Endotoxin depresses heart rate variability in mice: cytokine and steroid effects

Abstract

Heart rate variability (HRV) falls in humans with sepsis, but the mechanism is not well understood. We utilized a mouse model of endotoxemia to test the hypothesis that cytokines play a role in abnormal HRV during sepsis. Adult male C57BL/6 mice underwent surgical implantation of probes to continuously monitor electrocardiogram and temperature or blood pressure via radiotelemetry. Administration of high-dose LPS (Escherichia coli LPS, 10 mg/kg, n = 10) caused a biphasic response characterized by an early decrease in temperature and heart rate at 1 h in some mice, followed by a prolonged period of depressed HRV in all mice. Further studies showed that LPS doses as low as 0.01 mg/kg evoked a significant decrease in HRV. With high-dose LPS, the initial drops in temperature and HR were temporally correlated with peak expression of TNFalpha 1 h post-LPS, whereas maximal depression in HRV coincided with peak levels of multiple other cytokines 3-9 h post-LPS. Neither hypotension nor hypothermia explained the HRV response. Pretreatment with dexamethasone prior to LPS significantly blunted expression of 7 of the 10 cytokines studied and shortened the duration of depressed HRV by about half. Interestingly, dexamethasone treatment alone caused a dramatic increase in both low- and high-frequency HRV. Administration of recombinant TNFalpha caused a biphasic response in HR and HRV similar to that caused by LPS. Understanding the role of cytokines in abnormal HRV during sepsis could lead to improved strategies for detecting life-threatening nosocomial infections in intensive care unit patients.

Fig. 1.

Effect of vehicle treatment (A, B) and a high dose LPS (C, D) on heart rate. Continuous ECG, temperature, and activity data were acquired via radiotelemetry in 10 mice treated at time 0 with vehicle (sterile NS ip) or high-dose LPS (10 mg/kg ip) A: representative heart rate (HR; bpm) from a single mouse 9 h pre- to 24 h postvehicle administration. Mean HR for each 2-min interval for the 33-h period is shown. B: recording of activity (top, counts/min), HR (middle, bpm) and temperature (bottom, °C) for mouse shown in A from 0 to 12 h after vehicle treatment. C and D: representative heart rate from a single mouse before and after administration of LPS 10 mg/kg ip. Median HR of all mice before and after administration of 10 mg/kg LPS (n = 10) or vehicle (sterile NS, n = 4).

Fig. 2.

LPS causes an early (1 h) decrease in heart rate and temperature in 4 of 10 mice. Continuous ECG and temperature were recorded in 10 mice administered LPS 10 mg/kg ip at time 0. A: heart rate (bpm) and temperature 2 h pre- to 2 h post-LPS in a representative mouse, which showed decrease in HR and temperature 30–60 min after LPS. B: median heart rate (bpm) for each 2-min interval 2 h pre- to 2 h post-LPS in individual mice (gray lines) and average of all mice (black line) C: temperature curve for the same mice shown in B (gray lines) and average temperature of all mice (black line). Four of ten mice had decreased temperature <35°C and concurrent decrease in HR in the early period following LPS administration.

Fig. 3.

Time- and frequency-domain analyses show comparable endotoxin-induced depression of heart rate variability (HRV). ECG data were collected 24 h pre- and post-LPS in 10 mice administered 10 mg/kg LPS and analyzed using time- and frequency-domain methods. Median values for every 30-min interval are shown. Units are as follows: R-R interval (ms); SDNN, Standard deviation of normal R-R intervals (ms); coefficient of variance (SDNN/mean R-Ri); pNN6: percentage of normal R-R intervals differing from the previous one by >6 ms; low-frequency power (0.4–1.5 Hz) (AU); and high-frequency power (1.5–4 Hz) (AU).

Fig. 4.

