Recurrent exposure to subclinical lipopolysaccharide increases mortality and induces cardiac fibrosis in mice - PubMed (original) (raw)

Recurrent exposure to subclinical lipopolysaccharide increases mortality and induces cardiac fibrosis in mice

Wilbur Y W Lew et al. PLoS One. 2013.

Abstract

Background: Circulating subclinical lipopolysaccharide (LPS) occurs in health and disease. Ingesting high fatty meals increases LPS that cause metabolic endotoxemia. Subclinical LPS in periodontal disease may impair endothelial function. The heart may be targeted as cardiac cells express TLR4, the LPS receptor. It was hypothesized that recurrent exposure to subclinical LPS increases mortality and causes cardiac fibrosis.

Methods: C57Bl/6 mice were injected with intraperitoneal saline (control), low dose LPS (0.1 or 1 mg/kg), or moderate dose LPS (10 or 20 mg/kg), once a week for 3 months. Left ventricular (LV) function (echocardiography), hemodynamics (tail cuff pressure) and electrocardiograms (telemetry) were measured. Cardiac fibrosis was assessed by picrosirius red staining and LV expression of fibrosis related genes (QRT-PCR). Adult cardiac fibroblasts were isolated and exposed to LPS.

Results: LPS injections transiently increased heart rate and blood pressure (<6 hours) and mildly decreased LV function with full recovery by 24 hours. Mice tolerated weekly LPS for 2-3 months with no change in activity, appearance, appetite, weight, blood pressure, LV function, oximetry, or blood chemistries. Mortality increased after 60-90 days with moderate, but not low dose LPS. Arrhythmias occurred a few hours before death. LV collagen fraction area increased dose-dependently from 3.0±0.5% (SEM) in the saline control group, to 5.6±0.5% with low dose LPS and 9.7±0.9% with moderate dose LPS (P<0.05 moderate vs low dose LPS, and each LPS dose vs control). LPS increased LV expression of collagen Iα1, collagen IIIα1, MMP2, MMP9, TIMP1, periostin and IL-6 (P<0.05 moderate vs low dose LPS and vs control). LPS increased α-SMA immunostaining of myofibroblasts. LPS dose-dependently increased IL-6 in isolated adult cardiac fibroblasts.

Conclusions: Recurrent exposure to subclinical LPS increases mortality and induces cardiac fibrosis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Survival with weekly injections of saline or LPS.

Figure 2

Figure 2. Acute and chronic effects of LPS on left ventricular (LV) size and function by echocardiography.

Panel A: LV fractional shortening and heart rate (beats per minute, bpm) measured before (0 hours), and 6 and 24 hours after i.p. injection of saline (control), LPS 1, 10, or 20 mg/kg (each bar, mean+SEM, n = 6). There was a mild decrease in LV fractional shortening after 6 hours with LPS 10 and 20 mg/kg (*P<0.05 vs LPS 1 mg/kg or control) that resolved by 24 hours. Panel B: LV fractional shortening, heart rate, LV internal diameter in diastole (LVIDd), and LV mass measured after 2 months of weekly i.p. injections of saline (n = 11), LPS 10 mg/kg (n = 8) and LPS 20 mg/kg (n = 12), and after 3 months of weekly i.p. injections of saline (n = 13) or LPS 20 mg/kg (n = 5). There were a few minor changes with LPS 20 mg/kg (*P<0.05 compared with control), but no significant decrease in LV function, LV dilation, or change in LV mass.

Figure 3

Figure 3. Hemodynamics with weekly injections of LPS for 12 weeks.

Tail cuff systolic and diastolic blood pressure (SBP, DBP) and heart rate (HR, beats per minute, bpm) measured in conscious mice before (Pre) and 24 hours after (Post) weekly i.p. injections of saline (n = 7) or LPS (20 mg/kg, n = 6). Data (mean ± SEM) averaged over three weeks at 2 wk (1–3 weeks), 8 wk (7–9 weeks) and 12 wk (weeks 11–13). Hemodynamics were stable with no significant trends from 2 to 12 weeks. There was an interaction between LPS and the Pre-Post injection for SBP (P = 0.044), DBP (P = 0.015) and HR (P = 0.011) (mixed model analysis) reflecting small but consistent differences in HR and BP responses 24 hours after LPS compared with saline.

Figure 4

Figure 4. Bradycardia and high grade block develop just prior to death after weekly LPS.

Telemetry tracings from a mouse that died 57 days after weekly LPS 20 mg/kg i.p. Each strip is 2.5 seconds. The top panel shows the baseline ECG at 10 pm, 18 hours prior to death, with a heart rate of 640 beats per minute (bpm). The mouse had normal activity. The remaining panels show telemetry tracings during last two hours before death. At 3:09 pm, there was a junctional tachycardia at 720 bpm with a shift in the QRS axis, consistent with increased automaticity from another region or fascicle. One hour later at 4:04 pm, sinus rhythm returned (with p waves) at 540 bpm. However 1.5 hours later at 5:34 pm, episodes of high grade block occurred with sustained bradycardia at 384 bpm, then 10 minutes later at 5:44 pm progressive high grade AV block and an idioventricular escape rhythm just prior to death.

