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. 2013 Jan 18;132(2):467–477. doi: 10.1093/toxsci/kft008

Hypoxia Stress Test Reveals Exaggerated Cardiovascular Effects in Hypertensive Rats After Exposure to the Air Pollutant Acrolein

Christina M Perez *,✉, Allen D Ledbetter †, Mehdi S Hazari †, Najwa Haykal-Coates ‡, Alex P Carll §, Darrell W Winsett †, Daniel L Costa , Aimen K Farraj †,1
PMCID: PMC3937591  PMID: 23335627

Abstract

Exposure to air pollution increases the risk of cardiovascular morbidity and mortality, especially in susceptible populations. Despite increased risk, adverse responses are often delayed and require additional stress tests to reveal latent effects of exposure. The goal of this study was to use an episode of “transient hypoxia” as an extrinsic stressor to uncover latent susceptibility to environmental pollutants in a rodent model of hypertension. We hypothesized that exposure to acrolein, an unsaturated aldehyde and mucosal irritant found in cigarette smoke, diesel exhaust, and power plant emissions, would increase cardiopulmonary sensitivity to hypoxia, particularly in hypertensive rats. Spontaneously hypertensive and Wistar Kyoto (normotensive) rats, implanted with radiotelemeters, were exposed once for 3h to 3 ppm acrolein gas or filtered air in whole-body plethysmograph chambers and challenged with a 10% oxygen atmosphere (10min) 24h later. Acrolein exposure increased heart rate, blood pressure, breathing frequency, and minute volume in hypertensive rats and also increased the heart rate variability parameter LF, suggesting a potential role for increased sympathetic tone. Normotensive rats only had increased blood pressure during acrolein exposure. The hypoxia stress test after acrolein exposure revealed increased diastolic blood pressure only in hypertensive rats and increased minute volume and expiratory time only in normotensive rats. These results suggest that hypertension confers exaggerated sensitivity to air pollution and that the hypoxia stress test is a novel tool to reveal the potential latent effects of air pollution exposure.

Key Words: spontaneously hypertensive rat, hypoxia, acrolein, blood pressure.

Exposure to air pollution increases cardiovascular morbidity and mortality, especially in individuals with pre-existing cardiovascular diseases (Brook et al., 2010). Epidemiological studies indicate that these effects are not immediate, usually manifesting some time after exposure. For example, Peters et al. (2004) showed that humans exposed to traffic were more susceptible to myocardial infarction up to 24h after exposure. In addition, patients with implanted cardioverter defibrillators had increased incidence of life-threatening arrhythmias up to 2 days after air pollution exposure (Peters et al., 2000). These findings suggest that exposure to air pollution alters cardiovascular physiology and increases conditional susceptibility to triggers of thrombosis and arrhythmia. Moreover, the fact that these effects happen at lower exposure concentrations than those shown to elicit effects in animal studies indicates that the responses are more complicated than the standard monotonic dose-response relationship of traditional toxicology. The reduced capacity to compensate to daily stressors may account for the “lagged” responses associated with air pollution exposure. The study of the factors that determine the capacity to compensate fully presents a potentially informative approach when seeking to uncover and explain the latent effects of air pollution exposure.

A stress test is a common way to reveal enhanced sensitivity or the potential for adverse responses because increased cardiovascular effort causes reduced oxygen availability to tissues and may provoke autonomic imbalance (Goldberger et al., 2006). Numerous studies have shown that stress tests trigger many adverse cardiovascular effects including increased cardiac arrhythmias and deleterious changes in heart rate (HR), heart rate variability (HRV), an indirect indicator of cardiac autonomic tone, and electrocardiogram (ECG) parameters (Watanabe et al., 2001). Recently, exercise stress was used to demonstrate that exposure to diesel exhaust during exercise in men with coronary artery disease caused myocardial ischemia (Mills et al., 2007). Analogous effects, including increases in cardiac arrhythmia and myocardial ischemia, were demonstrated in rats infused with the sympathomimetic dobutamine 1 day after exposure to diesel exhaust (Hazari et al., 2012). In addition, rats exposed to particulate matter or acrolein had exaggerated sensitivity to aconitine, an agent that causes myocardial calcium loading (Hazari et al., 2009). These results are consistent with epidemiological evidence for a time lag in responses and highlight the value of stress tests in unmasking latent responses.

We have previously demonstrated that exposure to both particulate and gaseous air pollutants, including acrolein, causes exaggerated cardiovascular effects in rat models of hypertension and heart failure (Carll et al., 2012; Farraj et al., 2011; Hazari et al., 2009; Lamb et al., 2012). Whereas the mechanisms responsible for these effects are unclear, recent work by Wang et al. (2012) demonstrated that cardiac arrhythmias in particulate-exposed heart failure mice were in part due to altered sensitivity of the carotid body, a key organ involved in oxygen sensing and initiating reflex cardiopulmonary changes to maintain homeostasis. Furthermore, exposures to the air pollutants, tobacco smoke (Adgent, 2006), sulfur dioxide, and nitrogen dioxide (Hoppenbrouwers et al., 1981), have been linked to abnormal cardiopulmonary sensitivity responses to hypoxia. These findings suggest that the response to hypoxia may be useful in unmasking the latent effects of air pollution. The purpose of this study was to (1) examine the utility of a novel hypoxia stress test in revealing the potential latent effects of air pollution and (2) test the hypothesis that acrolein exposure will modify the response to hypoxia in hypertensive rats, but not in normal rats, given that hypertensive individuals have exaggerated sensitivity to low atmospheric oxygen (Yu et al., 1999). HR, blood pressure (BP), ECG, and HRV were measured during acrolein exposure and 1 day later during hypoxia challenge.

