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Am J Physiol Regul Integr Comp Physiol 295: R28-R37, 2008. First published May 7, 2008; doi:10.1152/ajpregu.00070.2008
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Physiological and Molecular Mechanisms Implicated in the Neural Control of Circulation

Dynamic time-varying analysis of heart rate and blood pressure variability in cats exposed to short-term chronic intermittent hypoxia

Sergio Rey,1,2 Mika P. Tarvainen,3 Pasi A. Karjalainen,3 and Rodrigo Iturriaga1

1Laboratorio de Neurobiología, Facultad de Ciencias Biológicas and 2Departamento de Nefrología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile; and 3Department of Physics, University of Kuopio, Kuopio, Finland

Submitted 30 January 2008 ; accepted in final form 2 May 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic intermittent hypoxia (CIH) contributes to the development of hypertension in patients with obstructive sleep apnea and animal models. However, the early cardiovascular changes that precede CIH-induced hypertension are not completely understood. Nevertheless, it has been proposed that one of the possible contributing mechanisms to CIH-induced hypertension is a potentiation of carotid body (CB) hypoxic chemoreflexes. Therefore, we studied the dynamic responses of heart rate, blood pressure, and their variabilities during acute exposure to different levels of hypoxia after CIH short-term preconditioning (4 days) in cats. In addition, we measured baroreflex sensitivity (BRS) on the control of heart rate by noninvasive techniques. To assess the relationships among these indexes and CB chemoreflexes, we also recorded CB chemosensory discharges. Our data show that short-term CIH reduced BRS, potentiated the increase in heart rate induced by acute hypoxia, and was associated with a dynamic shift of heart rate variability (HRV) spectral indexes toward the low-frequency band. In addition, we found a striking linear correlation (r = 0.97) between the low-to-high frequency ratio of HRV and baseline. CB chemosensory discharges in the CIH-treated cats. Thus, our results suggest that cyclic hypoxic stimulation of the CB by short-term CIH induces subtle but clear selective alterations of HRV and BRS in normotensive cats.

spectral analysis; baroreflex; carotid body

AUTONOMIC DYSREGULATION HAS been linked to chronic intermittent hypoxia (CIH) in animal models (22, 23) and is thought to be involved in the generation of hypertension and increased cardiovascular mortality in humans with obstructive sleep apnea (OSA) (42). Indeed, repetitive hypoxic episodes in OSA patients potentiate the cardiovascular and sympathetic responses induced by acute hypoxic stimulation of peripheral chemoreceptors (16, 33, 35) and impair the autonomic regulation of arterial blood pressure (29, 30) and the renin-angiotensin-aldosterone system (28).

The most currently accepted model for CIH-induced hypertension portrays carotid body (CB) chemoreceptor activation as one of the important mechanisms of ventilatory and cardiovascular potentiation to acute hypoxia (33). In fact, the cardioventilatory response to acute hypoxia in man and mammals is almost completely dependent on CB chemoreflexes(10, 15). Recently, Peng et al. (41) found that CIH increases the chemosensory responses of the rat CB to acute hypoxia. In the same line, we studied the early effects of CIH on cat chemosensory responses to acute hypoxia and found that cats exposed to CIH for 4 days showed enhanced CB chemosensory and ventilatory responses to acute hypoxia (43). However, the CB-mediated effects of CIH on cardiovascular hypoxic responses have been less extensively studied.