Figure 5

Figure 5. Recurrent LPS and arrhythmias prior to death.

Telemetry tracings from a mouse that died 39 days after weekly LPS 10 mg/kg i.p. Each strip is 2.5 seconds. The mouse was in sinus rhythm 552 beats per minute (bpm) at baseline at 1:00 p.m. Two hours later, an accelerated idioventricular rhythm (AIVR) or slow ventricular tachycardia (VT) with fusion complexes with sinus beats developed (3:01 pm), with an AIVR or slow VT at 648 bpm at 3:02 pm. The mouse returned to sinus rhythm at 625 bpm at 4:00 pm. One hour later, there was sinus exit block with slow idioventricular escape rhythm (4:56 pm), and then high degree AV block (5:02 pm) shortly before death.

Figure 6

Figure 6. Cardiac fibrosis induced by weekly subclinical LPS.

Panel A. Picrosirius staining was greater in a mouse injected with LPS 10 mg/kg/week for 12 weeks, compared with a control mouse injected weekly with saline. Panel B. H & E staining showed evidence of myofiber loss with replacement by fibrosis (arrow) after 15 weeks of LPS 20 mg/kg/week.

Figure 7

Figure 7. LPS dose-dependently increased percent collagen fraction area of the left ventricle.

Percent collagen fraction area (mean+SEM) measured by picrosirius staining of the left ventricle increased 12–15 weeks after weekly i.p. injections of low dose LPS (0.5±0.1 mg/kg, n = 15) or moderate dose LPS (12±1 mg/kg, n = 15) compared with saline (control, n = 17) (P<0.001, one way ANOVA). There was a greater increase with moderate dose LPS than with low dose LPS (P<0.05).

Figure 8

Figure 8. LPS dose-dependently increased expression of fibrosis-related genes in the left ventricle.

Expression of fibrosis-related genes in the left ventricle measured by QRT-PCR (mean+SEM) after 12–15 weeks of weekly i.p. injections of moderate dose LPS (12±1 mg/kg, n = 15), low dose LPS (0.5±0.1 mg/kg, n = 12) or saline (control, n = 17). Collagen 1α1, collagen IIIα1, MMP2, MMP9, TIMP1, and periostin increased with moderate dose LPS compared with low dose LPS or saline (P<0.05). Low dose LPS increased MMP9 compared with saline (P<0.05).

Figure 9

Figure 9. LPS increased IL-6 expression in the left ventricle.

Expression of the cytokines IL-1β, IL-6, TNF-α and TGF-β (mean+SEM percent of control) were measured in the left ventricle after 3 months of LPS (20 mg/kg/week i.p, n = 5) or saline (n = 6). There was a significant increase in IL-6 (P<0.001), but no significant change in IL-1β, TNF-α, or TGF-β.

Figure 10

Figure 10. LPS increased IL-6 expression in isolated adult cardiac fibroblasts.

Isolated adult cardiac fibroblasts were exposed to vehicle or LPS in concentrations of 0.1, 1.0, or 10 ng/ml for 48 hours. There was a dose dependent increase in IL-6 (P<0.001, ANOVA, each bar mean+SEM, n = 4). All LPS doses were higher than control and increased significantly with each increment in dose (P<0.05). LPS caused no significant change in TGF-β.

Figure 11

Figure 11. LPS activation of fibrosis-related genes attenuated by AT1R inhibitor losartan.

Expression of fibrosis-related genes in the left ventricle measured by QRT-PCR (mean+SEM) after 15 weeks of weekly i.p. injections of saline (control, n = 7), LPS (10 mg/kg, n = 6), saline with losartan (20 mg/kg/day in drinking water) (n = 6), or LPS with losartan (n = 5). LPS increased LV expression of fibrosis-related genes including collagen Iα1, collagen IIIα1, MMP2, MMP9, TIMP1 and periostin (P<0.05, 2 way ANOVA). Adding the AT1R inhibitor losartan to the drinking water prevented LPS-induced increases in collagen Iα1 and MMP2 and attenuated LPS-induced increases in collagen IIIα1 and MMP9 (P<0.05 for interaction between LPS and losartan), but had no effect on TIMP-1 or periostin. Losartan alone had no effect.

Figure 12

Figure 12. AT1R inhibitor losartan does not attenuate LPS-induced cardiac fibrosis.

Percent collagen fraction area in the left ventricle by picrosirius red staining (mean+SEM, n = 5) increased with LPS (P<0.001, 2 way ANOVA). LPS effects were not affected when the AT1R inhibitor losartan was added to the drinking water (P = 0.80). Losartan alone had no effect. There was no significant interaction between LPS and losartan (P = 0.70).

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This research was supported by the Medical Research Service, Department of Veterans Affairs (0007, P.I. WYWL) [www.research.va.gov], the Veterans Medical Research Foundation, San Diego, California (08254, P.I. WYWL) [www.vmrf.org], and the American Heart Association (11GRNT7610059, P.I. TT) [www.heart.org]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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