Role of β-adrenergic signaling in the development of Porphyromonas gingivalis lipopolysaccharide-induced cardiac dysfunction in mice

Figures

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  Fig. 1. Experimental procedure and effects of PG-LPS on body weight and cardiac muscle mass. (A) Experimental protocol. Male 12-week-old C57BL/6 mice were divided into four groups, normal control group (C), PG-LPS-treated group (L), a propranolol-treated group (P), and PG-LPS plus PPL (L + P) group, and various parameters of cardiac function were evaluated. (B) Consumed amounts of food and water were similar among all four groups during the experimental period. NS, not significantly different from the Control (P > 0.05) by ANOVA/Tukey–Kramer. (C) All groups showed similar body weight changes at 1 week after PG-LPS infusion. P = NS, not significantly different from the Control (P > 0.05) by ANOVA/Tukey–Kramer. (D) All groups showed similar values of cardiac muscle (CM) weight per body weight ratio at 1 week after PG-LPS infusion, NS, not significantly different from the Control (P > 0.05) by ANOVA/Tukey–Kramer. Data are presented as means ± SD and open circles show individual data.

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  Fig. 2. Effects of PG-LPS on LF/HF, nHF, mean HR and SDNN. (A) Experimental timeline. ECG was recorded for 24 h at 1 day before the PG-LPS treatment (Baseline) and at 1, 3 and 7 days after the PG-LPS treatment. (B) nLF, an index of sympathetic nervous activity, was significantly greater at all time points in the PG-LPS group, compared to the baseline. **P < 0.01 (Baseline vs. PG-LPS + 1 day; Baseline vs. PG-LPS + 3 days; Baseline vs. PG-LPS + 7 days) by one-way repeated-measures analysis of variance (ANOVA)/Bonferroni post hoc test. (C) nHF, an index of parasympathetic nervous activity, was significantly smaller at all time points in the PG-LPS group, compared to the baseline. **P < 0.01 (Baseline vs. PG-LPS + 1 day; Baseline vs. PG-LPS + 3 days; Baseline vs. PG-LPS + 7 days) by one-way repeated-measures analysis of variance (ANOVA)/Bonferroni post hoc test. (D) LF/HF, an index of sympatho-vagal balance, was significantly smaller at all time points in the PG-LPS group, compared to the baseline. **P < 0.01 (Baseline vs. PG-LPS + 1 day; Baseline vs. PG-LPS + 3 days; Baseline vs. PG-LPS + 7 days) by one-way repeated-measures analysis of variance (ANOVA)/Bonferroni post hoc test. (E) Mean HR…

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  Fig. 3. Effects of PG-LPS on time course of changes in HR and changes of mean HR at nighttime versus daytime. (A) Time course of changes in HR during daytime (8:00 AM to 8:00 PM) and nighttime (8:00 PM to 8:00 AM) for 48 h determined by ECG measurement at 1 day before (baseline; PG-LPS -1 day) and 7 days (LPS 7d; PG-LPS + 7 days) after PG-LPS treatment. **P < 0.01 by paired Student t-test (Baseline vs. LPS 7d). (B) Comparison of difference in HR (delta HR) between daytime and nighttime at 1 day before the PG-LPS treatment (baseline) and at 7 days after PG-LPS treatment (LPS 7d). **P < 0.01 by unpaired Student t-test. (C) Plasma chromogranin A levels at 7 days after PG-LPS treatment (L) was significantly greater than that in the control (C). **P < 0.01 by unpaired Student t-test. (D) TH phosphorylation at Ser-40 in the heart at 7 days after PG-LPS treatment (L) was significantly greater than that in the control. **P < 0.01 by unpaired Student t-test. Data are presented as means ± SD and open circles show individual data.