Heart rate (HRV) and blood pressure (BPV) variability indexes have been used as predictors of cardiovascular (5, 21, 35) and all-cause mortality (53). Spectral analysis of HRV depicts two major oscillatory components defined as a low-frequency (LF) band, thought to be related to combined sympathetic and parasympathetic influences (9), and a high-frequency (HF) band related to vagal influences and the respiratory sinus arrhythmia (9, 52). Thus, it has been proposed that the LF-to-HF ratio (LF/HF) of HRV and BPV are spectral indexes of the sympathovagal balance on heart rate (52) and blood pressure (20, 52), respectively. Spectral analysis has shown that acute hypoxia increases the LF component of HRV and BPV, which, in turn, is linearly related to peripheral sympathetic discharge frequency (37). Moreover, normotensive patients with recently diagnosed OSA showed an increased LF/HF ratio of HRV and BPV, associated with increased peripheral sympathetic discharges (29). Recently, Lai et al. (22) found that CIH increases arterial blood pressure, the LF component of BPV, and the LF/HF ratio of HRV in rats. These changes were parallel to a reduction of spontaneous baroreflex sensitivity (BRS) on heart rate. Thus, the available data suggest that CIH-induced hypertension is linked to an autonomic dysregulation, but it is not well known whether the changes of cardiovascular variability might precede the increase in arterial blood pressure and how they relate to CB chemosensory discharges. Therefore, we studied the changes induced by short-term CIH on the regulation of HRV and BPV in normotensive cats exposed to a protocol of brief exposure to CIH, which has been shown to enhance CB chemosensory and ventilatory responses to acute hypoxia (43). Accordingly, we measured the effect of various levels of inspired PO2 on heart rate and arterial blood pressure, along with HRV and BPV to assess short-term CIH induced cardiovascular changes in a noninvasive manner. In addition, we explored BRS over the control of heart rate in the same model.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Experiments were done in 30 male adult cats [3.5 ± 0.7 kg, mean (SD)]. The experimental protocols were approved by the Ethical Committee of the Facultad de Ciencias Biológicas of the Pontificia Universidad Católica de Chile and were performed according to the Guiding Principles for the Care and Use of Animals of the American Physiological Society.

Exposure to CIH. Twelve awake cats were housed in a cylindrical chamber, alternately flushed with 100% N2 and compressed air using timed solenoid valves (43). This gas alternation reduced the PO2 inside the chamber to a minimum of ~75 mmHg in 104 ± 1 s and returned to normoxia in 279 ± 2 s (n = 10 cycles); maintaining a PO2 below 100 mmHg for ~60 s. This hypoxic pattern was repeated nine times per hour during 8 h/day during four days. In situ experiments were done the morning of the day after the end of hypoxic exposures. As a control group, 10 cats were kept in normoxic conditions. Because noise produced by solenoid valves may induce startling reflexes that influence cardiovascular and autonomic responses (4), we also exposed eight cats to sham-CIH inside the chamber during 4 days, replacing N2 with compressed air.

Anesthesia and recordings of physiological variables. All surgical procedures and measurements were performed under anesthesia with sodium pentobarbital (40 mg/kg ip). Supplementary doses (8–12 mg iv) were applied to maintain a surgical level of anesthesia (stage III, plane 2) assessed by the absence of withdrawal reflexes to strong pressure on the paws, with persistence of patellar reflexes. At the end of the experiments, cats were euthanized with an overdose of anesthetic. Recordings were done as previously described (43). Cats were placed in a supine position, and the rectal temperature was maintained at 38.0 ± 0.5°C with a heated pad. The trachea was cannulated with a polyethylene tube to record the airflow signal with a pneumotachograph (model no. 00; Fleish, Lausanne, Switzerland), and the end-tidal CO2 pressure (PETCO2) was measured with a capnograph (Poet II; Criticare Systems, Waukesha, WI). One saphenous vein was cannulated for administration of drugs, and arterial blood pressure was recorded from the femoral artery with a pressure transducer (Statham P23; Oxnard, CA). The ECG was continuously recorded using the II lead. Data were fed to an analog-digital system (PowerLAB/8SP; AD Instruments, Castle Hill, Australia) and acquired in a computer. Respiratory rate (fR), breath-by-breath minute inspiratory volume (Vbb) and tidal volume (VT) were digitally obtained from the airflow signal. Heart rate (fH) was obtained from the ECG, and mean arterial pressure (Pa) from the arterial blood pressure recording. Data were analyzed using the Chart 5.5 software (AD Instruments). We studied the effect of short-term CIH on the cardiovascular (Pa, fH) variables at baseline (normoxia) and the responses elicited by several isocapnic levels of inspired PO2 (~20, 37, 74, 111, 370, and 740 mmHg), maintained for 1 min. We chose these inspired PO2 levels because they cover the same range that we used previously to study the effects of CIH on CB chemosensory discharges (43). Normally, cats were exposed to PO2 levels from hyperoxia to hypoxia, but we did not find any differences in the responses when the challenges were randomly applied. During the hypoxic challenges, there was no evidence of hypoxic ventilatory decline.