Materials and Methods

Animals.

Twelve-week-old male spontaneously hypertensive (SH) (n = 6) and Wistar Kyoto (WKY) normotensive (n = 6) rats (Charles River, Raleigh, NC) were housed in plastic cages (one per cage), maintained on a 12-h light/dark cycle at approximately 22°C and 50% relative humidity in our Association for Assessment and Accreditation of Laboratory Animal Care-approved facility, and held for a minimum of 1 week before exposure. Food (Prolab RMH 3000; PMI Nutrition International, St Louis, MO) and water were provided ad libitum, and all rats were randomized by weight. After all experiments were performed, rats were deeply anesthetized with an ip injection of Euthasol (200mg/kg Na pentobarbital and 25mg/kg phenytoin; Virbac Animal Health, Fort Worth, TX) and euthanized by exsanguination. The Institutional Animal Care and Use Committee of the U.S. Environmental Protection Agency (U.S. EPA) approved all protocols.

Telemeter implantation.

Animals were implanted with radiotelemetry transmitters to monitor HR, BP, and heart rhythms and electrocardiographic intervals (Model TL11M2-C50-PXT; Data Science International, Inc., St Paul, MN). Charles River Laboratories performed all telemetry implantation surgeries in accordance with methods specified by the vendor (Charles River Laboratories, 2005). Animals were shipped to the U.S. EPA 10 days after implantation surgeries, and animals were allowed to recover for 1 week at the U.S. EPA before exposures began.

A separate cohort of SH (n = 5) and WKY (n = 5) rats were implanted with femoral artery catheters in accordance with methods specified by the vendor (Charles River Laboratories, 2005). Animals were shipped to the U.S. EPA within 1 week of surgery. The catheters were flushed with saline, locked with heparin, and plugged immediately upon arrival and every 2 days thereafter until exposure began.

Acrolein exposure and hypoxia stress test.

A paired design was utilized to control for baseline differences in HR, BP, and ECG rhythms between rats. SH and WKY rats were first exposed to air control for 3h and then underwent a hypoxia stress test 24h later. The hypoxia stress test was performed 24h after acrolein exposure to uncover latent responses consistent with the delay between exposure and adverse health effects observed in some epidemiological studies (Peters et al., 2000, 2004). Exposure to subatmospheric oxygen has been used as a stress test in humans with hypertension to demonstrate exaggerated cardiorespiratory responses to hypoxia (Ledderhos et al., 2002). After 5 days, these same rats were exposed to 3 ppm acrolein for 3h and then underwent a second hypoxia stress test 24h later (please refer to Fig. 1A). We assumed that a period of 5 days between the initial hypoxia stress test and the subsequent exposures would allow residual effects, if any, of the initial hypoxia stress test to lapse. This was confirmed by the absence of any differences in air-exposed rats at baseline relative to the previous air and hypoxia exposures.

Fig. 1.

Fig. 1.

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A novel hypoxia stress test may reveal latent sensitivity to a gaseous pollutant. Panel (A) describes the experimental design for the study. Rats were exposed to air for 3h on day 1 and underwent a hypoxia stress test 24h after air exposure. These same rats were given a 5-day washout period and then exposed to 3 ppm acrolein for 3h followed by a hypoxia stress test 24h after acrolein exposure. The hypoxia stress test consists of a 30-min baseline, the descend period (time it takes for FIO2 to decrease from 20 to 10%), a 10-min hypoxia period (10% FIO2, an ascend period—time it takes for FIO2 to increase from 10 to 20%), and a 15-min period after the return to normal FIO2 (20%). Panel (B) represents the hypoxia stress test experimental setup. The rat is placed in a Buxco plethysmograph that sits over a radiotelemetry receiver. Ventilatory, ECG, HR, and BP data are captured during the entire stress test. For hypoxia, nitrogen is metered into the plethysmograph, and an oxygen sensor attached to the plethysmograph records FIO2 in real time.

