed the decrease in ST segment duration caused by low wDE. Values are mean ± SEM; * significantly different from controls; p < 0.05, n = 5-6.