Carotid body chemosensory recordings. After cardiovascular recordings, the CB chemosensory discharge was recorded, as previously described (43). In brief, one carotid sinus nerve and the ipsilateral ganglio-glomerular nerves were dissected and cut. The carotid sinus nerve was placed on a pair of platinum electrodes and covered with warm paraffin oil. The electroneurogram was preamplified (Grass P511; Grass Instruments, West Warwick, RI), bandpass filtered (10–500 Hz), amplified and fed to an electronic amplitude discriminator to assess the frequency of CB chemosensory discharges (f{chi}), expressed in Hertz. We normalized f{chi} to the maximum inhibition (f{chi}= 0 Hz) produced by breathing 100% O2 (Dejour's tests: a standard intervention). Carotid sinus barosensory activity was eliminated by crushing the common carotid arterial wall between the carotid sinus and the CB. The effectiveness of this maneuver was confirmed by the disappearance of large barosensory discharges associated with systolic blood pressure in the electroneurogram.

Cardiovascular variability signal acquisition and processing. We followed the guidelines from the Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology (52). Accordingly, 480s ECG and Pa recordings were digitally acquired at 2,000 Hz for processing with the Chart 5.5 software (AD Instruments). Baseline recordings were performed at the beginning of the experiments for HRV, BPV, and BRS analyses. Ectopic beats (extrasystoles) were deleted from the recording and interpolated. Cardiovascular responses to different inspired PO2 levels were recorded and used for time-varying analysis of HRV and BPV. Data were exported in ASCII format for processing with custom routines written in Matlab environment (Mathworks, Natick, MA), which were used to convert R-R interval (RRI) and systolic blood pressure (SBP) signal values to simultaneous RRI and SBP time series.

Spontaneous baroreflex sensitivity and baroreflex effectiveness index. BRS over heart rate was calculated using the sequence and spectral methods, which have been shown to be in good agreement with the pharmacological methods for baroreflex estimation (39). For the sequence method, we analyzed the RRI and SBP time series and searched for groups of sequentially increasing or decreasing RRI associated with increases or decreases in SBP (2). The up sequences were analyzed separately from down sequences. The criteria for sequences selection was 1) linear correlation coefficient higher than 0.85; 2) RRI changes above 0.5 ms; 3) SBP change above 1 mmHg; and 4) sequences equal or longer than three beats. The average slope of the regression lines provided the estimation of the BRS in ms/mmHg. In addition, we calculated the baroreflex effectiveness index (BEI), measured as the number of up or down SBP sequences associated with lengthening or shortening of RRI respectively, divided by the total of up or down SBP ramps in the recording (7). The spectral method is based on the calculation of BRS as the spectral transfer function between HRV and BPV in a specific frequency band, which requires the estimation of the coherence function between RRI and SBP. The spectral transfer function between HRV and BPV was calculated as the {alpha}-index described by Pagani et al. (38). The limits of agreement between measurements of BRS and BEI were assessed using the method described by Bland and Altman (3).

HRV and BPV calculation. The baseline cardiovascular variability and time-varying analysis of RRI and SBP were performed using customized HRV analysis software developed in Matlab environment (Mathworks, Natick, MA). The software used is based on a previous version of the HRV analysis software (34) developed by the Biosignal Analysis and Medical Imaging Group, Department of Physics, University of Kuopio, Finland.

Analysis parameters. We calculated time and frequency domain indexes of HRV and BPV. The bands for frequency domain analysis of cardiovascular data were adjusted according to the physiological ranges of fH and fR in cats. In all animals, fH was between 180 and 260 min–1 and fR was in the range of 10–50 min–1 (equivalent to 0.167–0.833 Hz). This fR range included the maximum values of fR attained during hypoxia. Hence, the HF band was fixed between 0.150 and 0.833 Hz, and the LF band was fixed between 0.025 and 0.150 Hz, according to DiRienzo et al. (6). Both RRI and SBP time series were transformed to evenly sampled time series by using a cubic spline interpolation with a 4-Hz sampling rate.

Short cardiovascular registers lack sufficient resolution to accurately estimate the variability of the very-low-frequency (VLF < 0.025 Hz) components. These trendlike components affect the analysis parameters in an undesirable way when only the short-term variability is of interest, and thus they were removed by using a smoothness priors based detrending method (51). This method functions as a time-varying finite-impulse response high-pass filter. The cutoff frequency of the filter was set to 0.04 Hz.