All exposures (air and acrolein) and the hypoxia stress test took place in whole-body plethysmography (WBP) chambers (Model PLY3213, Buxco Electronics, Inc., Wilmington, NC), which continuously and noninvasively monitor ventilatory parameters in conscious animals. All rats were acclimated to the chambers for 1h two days prior to both air and acrolein exposure. On exposure days, rats were allowed to acclimate to exposure chambers for 30min, and then baseline data were recorded for the next 30min. For air exposure, rats were exposed to filtered air in WBP chambers for 3h. HR, systolic and diastolic BP, ECG waveforms, and ventilatory data were collected during the entire exposure period. After exposure, animals were returned to their home cages. Twenty-four hours after air exposure, animals underwent a hypoxia stress test. Animals were placed in WBP chambers and allowed to acclimate for 30min. A 30-min baseline was then recorded. After baseline measurements, animals were kept at 20% oxygen concentration (ambient O2 concentration) for 5min before the hypoxia stress test. To reduce the PO2 in the chamber, 100% nitrogen (N2) gas was delivered to a glass mixing chamber where the gas was mixed with dry filtered air to achieve a fraction of inspired O2 (FIO2) of 10% at a flow rate of 2 L/min. Oxygen levels were monitored continuously with an O2 sensor attached directly to the chamber, and an FIO2 of 10% was maintained for 10min. Nitrogen was then turned off, and the FIO2 oxygen saturation rose to 20% (Fig. 1B). Rats were then kept in the chambers for an additional 15min. HR, systolic and diastolic BP, ECG waveforms, and ventilatory data were collected continuously during the stress test.

Five days after air exposure and the hypoxia stress test when all latent preconditioning was absent, these same rats were exposed to 3 ppm acrolein for 3h in the same WBP chambers. The concentration of acrolein (3 ppm) is higher than ambient levels, but it may represent concentrations in high combustion areas (Hazari et al., 2008). In addition, cigarette smoke contains up to 90 ppm acrolein (Esterbauer et al., 1991), and acrolein levels in sidestream tobacco smoke are as high as 10 ppm (Esterbauer et al., 1991). Acrolein gas was metered from a 1000 ppm cylinder into a glass mixing chamber where the gas was mixed with dry filtered dilution air to achieve a final concentration of 3 ppm of acrolein with a total flow of 6 L/min. The actual chamber concentration was measured using an HP5890 gas chromatograph (GMI Inc., Ramsey, MN) equipped with manual injection, a flame ionization detector, and a DB-VRX capillary column. The plethysmograph pressure was monitored using Biosystems XA software (Buxco Electronics, Inc.). Using respiratory-induced fluctuations in ambient pressure, respiratory parameters including tidal volume, breathing frequency, inspiratory time, and expiratory time were calculated and recorded on a breath-by-breath basis and averaged over 10-s intervals. HR, systolic and diastolic BP, ECG waveforms, and ventilatory data were collected during the exposure, and animals were returned to their home cages after exposure. Rats underwent a hypoxia stress test 24h later as previously described. Because the data collected were repeated measures, the data during acrolein exposure and the subsequent hypoxia stress test are presented as % change from air exposure and the initial hypoxia stress test, respectively.

Validation of hypoxia stress test.

The hypoxia stress test was validated by measuring arterial blood O2 saturation (SaO2) during hypoxia challenge in a separate cohort of rats. Rats implanted with femoral artery catheters were acclimated to WBP chambers (Model PLY3213, Buxco Electronics, Inc.) for 1h two days prior to hypoxia validation. On the day of validation, the exteriorized catheter was extended with a 1-ml PE50 catheter attached to a 23-gauge adapter and run through a premeasured hole in the wall of the plethysmograph to allow for blood draws outside of the sealed exposure chamber. Rats were acclimated to the exposure chambers for 30min and then underwent a 250-µl blood draw while conscious and unrestrained within the chamber. This served as the baseline blood sample. Nitrogen was metered into the chamber to reduce the FIO2 to 10%, and a second 250-µl blood sample was taken after the FIO2 stabilized at 10%. The addition of N2 was discontinued, and a final 250-µl blood sample was taken 15min after the oxygen content of the chamber stabilized at an FIO2 of 20%. For each blood draw, the pin was removed from the tip of the catheter, and the heparin was allowed to exit the catheter. When only blood remained, a 23-gauge adapter attached to a 1-ml syringe was placed at the end of the catheter and used to draw a 250-µl sample. The blood sample was immediately read on an OPTI CCA-TS Blood Analyzer (OPTI Medical Systems, Inc.) using the OPTI Cassette E-Ca, which measures blood pH, PCO2, PO2, Na+, K+, Ca2+, total hemoglobin, oxygen saturation (SaO2), and hematocrit. The catheter was flushed with saline and locked with heparin after each blood draw.

WBP data acquisition and analysis.

All exposures were performed in WBP chambers (Buxco Electronics, Sharon, CT). The plethysmography methodology permitted continuous monitoring of breathing frequency, tidal volume, minute volume, inspiratory time, and expiratory time. Animals were acclimated to plethysmographs for 1h each day two days prior to exposure before both air and acrolein exposure. Plethysmography chambers (model PLY3213; Buxco Electronics) were calibrated each day before every animal loading. A bias flow regulator delivered fresh air (1.8 L/min) to each cylindrical chamber, preventing CO2 buildup within the chamber. Unrestrained animals were placed in individual cylindrical plethysmograph boxes containing a built-in reference chamber for measuring respiration-induced pressure fluctuations. Data were channeled to computer software (BioSystem XA; Buxco Electronics) that calculated respiratory parameters. Data were collected continuously for each parameter, and automated breath-by-breath analyses were performed using a rejection algorithm to eliminate breaths that were outside a given range. On exposure day, animals were acclimated to the chambers for 30min before any readings were taken. After acclimation, 30min of baseline data were taken, and animals were exposed to either control air or 3 ppm acrolein for 3h. After exposure, rats were removed and returned to home cages.