LPS effect on heart rate variability is dose dependent. A: median standard deviation of normal R-R intervals (SDNN) normalized to pre-LPS baseline (“1” indicated by horizontal line). Mice received vehicle or low-, medium-, or high-dose LPS (0.01, 1, and 10 mg/kg ip; n = 4–10 each). B: dose-response curve. Median normalized SDNN from 1.5–2 h following injection of LPS at doses ranging from 0.001 to 10 mg/kg (n = 4–10 per group). Vehicle-treated group is represented by leftmost point. *P < 0.05 at each LPS dose vs. vehicle.

Fig. 5.

LPS-induced hypotension and decreased heart rate variability are not correlated. Blood pressure and heart rate were continuously measured in mice before and after administration of LPS 10 mg/kg ip (n = 5). A: mean blood pressure (mmHg) for each mouse from 2 h before to 6 h after LPS administration. B: mean blood pressure vs. standard deviation of normal R-R intervals (SDNN, s) for 6 h after LPS administration in all five mice. There is no significant correlation between blood pressure and SDNN (Spearman's rank correlation coefficient: r = 0.22).

Fig. 6.

Dexamethasone shortens the duration of LPS-induced HRV depression and increases HRV. Mice were given dexamethasone alone 0.7 mg/kg (n = 4), dexamethasone followed 90 min later by LPS (n = 5), LPS 10 mg/kg alone (n = 10), or vehicle (n = 4). A: median SDNN for every 2-min interval 9 h pre- to 24 h post-LPS, normalized to pre-LPS baseline (horizontal line at “1”) B: median low- (0.4–1.5 Hz) and high (1.5–4 Hz) -frequency power spectra from 24 h predexamethasone to 24 h postdexamethasone alone (n = 4). Arbitrary units are shown.

Fig. 7.

Profiling the cytokine response to LPS ± dexamethasone. Cytokines were analyzed in mouse plasma from 0 to 9 h following LPS 10 mg/kg alone (LPS, n = 3–6) or 0.7 mg/kg dexamethasone followed 90 min later by LPS 10 mg/kg (DEX, n = 3). A: hierarchical clustering analysis of 23 cytokines was used to generate a dendrogram depicting the similarity of individual cytokine profiles and identify the cytokines most responsive to LPS. B: time course of expression (pg/ml) of select cytokines to LPS (solid lines) or DEX followed by LPS (dotted lines). Six cytokines peaked at 3 h post-LPS, whereas two peaked at 1 h and two peaked at 6–9 h. *P < 0.05 LPS vs. time 0. **P < 0.05 LPS vs. time 0 and Dex+LPS vs. LPS alone.

Fig. 8.

TNFα causes early (1 h) decrease in heart rate and temperature in 3 of six mice. Continuous ECG and temperature were recorded in six mice administered recombinant murine TNFα 0.2 mg/kg ip at time 0. A: heart rate (bpm) and temperature 2 h pre- to 2 h post-TNFα in a representative mouse, which showed decrease in HR and temperature 30–60 min after TNFα. B: median heart rate (bpm) for each 2-min interval 2 h pre- to 2 h post-TNFα in individual mice (gray lines) and average of all mice (black line) C: temperature curve for the same mice shown in B (gray lines), and average of all mice (black line). Three of six mice had decreased temperature <35°C and concurrent decrease in HR in the early period following TNFα administration.

Fig. 9.

TNFα causes decreased HRV and increased cytokines similar to LPS. A: recombinant murine TNFα 0.2 mg/kg ip was administered to mice undergoing continuous radiotelemetry (n = 6). Median heart rate variability for all mice from 9 h pre- to 24 h post-TNFα. Data points represent median SDNN normalized to pre-TNFα baseline (“1” indicated by horizontal line). B: recombinant murine TNFα 0.2 mg/kg ip was administered to a separate group of mice not undergoing telemetry (n = 6), and blood was drawn 6 h after TNFα administration for quantitation of 10 cytokines. Fold increase at 6 h post-TNFα compared with baseline is shown (means ± SE) *P < 0.05 at 6 h vs. baseline.