The frequency-domain parameters were assessed through parametric spectral estimation based on an autoregressive model. The optimal order of the model was determined by using three different selection criteria; final prediction error, Akaike's information criteria, and minimum description length (11). On the basis of these criteria the model order was set at 20.

HRV analysis. At baseline, time-domain measurements were the standard deviation of R-R intervals (SDRR) and systolic blood pressure (SDSBP). Frequency-domain measurements of HRV and BPV included the absolute powers of VLF, LF, and HF frequency bands (expressed in ms2 and mmHg2) and center frequencies for each band in Hertz. For analyses, the powers of LF and HF bands were transformed into normalized units (nu), that is, the absolute power values were divided by the total spectral power subtracted by the VLF power. In addition, the ratio between the LF and HF powers, that is, LF/HF, was calculated.

Time-varying analysis. The time-varying analysis of RRI and SBP time series was performed using a short-time Fourier transform (also known as spectrogram) for time-varying spectrum estimation. In this method, a window is slid over the whole recording and at each window location, a spectrum estimate is calculated by using fast Fourier transform. Here, a 60-s window was used for obtaining the time-varying spectrum. All spectral indexes were calculated for each window, as well as the maximum LF/HF ratio, which refers to the largest ratio among these consecutive spectra.

Statistical analysis. All data were expressed as means ± SE, unless otherwise indicated. Paired comparisons between two groups were done using the Wilcoxon paired-rank test. The differences between three or more groups were assessed with the Kruskal-Wallis test, followed by post hoc comparisons with the Dwass-Steel-Critchlow-Fligner test. Heart rate and blood pressure changes induced by different PO2 levels were assessed with two-way ANOVA of the logarithm-converted measurements followed by post hoc comparisons with the Bonferroni test. Linear regression analysis was performed on the normalized logarithm-converted f{chi}, LF, HF, LF/HF, and BRS data. F-tests were used to determine whether slopes were statistically different from zero. All analyses were done with the StatsDirect v.2.6.2 software (StatsDirect, Cheshire, UK). Statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of CIH on baseline physiological variables. We did not detect statistical differences in the baseline physiological variables measured among control, sham-CIH, and CIH cats (P > 0.05), except fR, which was lower in the CIH-exposed cats (Table 1, P < 0.05). There seemed to be a slightly higher basal VT in the CIH group; however, this apparent difference did not attain statistical significance. Moreover, Vbb was unchanged in the CIH-exposed cats (P > 0.05).


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  		Table 1. Effects of short-term CIH on baseline physiological values

 
Baroreflex sensitivity and levels of agreement among methods. Table 2 summarizes the effect of short-term CIH on spontaneous BRS evaluated with the sequence and spectral methods. Baroreflex sensitivity was reduced in CIH-treated cats compared with control and sham-CIH animals when up sequences were analyzed (P < 0.01). The {alpha}-index was calculated in the frequency range in which the squared coherence modulus between RRI and SBP was maximum and above 0.7 (k2 = 0.86 ± 0.03 at 0.43 ± 0.03 Hz; n = 30). Values of the {alpha}-index were significantly lower in the CIH-treated group (P < 0.01, Table 2), but we did not detect differences within BRS down sequences. The BEI for up and down sequences was not different among all groups (P > 0.05, Table 2). Bland-Altman analysis showed statistically significant levels of agreement and strong correlation coefficients between BRS, {alpha}-index, and BEI, with the exception of the comparison between BRSD and the {alpha}-index method that showed a lower correlation coefficient (Table 3).


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  		Table 2. Baroreflex sensitivity and effectiveness indexes in cats exposed to short-term CIH

 

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  		Table 3. Levels of agreement between sequence and spectral indexes of baroreflex sensitivity using the Bland-Altman method (n = 30)