Radiotelemetry data acquisition.

Radiotelemetry methodology (Data Sciences International, Inc.) enabled constant monitoring of ECG data in unrestrained, unanesthetized rats from implantation until euthanasia. Remote receivers (DataART3.01; Data Sciences International, Inc.) were positioned under the home cages and under the plethysmographs during exposure, which collected the ECG data and transferred them to the computer for storage. In home cages, sixty-second segments of ECG waveforms were acquired and saved at 15-min intervals from surgical recovery through euthanasia not including the exposure period. Pre-exposure baseline data were collected from home cages and a 30-min baseline in exposure cages after a 30-min acclimation period. During the 3-h exposure, sixty-second segments were acquired and saved at 5-min intervals. After exposure, rats were monitored in home cages until euthanasia, approximately 18h after the end of the final hypoxia challenge. HR and BP were automatically obtained from the ECG and BP waveforms, respectively, with data acquisition software (DataART3.01; Data Sciences International, Inc.).

ECG, arrhythmia identification, and HRV analysis.

ECGAuto software (EMKA Technologies, Falls Church, VA) was used for automated analyses of ECG wave amplitudes and segment durations and areas, as well as for the visual identification and enumeration of cardiac arrhythmias. Several parameters were determined for each ECG waveform: PR interval; R amplitude and duration; QRS area; ST interval, amplitude, and area; and T-wave amplitude and area; QT interval; and heart rate–corrected QT interval. ECG parameters during exposure were analyzed in terms of baseline (30min recordings while in the exposure chambers immediately before the beginning of exposure) and hours 1–3 (constituting the entire exposure period between 8:00 a.m. and 11:00 a.m.).

Cardiac arrhythmic events were identified in part by using the Lambeth conventions (Walker et al., 1988) as a guideline for the identification of arrhythmias in rats. Arrhythmias were identified as atrial premature beats, ventricular premature beats, sinoatrial blocks, atrioventricular blocks, or ventricular tachycardia. Arrhythmias were quantified, counted, and totaled over an 18-h period prior to exposure (this corresponded to the same times assessed after exposure), during the 4-h exposure period, or during the 18-h period beginning immediately after exposure. Total arrhythmia counts during exposure were quantified (total of forty-eight 2-min segments during 3-h exposure period) and expressed as counts per minute.

For the analysis of HRV, thorough visual inspection was conducted to identify and exclude arrhythmias, artifacts, and 1-min ECG waveforms lacking distinguishable R waves for more than 30 s. The analysis of HRV generated HR and time-domain measures, including mean time between adjacent QRS complex peaks (the RR interval), a standard deviation of the RR interval (SDNN), SDNN normalized for the effects of HR (SDNN/[RR interval × 100]), and the square root of the mean of squared differences of adjacent RR intervals (RMSSD). The SDNN represents overall HRV, whereas RMSSD represents parasympathetic influence over HR. The analysis of HRV also calculated frequency domain parameters, particularly low frequency (LF) and high frequency (HF), and the ratio of these two frequency domains (LF/HF). LF is generally believed to represent a mixture of sympathetic and parasympathetic tone, whereas HF indicates cardiac parasympathetic (vagal) tone, and LF/HF serves as an index of sympathovagal balance.

Statistics.

The statistical analyses of the Buxco, ECG, and HRV data in this study used SAS version 9.2 software (SAS Institute Inc., Cary, NC). We used PROC MIXED of SAS because it offers greater flexibility for the modeling of repeated measures data than PROC GLM. It is also suitable for analysis of large, unbalanced data with missing data at random. A linear mixed model with restricted maximum-likelihood estimation analysis, least squares means, and repeated measures ANOVA was used to determine which TIME × TRT interactions were statistically significant between baseline and exposure. Multiple comparison adjustment for the p values and confidence limits for the differences between the least squares means was done using adjust = Tukey HSD test. All blood gas data were analyzed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA) with a one-way ANOVA model examining the main effects of each model. All strain comparisons were performed using GraphPad Prism (GraphPad Software, Inc.) with a Student t-test. Trend analysis of SDNN data during exposure and HR data during hypoxia challenge was performed using GraphPad Prism (GraphPad Software, Inc.) with a one-way ANOVA followed by a post hoc test for linear trend between mean and column number. A value of p < 0.05 was considered statistically significant.