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  Fig. 4. Effects of PPL on PG-LPS-induced heart fibrosis and cardiac myocyte apoptosis. (A) Representative images of Masson-trichrome-stained sections of cardiac muscle in the Control (C; upper left), PG-LPS (L; upper right), PPL (P; lower right) and PG-LPS + PPL (L + P; lower right) groups. Scale bar = 50 μm. (B) The area of fibrosis was significantly increased in the L group (n = 5), but this increase was suppressed in the L + P group (n = 5). **P < 0 .01 by ANOVA/Tukey–Kramer. (C) Expression of α-SMA (normalized to GAPDH expression) as a marker of cardiac fibrosis was significantly increased in the P group (n = 7), but this increase was suppressed in the L + P group (n = 7). (D) Representative images of TUNEL-positive nuclei (black-brown) in TUNEL-stained sections of cardiac muscle. TUNEL-positive nuclei were counted in the Control (C; upper left), PG-LPS (L; upper right), PPL (P; lower left) and PG-LPS + PPL (L + P; lower right) groups. Scale bar = 20 μm. Black arrows: TUNEL-positive myocytes. (E) The number of TUNEL-positive nuclei was significantly increased in the L group (n = 4), but this increase was suppressed in the L + P group (n = 4). **P < 0.01 by ANOVA/Tukey–Kramer. (F) The…

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  Fig. 5. Effects of PPL on PG-LPS-induced oxidative stress in the heart. (A) Representative images of 8-OHdG-immunostained sections of cardiac muscle in the Control (C; upper left), PG-LPS (L; upper right), PPL (P; lower left) and PG-LPS + PPL (L + P; lower right) groups. Scale bar = 20 μm. Black arrows: 8-OHdG-positive myocytes. (B) The number of 8-OHdG-positive myocytes was significantly increased in the L group (n = 4), but this increase was suppressed in the L + P group (n = 4). **P < 0 .01 by ANOVA/Tukey–Kramer. (C) Expression of XO (normalized to GAPDH) was significantly increased in the L group (n = 10), but this increase was suppressed in the L + P group (n = 10). **P < 0 .01 by ANOVA/Tukey–Kramer. (D) Expression of NOX4 (normalized to GAPDH) was significantly increased in the L group (n = 10), but this increase was suppressed in the L + P group (n = 10). **P < 0 .01 by ANOVA/Tukey–Kramer. Data are presented as means ± SD and open circles show individual data.

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  Fig. 6. Effects of PPL on RIP3, CaMKII, RyR2, PLB in the heart. (A) RIP3 expression (normalized to GAPDH) was significantly increased in the L group, and this increase was significantly attenuated in the L + P group. **P < 0 .01 by ANOVA/Tukey–Kramer. (B) CaMKII phosphorylation (Thr-286) was significantly increased in the L group, and this increase was significantly attenuated in the L + P group. **P < 0 .01 by ANOVA/Tukey–Kramer. (C) CaMKII oxidation (Met-281/282) was significantly increased in the L group, and this increase was significantly attenuated in the L + P group. **P < 0 .01 by ANOVA/Tukey–Kramer. (D) RyR2 phosphorylation (Ser-2814) was significantly increased in the L group, and this increase was significantly attenuated in the L + P group. *P < 0 .05, **P < 0 .01 by ANOVA/Tukey–Kramer. (E) PLB phosphorylation (Thr-17) was significantly increased in the L group, and this increase was significantly attenuated in the L + P group. **P < 0 .01 by ANOVA/Tukey–Kramer. (F) NLRP3 was significantly increased in the L group, and this increase was significantly attenuated in the L + P group. **P < 0 .01 by ANOVA/Tukey–Kramer. Bar plots represent means ± SD and open circles show…

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  Fig. 7. Proposed role of β-AR signaling in the heart of PG-LPS-treated mice. β-AR/Gsα/AC signaling is activated by the PG-LPS treatment, leading to ROS production through XO and NOX4 and Ca2+ leakage, which mediates RyR2 phosphorylation at Ser-2814 and PLB phosphorylation at Thr-17 via activation of ROS/RIP3/CaMKII signaling. These changes might cause activation of NLRP3 inflammasome pathway, leading to fibrosis, myocyte apoptosis, oxidative stress and cardiac dysfunction. β-AR, β-adrenoreceptor; Gsα, α subunit of Gs; AC5, type 5 adenylyl cyclase; SERCA, sarcoendoplasmic reticulum ATPase; SR, sarcoendoplasmic reticulum; RyR2, ryanodine receptor 2; PLB, phsopholamban; NLRP3, NOD-like receptor family, pyrin domain-3.