 
Cardiovascular variability at baseline. Fig. 1 shows the effects of short-term CIH on the HRV and BPV baseline values. The overall HRV was reduced in CIH-treated cats, as shown by SDRR values (P > 0.05, Fig. 1B). Exposure to CIH did not change the center frequencies in the LF band (P > 0.05, not shown). However, CIH-treated cats showed lower central frequencies in the HF band (0.326 ± 0.037 Hz, P < 0.01) compared with control and sham-CIH animals (0.467 ± 0.041 Hz and 0.506 ± 0.030 Hz, respectively). For comparison of the center frequencies between control and CIH cats, see the example in Fig. 1, C and D. We found a linear correlation between the central frequency of the HF band of HRV and fR (r = 0.95, P < 0.0001) in all pooled experiments (n = 30). The absolute variability of the HF band was reduced in the CIH-treated group (0.16 ± 0.04 ms2), compared with control (1.61 ± 0.44 ms2) and sham-CIH cats (1.21 ± 0.26 ms2; n = 8–12; P < 0.05). In contrast, absolute LF band variabilities were not statistically different among the three experimental groups (0.30 ± 0.09 ms2, control; 0.25 ± 0.09 ms2, sham-CIH; 0.14 ± 0.02 ms2, CIH; n = 8–12; P > 0.05). Normalized LF and HF variabilities showed that CIH increased HRV in the LF band and reduced HF variability (Fig. 1E), thus increasing LF/HF from 0.33 ± 0.09 and 0.35 ± 0.12 in control and sham-CIH cats to 1.09 ± 0.15 in the CIH-treated group (n = 8–12, P < 0.05). On the contrary, CIH did not modify either the time (Fig. 1B) or the frequency domain measurements of BPV at baseline (Fig. 1, D and F). Center frequencies of each BPV band yielded identical results compared with HRV analyses (see examples in Fig. 1, C and D).


Figure 1
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  		Fig. 1. Effects of short-term chronic intermittent hypoxia (CIH) on heart rate variability (HRV) and blood pressure variability (BPV). A: example of simultaneous recording of R-R interval (RRI) and systolic blood pressure (SBP). B: for time-domain analysis, standard deviation of RRI (SDRR) and SBP (SDSBP); For spectral analysis, representative power spectral density graphs of HRV (C) and BPV (D) are shown in one control (continuous line) and one CIH-treated cat (segmented line). Summary of the spectral analysis of HRV (E) and BPV (F). PSD, power spectral density; LF, low-frequency band; HF, high frequency band. *P < 0.05, {dagger}P < 0.01 CIH vs. control and sham-CIH; Dwass-Steel-Critchlow-Fligner post hoc test after Kruskal-Wallis comparisons (n = 8–12).

 
Relationship between baseline chemosensory discharges and heart rate variability spectral indexes. Baseline f{chi} was higher in the CIH-treated group compared with sham-CIH and controls (67.8 ± 9.7 Hz vs. 37.0 ± 5.6 Hz and 38.1 ± 4.0 Hz, respectively; P < 0.05, n = 6). A statistically significant linear correlation (r > 0.85, P < 0.05) was found between baseline f{chi} and LF/HF, LF and HF only in the CIH-treated cats (Fig. 2, right). This correlation was positive between f{chi}, LF, and LF/HF and negative between f{chi} and HF (Fig. 2). On the contrary, in the control and sham-CIH groups, the correlation coefficients were low and lacked statistical significance (Fig. 2, left and middle).


Figure 2
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  		Fig. 2. Correlation between basal carotid body chemosensory discharges (f{chi}) and spectral indexes of HRV in six control (left), sham-CIH (middle), and CIH-treated cats (right). Low-frequency band (LF; top), high-frequency band (HF; middle); LF-to-HF ratio (LF/HF; bottom); r, correlation coefficient; nu, normalized units; segmented line, 95% confidence interval of the regression lines.

 
Effect of acute hypoxia on cardiovascular variables. fH and Pa were expressed as variations from the baseline (fH, Pa). The reduction of inspired PO2 from 740 to 20 mmHg increased fH and FormulaPa in all experimental groups (Fig. 3, A and B; P < 0.001). In addition, we found that the tachycardic responses were larger in the CIH-treated group. Two-way ANOVA showed enhanced overall fH in the CIH-treated group compared with control and sham-CIH (Fig. 3A, P < 0.001). Bonferroni test showed that the tachycardia induced by severe hypoxia (PO2 ~22–37 mmHg) was more pronounced in the CIH-exposed cats, compared with control and sham-CIH groups (P < 0.01). Despite the significant pressor effect of decreasing inspired PO2 (P < 0.001), no differences were detected in the FormulaPa response among the experimental groups (P > 0.05; Fig. 3B).