Results

Effect of Acrolein on HR, BP, and HRV

HRs increased in SH rats during all 3h of acrolein exposure (Fig. 2A). At hour 1, mean heart rates increased from baseline levels of 294.4±5.4 beats per minute (bpm) to 313.3±15.3 bpm. At hour 2, mean heart rates increased from baseline levels of 269.2±3.9 bpm to 297.7±16.0 bpm. At hour 3, mean heart rates increased from baseline levels of 274.1±6.7 bpm to 305±16.7 bpm. When each acrolein-exposed rat is compared with itself during air exposure, these changes constituted a 10.3±4.22% increase from air at hour 1, a 10.8±4.57% increase from air at hour 2, and a 14.6±3.17% increase from air at hour 3 (p < 0.05; Fig. 2A). HR did not change in WKY rats during acrolein exposure.

Fig. 2.

Fig. 2.

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Acrolein exposure causes increases in HR, mean arterial BP, and the LF component of HRV in SH rats. Panels (A), (B), (C), and (D) relate to HR, BP, LF, and SDNN values at baseline and all 3h of acrolein exposure, respectively. Panels (A), (B), (C), and (D) are presented as % change from air. Percent change baseline values are compared with the average % change values during each hour of exposure. Means and standard errors are reported. Significant differences from baseline percent change values (p < 0.05) are denoted with *. Significant differences (p < 0.05) between SH and WKY strains at each hour are denoted with #. There was a significant negative trend in the SDNN data (p = 0.0275; r 2 = 0.2076).

Before exposures commenced, the baseline BP values for all rats were calculated, and it was determined that SH rats had an average of 44 mmHg higher baseline mean arterial BP level than WKY rats (162.46±3.71 mmHg vs. 118.57±4.26 mmHg, respectively), consistent with their hypertensive phenotype. Mean arterial BP increased during hour 2 of acrolein exposure in SH rats and during hour 3 of acrolein exposure in both SH and WKY rats (Fig. 2B). In SH rats, mean arterial BP increased from baseline levels of 150.20±2.35 mmHg to 169.65±4.04 mmHg at hour 2, constituting a 15.19±2.51% increase from air (p < 0.05; Fig. 2B). At hour 3, mean arterial BP increased from baseline levels of 154.14±2.07 mmHg to 189.32±4.06 mmHg, constituting a 23.67±2.79% increase from air in SH rats (p < 0.05; Fig. 2B). The increases in mean arterial BP at hour 3 were accompanied by 22.86±3.38% and 23.31±3.30% increases in systolic and diastolic BP, respectively. In WKY rats, BP significantly increased from baseline levels of 120.15±2.74 mmHg to 130.24±5.38 mmHg at hour 3, constituting a 14.44±2.55% increase from air (p < 0.05; Fig. 2B).

SH rats also had a significant increase in the LF HRV parameter during hour 3 of acrolein exposure (Fig. 2C). LF increased from baseline levels of 1.55±0.37 to 3.07±0.51ms2, constituting a 109.39±29.74% increase from air (p < 0.05; Fig. 2C). In addition, SH rats had a nonsignificant decrease in SDNN during all hours of acrolein exposure, but there was a significant negative trend in the data (p = 0.0275; r 2 = 0.2076) (Fig. 2D). There were no significant changes in RMSSD, HF, and LF/HF in SH rats, and there were no significant changes in any HRV parameter in WKY rats.

Ventilatory Changes During Acrolein Exposure

SH rats had a significant increase in breathing frequency and minute volume in the third hour of acrolein exposure (Figs. 3A and B). Breathing frequency increased from baseline levels of 101.33±8.86 breaths per minute at baseline to 122.95±12.96 breaths per minute at hour 3, constituting a 34±8.61% increase from air (p < 0.05; Fig. 3A). Minute volume increased from 149.87±11.4ml at baseline to 181.4±20.85ml at hour 3 of acrolein exposure, constituting an increase of 25±4.27% from air (p < 0.05; Fig. 3B). There were no significant changes in ventilatory parameters during acrolein exposure in WKY rats.

Fig. 3.

Fig. 3.

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Acrolein exposure causes significant increases in breathing frequency and minute volume in SH rats. Panels (A) and (B) relate to breathing frequency and minute volume, respectively, measured at baseline, 1, 2, and 3h during acrolein exposure. All values are presented as % change from air. Percent change baseline values are compared with the average % change values during each hour of exposure. Significant differences from baseline percent change values (p < 0.05) are denoted with *. Significant differences (p < 0.05) between SH and WKY strains at each hour are denoted with #.

Electrocardiographic Changes and Arrhythmogenesis During Acrolein Exposure

There were no significant changes in PR interval, QRS interval or area, and ST- or T-wave area in SH and WKY rats exposed to acrolein (Supplementary table I). WKY rats did have a significant increase in the ST segment, but this change occurred during exposure to air and is not related to acrolein exposure (Supplementary table I). There were no significant changes in ECG, HRV, or arrhythmia after either hypoxia challenge. There were no significant changes in the frequency of spontaneous arrhythmia during or after acrolein exposure.

