Exercise Training Attenuates Acute β-Adrenergic Receptor Activation-Induced Cardiac Inflammation via the Activation of AMP-Activated Protein Kinase

Figures

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  Figure 1. Exercise training could inhibit ISO-induced cardiac inflammation: (A) a pattern diagram of the experimental exercise training protocol; (B,C) representative immunostainings and quantifications of Mac-3 (macrophage marker) in the hearts on the third day after ISO treatment (bar = 500 μm); (D–F) the concentrations of MCP-1 (D), MCP-5 (E) and IL-18 (F) in the myocardium of the mice, which were determined using ELISA kits. Note: ISO, isoprenaline; WT, wild-type; Running, exercise training; MCP-1, monocyte chemoattractant protein-1; MCP-5, monocyte chemoattractant protein-5; IL-18, interleukin-18; n = 6. The data are the mean ± SEM from a one-way ANOVA with a Tukey’s post-hoc test.

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  Figure 2. Exercise training inhibited the ISO-induced activation of NLRP3 inflammasomes and increases in ROS and ICAM-1 production: (A) the Western blot analysis of pro-caspase-1, caspase-1 and NLRP3 levels in WT mice heart tissue; (B–D) the quantitative analysis of the relative protein expression of (B) pro-caspase-1, (C) caspase-1 and (D) NLRP3 in the mouse heart tissue; (E,F) (E) the fluorescent microscopy of representative DHE staining for ROS levels in the left ventricle (LV) sections from wild-type mice (bar = 50 μm) and (F) the quantification of the fluorescence intensity of DHE in the heart sections; (G,H) (G) the fluorescent microscopy of representative staining for ICAM-1 levels in the left ventricle (LV) sections from wild-type mice (bar = 50 μm) and (H) the quantification of the fluorescence intensity of ICAM-1 in the heart sections. Note: ISO, isoprenaline; Running, exercise training; WT, wild-type; LV, left ventricle; CD31, vascular endothelial cell maker; ICAM-1, intercellular adhesion molecule 1; n = 6. The data are the mean ± SEM from a one-way ANOVA with a Tukey’s post-hoc test.

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  Figure 3. Exercise could activate AMPK, and AMPK activation could inhibit ISO-induced ROS production and NLRP3 inflammasome activation in NMCMs: (A,B) (A) the Western blot analysis of the protein levels of phosphorylated and total AMPK in wild-type mice under sedentary or exercise training conditions and (B) the quantification of phosphorylated AMPK relative to total AMPK; (C) a pattern diagram of the experimental protocol for the NMCMs (the NMCMs were pretreated with an AMPK activator (1 mM of metformin), AMPK inhibitor (0.1 μM of compound C) or AMPK activator (metformin) + AMPK inhibitor (compound C) for 30 min and then treated with ISO (10 μM) for 1 h); (D–G) (D) the protein levels of p-AMPK, NLRP3 and caspase-1 (p20) at 1 h after ISO (10 μM) treatment in mouse cardiomyocytes that were pretreated with the AMPK activator (1 mM of metformin) or AMPK inhibitor (0.1 μM of compound C) for 30 min and the quantitative analysis of the relative protein expression of (E) p-AMPK, (F) NLRP3 and (G) caspase-1 in the NMCMs; (H,I) the average ROS signal intensity in the NMCMs, which was measured after DHE staining (bar = 100 μm). Note: ISO, isoprenaline; Met, metformin; Comp C, compound C; Running,…

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  Figure 4. AMPKα2−/− mice failed to reduce ISO-induced cardiac inflammation: (A) the protocol that was used for the evaluation of the effects of exercise training on ISO-induced cardiac inflammation in AMPKα2−/− mice; (B,C) Mac-3 (macrophage marker) staining, which was used to calculate the area of macrophage infiltration (compared to the sedentary group, the area of macrophage infiltration did not decrease in AMPKα2−/− mice after exercise training (bar = 500 μm)); (D–F) the concentrations of (D) MCP-1, (E) MCP-5 and (F) IL-18 in the myocardium of the AMPKα2−/− mice, which was determined using an ELISA. Note: ISO, isoprenaline; Running, exercise training; MCP-1, monocyte chemoattractant protein-1; MCP-5, monocyte chemoattractant protein-5; IL-18, interleukin-18; n = 6. The data are the mean ± SEM from a one-way ANOVA with a Tukey’s post-hoc test.

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  Figure 5. Exercise training reduced ISO-induced cardiac inflammation in an AMPK-dependent manner: (A–D) heart tissue was collected and Western blotting was used to detect and quantitatively analyze the relative protein expression of (B) pro-caspase-1, (C) caspase-1 and (D) NLRP3 in the heart tissue; (E,F) (E) the fluorescent microscopy of representative DHE staining for ROS levels in the left ventricle (LV) sections from AMPKα2−/− mice (bar = 50 μm) and (F) the quantification of the fluorescence intensity of DHE in the AMPKα2−/− sections (n = 6); (G,H) (G) the fluorescent microscopy of representative staining for ICAM-1 levels in the left ventricle (LV) sections from AMPKα2−/− mice (bar = 50 μm) and (H) the quantification of the fluorescence intensity of ICAM-1 in the heart sections. Note: ISO, isoprenaline; Running, exercise training; WT, wild-type; LV, left ventricle; CD31, vascular endothelial cell maker; ICAM-1, intercellular adhesion molecule 1; n = 6. The data are the mean ± SEM from a one-way ANOVA with a Tukey’s post-hoc test.

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  Figure 6. The working model showing how exercise training alleviated ISO-induced cardiac inflammation by activating AMPK. Note: β-AR, β-adrenergic receptor; p-AMPK, phosphorylated AMP-activated protein kinase; ROS, reactive oxygen species; NLRP3, NLR Family, pyrin domain-containing 3; ASC, apoptosis-associated speck-like protein containing a CARD (caspase activation and recruitment domain); IL-18, interleukin-18.