Figure 3
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  		Fig. 3. Effect of different inspired PO2 on (A) and FormulaPa (B). A progressive reduction of PO2 increased fH and FormulaPa in all experimental groups (P < 0.001). Comparison of CIH, sham-CIH, and control data within each PO2 showed larger tachycardic responses to acute hypoxia in CIH-treated cats. {dagger}P < 0.01; {ddagger}P < 0.001 CIH vs. control and sham-CIH; Bonferroni test after two-way ANOVA (n = 8–12).

 
Dynamic time-varying analysis of heart rate and blood pressure variability. The dynamic study of the power spectral density of HRV and BPV using time-varying analysis showed that acute severe hypoxia increases the center frequency of the HF band, consistent with an increase of fR (tachypnea) in all animals (Fig. 4, A and B). Spontaneous gasps produced interruptions in the variability of the HF band (Fig. 4, A and B). The decrease of inspired PO2 increased LF and HF power spectral density. When the relationship between the spectral bands was studied at different PO2, we found that decreasing inspired PO2 increases LF/HF, indicating a predominance of LF band variability on HRV (Fig. 5A) and BPV (Fig. 5B) recordings (P < 0.001, two-way ANOVA). However, short-term CIH selectively potentiated HRV hypoxic responses since the maximum LF/HF values over time were significantly higher in the CIH-treated cats compared with control and sham-CIH at a PO2 range within 22 and 150 mmHg (Fig. 5A, P < 0.05). In contrast, time-varying analysis of BPV data did not detect any difference among the three experimental groups (Fig. 5B, P > 0.05).


Figure 4
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  		Fig. 4. Dynamic time-varying analysis of HRV and BPV during acute hypoxia in one control cat. Power spectral density is coded with a pseudocolor scale. Notice that spontaneous gasps (triangles) elicit changes in the HF band of both HRV (A) and BPV (B). Acute hypoxia increased the central frequency of the HF band and power of both LF and HF bands in addition to the increments of heart rate and arterial pressure. PO2 levels: {square}, normoxia (150 mmHg); {blacksquare} hypoxia (22 mmHg).

 

Figure 5
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  		Fig. 5. Effect of different PO2 on the maximum LF/HF of HRV (A) and BPV (B). The progressive reduction of inspired PO2 increased maximum LF/HF of HRV and BPV over time in all experimental groups (P < 0.001). Comparison of CIH, sham-CIH, and control data within each PO2 showed larger increases in the maximum LF/HF of HRV elicited by acute hypoxia in CIH-treated cats, without affecting BPV. *P < 0.05; {dagger}P < 0.01; CIH vs. control and sham-CIH; Bonferroni test after two-way ANOVA (n = 8–12).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of this study showed that exposure of cats to cyclic episodes of mild hypoxia (PO2 ~75 mmHg) repeated during 8 h for 4 days: 1) decreased baroreflex sensitivity; 2) decreased total HRV predominantly reducing HF band absolute variability, resulting in predominance of the LF band; 3) did not affect BPV; 4) induced a linear coupling between baseline CB chemosensory discharges and spectral indexes of HRV; and 5) enhanced the tachycardic responses to acute severe hypoxia, without affecting the pressor response.

Previously, we found that cats exposed to CIH for 4 days presented enhanced chemosensory and ventilatory responses to acute hypoxia (43). In this study, we show that baseline cardiovascular and ventilatory parameters in CIH-treated cats were similar to normoxic controls and sham-CIH-treated groups (see Table 1). However, fR was lower in the CIH-treated cats, without changes in Vbb due to an increased—but not statistically significant—tidal volume. Similar Vbb values in all groups may seem to be contradictory with an increased f{chi} during normoxia in CIH-treated cats. However, we previously showed that the enhancement of the CB ventilatory chemoreflex was evident in the hypoxic range (PO2 < 150 mmHg) with little effect of the increased f{chi} on basal fR ventilation, probably due to the action of other chemosensory buffer systems (i.e., aortic bodies and central chemoreceptors) (43). In fact, fR has a poor relationship with CB chemosensory drive in cats, since hypoxic CB chemostimulation is followed by increased VT rather than tachypnea (46), as opposed to the rat model, in which CB stimulation results in a simultaneous increase of both VT and fR (26). We did not find evidence of transient posthypoxic ventilatory (VT and fR) decline after acute hypoxic exposure in any group, thus bradypnea in the CIH-treated cats may be the result of vagally mediated control such as the Hering-Breuer reflex, which can be reduced by hypoxia or hypercapnia (25).