Validation of the Hypoxia Stress Test

All data for hypoxia validation are presented in Table I. A separate cohort of SH and WKY rats implanted with femoral artery catheters underwent the hypoxia stress test, and blood samples were analyzed before exposure to the 10% chamber O2 atmosphere, during exposure to the 10% chamber O2 atmosphere, and after the return to normal 20% chamber O2 concentration. Exposure to 10% chamber O2 atmosphere caused decreases in pO2, pCO2, and arterial blood O2 saturation in both strains of rats. In SH rats, pO2 levels decreased from 98.6±2.42 mmHg at baseline to 50.20±1.71 mmHg during exposure to 10% chamber O2 and increased again to 94.60±4.53 mmHg after O2 levels were returned to 20% (p < 0.05; Table I). In WKY rats, pO2 levels decreased from 99.80±5.16 mmHg at baseline to 54.80±2.60 mmHg during exposure to 10% chamber O2 and increased again to 95.40±1.89 mmHg after O2 levels were returned to 20% (p < 0.05; Table I).

Table 1.

Arterial Blood Parameters During the Hypoxia Stress Test

Exposure Timing pO2 (mmHg) pCO2 (mmHg) SaO2 (%) pH K+ (mmol/l)
SH rat Baseline 98.60±2.42 37.00±1.00 93.60±0.51 7.31±0.033 7.66±1.10
10% chamber O2 50.20±1.71* 31.00±0.84* 79.40±0.68* 7.47±0.044 7.36±0.81
20% chamber O2 94.60±4.53 38.20±1.80 94.20±0.37 7.39±0.048 6.70±0.72
WKY rat Baseline 99.80±5.16 41.40±2.86 94.60±0.68 7.41±0.045 5.70±0.49
10% chamber O2 54.80±2.60* 28.60±1.25* 82.40±0.68* 7.43±0.070 5.80±0.87
20% chamber O2 95.40±1.89 41.80±1.80 93.40±0.40 7.40±0.023 5.76±0.69
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Notes. The hypoxia stress test successfully decreases pO2, pCO2, and blood SaO2 in arterial blood in SH and WKY rats. pH and K+ concentrations are also reported. Means and standard errors are reported.

Significant differences (p < 0.05) are denoted with *.

In SH rats, pCO2 levels decreased from 37.00±1.00 mmHg at baseline to 31.00±0.84 mmHg during exposure to 10% chamber O2 and increased again to 38.20±1.80 mmHg after O2 levels were returned to 20% (p < 0.05; Table I). In WKY rats, pCO2 levels decreased from 41.40±2.86 mmHg at baseline to 28.60±1.25 mmHg during exposure to 10% chamber O2 and increased again to 41.80±1.80 mmHg after oxygen levels were returned to 20% (p < 0.05; Table I).

In SH rats, SaO2 levels decreased from 93.60±0.51% at baseline to 79.40±0.68% during exposure to 10% chamber O2 and increased again to 94.20±0.37% after O2 levels were returned to 20% (p < 0.05; Table I). In WKY rats, O2 saturation levels decreased from 94.60±0.68% at baseline to 82.40±0.68% during exposure to 10% chamber O2 and increased again to 93.40±0.40% after O2 levels were returned to 20% (p < 0.05; Table I).

BP and HR Changes During Hypoxia Stress Test

Hypoxia stress test was initially performed 24h after a 3-h exposure to air. The 10% chamber O2 concentration caused similar increases in HR in both the SH and WKY rats during the hypoxia period but did not affect systolic BP in either strain (data not shown).

Hypoxia stress test after acrolein exposure caused a significant increase in diastolic BP during exposure to 10% chamber O2 in SH rats (p < 0.05; Fig. 4A). Diastolic BP increased from 124.99±5.16 mmHg at baseline to 135.99±2.51 mmHg during exposure to 10% chamber O2, constituting a 12.11±2.23% increase from air (p < 0.05; Fig. 4A). The hypoxia stress test caused a nonsignificant decrease in HR during the descend period, the time during which chamber O2 saturation decreases from 20 to 10%, in the SH rat and a nonsignificant increase in HR during exposure to 10% chamber O2 in WKY rats (Fig. 4B). In addition, SH rats displayed a higher baseline BP than WKY rats during the entire hypoxia stress test.

Fig. 4.

Fig. 4.

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Hypoxia stress test after acrolein exposure causes significant increases in diastolic BP in SH rats. Panels (A) and (B) refer to diastolic BP and HR, respectively, measured at baseline, the descend period, 10% FIO2, the ascend period, and the return to 20% FIO2. All values are presented as % change from hypoxia challenge after air exposure. Percent change baseline values are compared with the average % change values during the different segments of the hypoxia challenge. Means and standard errors are reported. Significant differences from baseline percent change values (p < 0.05) are denoted with *. Significant differences (p < 0.05) between SH and WKY strains at each hour are denoted with #.

Respiratory Changes During Hypoxia Stress Test

Hypoxia stress test following air exposure increased breathing frequency and minute volume in SH and WKY rats during the descent and hypoxia period of the stress test and decreased expiratory time in SH and WKY rats during the descend period (data not shown). The magnitudes of these changes were similar between the two strains.

WKY rats, but not SH rats, showed significant increases in minute volume and expiratory time during hypoxia stress test performed after acrolein exposure. Minute volume increased from baseline levels of 125.00±11.74 to 187.36±9.49ml, constituting a 57.93±23.8% increase from air (p < 0.05; Fig. 5A). Expiratory time increased from baseline levels of 0.30±0.02 to 0.72±0.03 s, constituting a 130.02±31.60% change from air during the ascend period (p < 0.05; Fig. 5B).