Despite the effects of short-term CIH on HRV described in this study, it is worth to note that the physiological significance of the spectral components of HRV is a matter of active debate (24, 40). In fact, the main methodological caveat of the present study is that we did not measure sympathetic or parasympathetic discharges to the heart, precluding the direct assessment of the relationship between the spectral HRV bands and cardiac autonomic control. Moreover, this relationship seems to be dependent on the model used and methodologies involved (40). Most evidence supporting the hypothesis that spectral HRV and BPV bands represent indexes of autonomic control of circulation, comes from animal and human studies in which BPV or HRV changes were induced by manipulation of autonomic cardiovascular control through drug administration or physiological stimuli (40). Pagani et al. (37) found a positive correlation between the LF component of HRV/BPV and muscle sympathetic nerve activity. In addition, Kuo et al. (19) found a high correlation between the HF component of HRV and the vagus nerve activity in anesthetized rats. Lusina et al. (23) measured the hypoxic ventilatory response in healthy humans exposed to a CIH for days and found a direct relationship between the magnitude of the hypoxic ventilatory response and the sympathetic peroneal nerve activity, which was attributed to an augmented CB chemosensory drive after returning to normoxia. In addition, several studies have found a combination of cardiovascular adjustments, such as increased arterial blood pressure and heart rate (13, 23) or augmented pressor and tachycardic responses to acute hypoxia (18), depending on each particular CIH protocol. In the present study, we found that short-term CIH induced a reduction in overall HRV without changes in BPV or Pa. When power spectral density was expressed as normalized units, we detected a shift in the LF/HF relationship toward the LF band, due to a reduction of the HF band variability. A novel finding of this study, using dynamic time-varying analysis of HRV, was that short-term CIH selectively enhanced the maximum LF/HF values over time during acute hypoxia challenges. It is known that repetitive hypoxic episodes induce hyperventilation, increasing stroke volume, producing tachycardia (49), and sympathetic activation (22, 44). Present data do not allow us to ascertain the underlying mechanisms of the LF/HF changes hereby described. However, we found that the maximum value of LF/HF ratio over time increased as the inspired PO2 was reduced. Moreover, to our knowledge, this is the first study that shows direct measurements of CB chemosensory discharges in relation to spectral indexes of HRV and BPV. A striking finding in our study was that a high linear relationship (r = 0.97) between basal f{chi} and LF/HF ratio of HRV was found only in the CIH-treated cats. Although a high correlation is not indicative of a cause-effect relationship, these results suggest that potentiation of CB chemosensory discharges may be linked to early changes in the reflex control of heart rate in cats exposed to short-term CIH, before any alteration in baseline arterial pressure or BPV. Dick et al. (8) showed that brief exposure of rats to CIH induces a parallel potentiation of phrenic and splanchnic sympathetic discharges, suggesting that the CIH-induced enhancement of cardioventilatory and sympathetic responses is coupled probably at the level of the central nervous system, since this potentiation is prevented by serotonin antagonist administration (27). Our time-varying analysis data agree with and extend our previous findings (43), which were obtained using a fast Fourier transform method to study baseline HRV. It is worthwhile to mention that recent reports show that reduced overall HRV and increased LF spectral power are associated with increased mortality (53), hypertension (36), and sympathetic overactivity (12, 36). Moreover, studies performed in humans with OSA detected an increased peripheral chemoreflex drive (31, 32) and reduced HRV with LF predominance (29). Interestingly, Narkiewicz et al. (29) reported that patients with mild OSA presented decreased overall HRV without changes in BPV, while patients with moderate to severe OSA showed increased BPV. None of the OSA patients studied were hypertensive. Thus, HRV and BPV data hereby presented indicate a clear effect of CIH in cats that resembles what is observed in OSA patients.