Fig. 5.

Fig. 5.

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Hypoxia stress test after acrolein exposure causes significant increases in minute volume and expiratory time in WKY rats. Panels (A) and (B) relate to minute volume and expiratory time, respectively, measured at baseline, the descend period, 10% FIO2, the ascend period, and the return to 20% FIO2. All values are presented as % change from hypoxia challenge after air exposure. Percent change baseline values are compared with the average % change values during the different segments of the hypoxia challenge. Means and standard errors are reported. Significant differences from baseline percent change values (p < 0.05) are denoted with *. Significant differences (p < 0.05) between SH and WKY strains at each hour are denoted with #.

Arrhythmogenesis, Electrocardiographic, and HRV Changes During Hypoxia Stress Test

There were no significant changes in frequency of spontaneous arrhythmia, electrocardiographic, or HRV parameters during the hypoxia stress test after acrolein exposure in SH or WKY rats (Supplementary tables II and III, respectively).

Discussion

This study demonstrates that acrolein exposure alone increases HR, mean arterial BP, breathing frequency, and minute volume in the hypertensive SH rat, with limited effects in the normotensive WKY rat. The cardiovascular responses in the SH rat during acrolein exposure were coupled with increases in the HRV parameter LF indicative of altered autonomic tone. The hypoxia stress test performed after acrolein exposure revealed additional strain-dependent cardiopulmonary responses including increased diastolic BP only in the SH rat and increased minute volume and expiratory time only in WKY rats.

The increases in HR and BP during acrolein exposure in the hypertensive rat may be related to altered autonomic tone during exposure. Chronic exposure to acrolein in mice has been linked to left ventricular dilation (Ismahil et al., 2011) and progression of atherosclerosis (Srivastava et al., 2011), whereas acute exposure to 3 ppm acrolein in this study caused significant increases in HR in SH rats during all 3h of exposure. This was accompanied by increases in BP and the LF component of HRV and, although not statistically significant, a decrease in SDNN. Although the implications of these changes in rats are unclear, similar changes in humans are consistent with elevated sympathetic tone and an increased risk of heart disease (Pope et al., 2001). In addition, iv injection of acrolein causes significant increases in BP that are reversed with guanethidine, a sympatholytic drug (Green and Egle, 1983). Despite these findings, there were no significant changes in LF/HF, a measure of sympathetic and parasympathetic balance. Furthermore, LF alone may not be a reliable indicator of sympathetic tone and may instead reflect an interaction of the sympathetic and parasympathetic nervous systems (Houle and Billman, 1999). Thus, further work is required to confirm linkages with altered autonomic tone. Although WKY rats had a significant increase in BP in the third hour of acrolein exposure, this response was not accompanied by increases in HR or any significant changes in HRV.

The hypoxia stress test revealed exaggerated cardiovascular sensitivity, characterized by increased diastolic BP, only in acrolein-exposed SH rats. This response may be due to increased vascular resistance as in vivo acrolein exposure in SH rats has been shown to increase ex vivo aortic reactivity (Conklin et al., 2006). Hypoxia has been shown to heighten sympathetic activation in healthy men during periods of submaximal exercise (Al Haddad et al., 2012). In addition, chronic intermittent hypoxia has been shown to increase arterial BP and impair vasodilation through increased levels of angiotensin II (Marcus et al., 2012). Carotid body–mediated sensation of low blood SaO2 levels triggers reflex cardiovascular responses including tachycardia, hypertension, and increased cardiac output (Downing et al., 1962). SH rats have larger carotid bodies (Habeck et al., 1985) and are more sensitive to the effects of hypoxia as evidenced by increased carotid sinus nerve activity and intracellular Ca2+ changes (Weil et al., 1998). Thus, differential carotid body sensitivity may in part underlie the differences in responses to acrolein and hypoxia in normotensive and hypertensive rats. Recent work by Wang et al. (2012) demonstrated that particulate matter exposure–induced cardiac arrhythmias in mice with heart failure were in part due to dysregulation of carotid body sensitivity. Further research is required to examine the impact of carotid body sensitivity and the potential effects of air pollutant exposure on carotid body–mediated cardiovascular responses. Although the ventilatory responses to hypoxia after the mock air exposure were similar in SH and WKY rats, only WKY rats exposed to acrolein had significant increases in minute volume and expiratory time during the hypoxia stress test. It is unclear exactly why these ventilatory responses occurred only in WKY rats and not in SH rats, but it may relate to the increased respiratory sensitivity to air pollutant inhalation in the SH rat compared with the WKY rat. Previous studies (Farraj et al., 2011) have shown that SH rats respond with greater airway inflammation and injury after air pollution exposure than WKY rats, and underlying susceptibility in the SH rat may result in the nerve fibers being overloaded and unable to illicit a normal response to acrolein. An acrolein-induced airway lesion in the SH rat may have precluded a normal ventilatory response to hypoxia during the hypoxia stress test (Leikauf et al., 1989).

These findings demonstrate that the hypoxia stress test is a novel approach to reveal latent responses to air pollutants. Researchers are becoming increasingly interested in latent responses to air pollution exposure, and stress tests provide a useful model to study latent effects that would be missed in an acute exposure setting. Traditionally, epidemiological studies have estimated morbidity and mortality within 24h of air pollution events. Although this time frame reveals significant increases in cardiovascular events after air pollution exposure, it may miss equally severe delayed responses that can occur up to 3 days (Faustini et al., 2011) and even 2 weeks after exposure (Goodman et al., 2004). Several stress tests have been used to determine enhanced sensitivity to air pollutants including exercise stress test in humans (Gold et al., 2005), dobutamine stress test in rats (Hazari et al., 2012), and isoproterenol cardiac stress test in rats (André et al., 2011). Intermittent hypoxia has been used to model nocturnal desaturation in sleep apnea patients (Norton et al., 2011). We designed a new hypoxia stress test with one prolonged (10min) acute session of hypoxia followed by a return to normoxic conditions. The effects of the 10% O2 atmosphere were validated by measuring blood O2 saturation in arterial blood drawn before, during, and after hypoxia challenge. Arterial O2 saturation, pO2, and pCO2 decreased significantly during hypoxia in both strains of rats. In addition, pH was not significantly affected by hypoxia challenge, demonstrating that the rat completely compensates for the drop in pCO2, suggesting that the effects are not due to metabolic acidosis. Normal pO2 levels in humans range from 80 to 100 mmHg, and a drop in partial pressure below 60 mmHg is required for hypoxemia. Normal pCO2 levels in the humans range from 35–45 mmHg, and the hypoxia stress test reduced pCO2 levels well below this level. Although the stress test was used in this context to examine susceptibility after air pollution exposure, the data suggest that it can easily be applied to different models to examine overall cardiovascular sensitivity.

The exaggerated effects of acrolein in the SH rats in this study are consistent with our previous studies with other air pollutants indicating enhanced sensitivity in this strain (Farraj et al., 2011; Lamb et al., 2012). In this study, SH rats had an average of 44 mmHg higher BP than WKY rats. It has long been known that hypertension increases the risk of cardiovascular disease and stroke (Brook et al., 2010). In addition to higher baseline BP, SH rats have increased arterial wall thickness (Mulvany and Halpern, 1977) and undergo left ventricular remodeling characterized by hypertrophy, fibrosis, and changes in membrane channels, cellular energetics, and ion regulation that combine to heighten myocardial sensitivity (Bernardo et al., 2010). SH rats were not only more susceptible to HR and BP increases during acrolein exposure, they also had significant increases in breathing frequency and minute volume in the third hour of exposure. Previous studies have found that acrolein, like other pulmonary irritants, causes decreases in respiratory rate (Hazari et al., 2008). It is unclear why SH rats had increased breathing frequency at hour 3 of acrolein exposure, but it is possible that these responses were secondary to the significant increases in HR and BP that were initiated during hours 1 and 2 of acrolein exposure. Minute volume tends to increase with exercise or stress, and it has been shown to increase with acrolein exposures (Linhart et al., 1996). Taken together, the results demonstrate that the SH rat has increased susceptibility to the adverse effects of acrolein exposure, including cardiovascular effects that appear directly and immediately and priming effects through homeostatic control pathways perhaps involving autonomic reflexes that set the stage for latent responsiveness to various stimuli.

The present findings demonstrate that acrolein-induced cardiovascular responses may result from modulation of autonomic tone, specifically increased sympathetic input to the heart. These findings also highlight the utility of the hypoxia stress test as a tool to unmask the potential for latent effects of air pollution via less appreciated mechanisms that mediate cardiovascular dysfunction associated with exposure to air pollution. To further examine the utility of this hypoxia stress test, we will assess the effects of exposure to other gaseous and particulate air pollutants (e.g., ozone and diesel exhaust) in future studies. Finally, the results further show that pre-existing cardiovascular disease confers exaggerated sensitivity to the effects of a model irritant, acrolein, that are consistent with the temporal relationship between exposure and clinical outcomes observed in epidemiological studies showing “delayed or latent” outcomes of air pollutant exposures.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.oxfordjournals.org/.

FUNDING

National Science Foundation Graduate Research Fellowship (to C.M.P.); the EPA-NHEERL/UNC-DESE Cooperative Training in Environmental Sciences & Research (CR83323601 to A.P.C.); the EPA/UNC Toxicology Training Agreement (CR-83515201-0 to A.P.C.).

Supplementary Material

Supplementary Data
supp_132_2_467__index.html (1.2KB, html)

ACKNOWLEDGMENTS

The authors thank the following colleagues at the United States Environmental Protection Agency (Research Triangle Park, NC): Dr Wayne E. Cascio, Dr Bob Luebke, and Dr Robert MacPhail for reviewing the manuscript before submission. We would also like to thank John Havel for his wonderful help in creating the hypoxia stress test image.

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