Baroreflex activation attenuates hyperventilation (14) and sympathetic activation (50) induced by acute hypoxia in humans, providing a physiological negative feedback counteracting hypoxia-induced sympathoexcitation. Lai et al. (22) found decreased BRS over heart rate and increased LF components of HRV and BPV in rats exposed to CIH for 5 days. In their experiments, the Pa of rats became significantly elevated (~15 mmHg) after 5 days of CIH exposure, characterized by a hypoxic pattern that reduced the inspired O2 fraction to 2–6% 48 times/h during 6 h. As opposed to this study, our data show that cats exposed to mild hypoxic episodes (PO2 ~ 75 mmHg) repeated during 8 h for 4 days present early changes in HRV and decreased BRS over heart rate without alterations in baseline Pa and fH. Thus, cardiovascular effects induced by periodic hypoxia seem to be highly dependent on the animal model, as well as the pattern and severity of the hypoxic episodes. The length of exposure to CIH necessary to produce a persistent hypertensive state might also vary among preparations and animal models.

We performed all experiments in anesthetized animals because it was necessary for recording CB chemosensory discharges in situ. However, it is worth noting that anesthesia may influence HRV, BPV, baroreflex indexes, and sympathetic outflow to target organs; highlighting an important difference between this study and the others mentioned above. Particularly, pentobarbital anesthesia can induce autonomic depression (54) and reduction of the arterial baroreflex gain (47). Therefore, despite the fact that our HRV data show similar values to awake cats (1), we cannot rule out a depressor effect of pentobarbital sodium. Nevertheless, clinical assessment of the depth of anesthesia did not show apparent differences among our experimental groups.

Previously, we reported that exposure of cats to short-term CIH for 4 days did not potentiate fH or Pa changes induced by acute severe hypoxia (43), comparing the absolute values between CIH-treated and control cats (n = 6). In this report, we further evaluated fH and FormulaPa at seven different PO2 levels in control, sham, and CIH-treated cats and found that only CIH-treated cats showed a significantly enhanced {Delta}fH induced by acute severe hypoxia (PO2 ~22 mmHg) without changes of FormulaPa. Interestingly, other studies have shown that tachycardia predicts later development of hypertension and indicates increased sympathetic tone in normotensive (17, 45) and essential hypertensive patients (17). Our data suggest that acute hypoxia-induced tachycardia is an early event in the cardiovascular adjustments to short-term CIH preconditioning in cats.

Perspectives and Significance

The major goal of this study was to assess the early cardiovascular changes after exposure to short-term CIH in cats. We studied the tachycardic and pressor effects of graded levels of hypoxia after CIH preconditioning for 4 days using direct recordings of CB chemosensory activity and simultaneous measurements of HRV, BPV, and BRS. Our results suggest that CB chemoreflex activation by brief exposure to CIH induces early selective changes in the regulation of heart rate, HRV, and BRS, without modifying the baseline arterial blood pressure and BPV in anesthetized cats. We found a linear correlation between baseline CB chemosensory discharges and the spectral indexes of HRV only in the CIH preconditioned cats. Therefore, it is likely that periodic hypoxic stimulation of the CB may contribute to modulate the spectral indexes of HRV and enhance the hypoxic tachycardic response. Our data obtained from the dynamic time-varying HRV analysis showed that spectral indexes of HRV and BPV change proportionally to the intensity of the acute hypoxic challenges. Strikingly, these findings resemble the alterations observed in normotensive patients with mild OSA (29), who are at increased risk for hypertension and share a shift of LF/HF with essential and borderline hypertensive patients (17). However, because we did not measure sympathetic discharges, and according to the controversy around the physiological significance of spectral analysis of cardiovascular variability (24, 40), we cannot conclude that potentiation of CB chemoreflexes by CIH is the cause of changes in heart rate and blood pressure oscillations in cats. Nevertheless, our data strongly suggest that CB stimulation by short-term CIH is associated with clear and selective changes in acute hypoxia-induced tachycardia, HRV, and BRS on the control of heart rate. Future efforts should focus on investigating the physiological mechanisms underlying these changes and to ascertain the cardioventilatory modifications induced by CIH.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Chilean National Fund for Scientific and Technological Development Grants 1030330 and 1070584.

Current address of S. Rey: Vascular Biology Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, 733 N Broadway St., Suite 663, Baltimore, MD 21205, USA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Iturriaga, Laboratorio de Neurobiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile (e-mail: riturriaga{at}bio.puc.cl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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