NLRP3 inflammasome promotes myocardial remodeling during diet-induced obesity

Background: Obesity is an increasingly prevalent metabolic disorder in the modern world and is associated with structural and functional changes in the heart. The NLRP3 inflammasome is an innate immune sensor that can be activated in response to endogenous danger signals and triggers activation of interleukin (IL)-1β and IL-18. Increasing evidence points to the involvement of the NLRP3 inflammasome in obesity-induced inflammation and insulin resistance, and we hypothesized that it also could play a role in the development of obesity induced cardiac alterations. Methods and Results: WT, Nlrp3−/−, and ASC−/− (Pycard−/−) male mice were exposed to high fat diet (HFD; 60 cal% fat) or control diet for 52 weeks. Cardiac structure and function were evaluated by echocardiography and magnetic resonance imaging, respectively. Whereas, NLRP3 and ASC deficiency did not affect the cardiac hypertrophic response to obesity, it was preventive against left ventricle concentric remodeling and impairment of diastolic function. Furthermore, whereas NLRP3 and ASC deficiency attenuated systemic inflammation in HFD fed mice; long-term HFD did not induce significant cardiac fibrosis or inflammation, suggesting that the beneficial effects of NLRP3 inflammasome deficiency on myocardial remodeling at least partly reflect systemic mechanisms. Nlrp3 and ASC (Pycard) deficient mice were also protected against obesity-induced systemic metabolic dysregulation, as well as lipid accumulation and impaired insulin signaling in hepatic and cardiac tissues. Conclusions: Our data indicate that the NLRP3 inflammasome modulates cardiac concentric remodeling in obesity through effects on systemic inflammation and metabolic disturbances, with effect on insulin signaling as a potential mediator within the myocardium.publishedVersio

Sustained Toll-like receptor 9 activation promotes systemic and cardiac inflammation, and aggravates diastolic heart failure in SERCA2a KO mice

Aim Cardiac inflammation is important in the pathogenesis of heart failure. However, the consequence of systemic inflammation on concomitant established heart failure, and in particular diastolic heart failure, is less explored. Here we investigated the impact of systemic inflammation, caused by sustained Toll-like receptor 9 activation, on established diastolic heart failure. Methods and Results Diastolic heart failure was established in 8–10 week old cardiomyocyte specific, inducible SERCA2a knock out (i.e., SERCA2a KO) C57Bl/6J mice. Four weeks after conditional KO, mice were randomized to receive Toll-like receptor 9 agonist (CpG B; 2μg/g body weight) or PBS every third day. After additional four weeks, echocardiography, phase contrast magnetic resonance imaging, histology, flow cytometry, and cardiac RNA analyses were performed. A subgroup was followed, registering morbidity and death. Non-heart failure control groups treated with CpG B or PBS served as controls. Our main findings were: (i) Toll-like receptor 9 activation (CpG B) reduced life expectancy in SERCA2a KO mice compared to PBS treated SERCA2a KO mice. (ii) Diastolic function was lower in SERCA2a KO mice with Toll-like receptor 9 activation. (iii) Toll-like receptor 9 stimulated SERCA2a KO mice also had increased cardiac and systemic inflammation. Conclusion Sustained activation of Toll-like receptor 9 causes cardiac and systemic inflammation, and deterioration of SERCA2a depletion-mediated diastolic heart failure

Quantitative PCR on left ventricle myocardial tissue of mice with HF 8 weeks after SERCA2a gene excision and 4 weeks after initiation of sustained TLR9 stimulation.

<p>(A) CXCL2, Chemokine C-X-C motif ligand 2 (B) CXCL10, Chemokine C-X-C motif ligand 10 (C) MCP-1, Monocyte chemotactic protein-1 (D) TNF, Tumor necrosis factor. Statistics were done using Mann Whitney U- test (n = 7–12 per group). Data are mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01 vs. SERCA2a KO. ^#<i>P</i><0.05, ^##<i>P</i><0.05 vs. control with same intervention.</p

Echocardiographic parameters in SERCA2a KO and control mice 8 weeks after gene excision and 4 weeks after initiation of sustained TLR9 stimulation.

<p>LV, Left ventricle; EF, Ejection fraction; CO, Cardiac output; SV, Stroke volume; d, diastolic; s, systolic; TL, Tibia length (mm); IVS, inter ventricular septum thickness; LVPW, LV posterior wall thickness; LVvol, LV volume; RWT, relative wall thickness (Formula: IVS;d+LVPW;d/ LVID;d); Data are expressed as the mean±SD.</p><p>*<i>P</i><0.05 vs. SERCA2a KO mice.</p><p>^#<i>P</i><0.05</p><p>^##<i>P</i><0.01</p><p>^<i>###</i><i>P</i><0.001 vs. control with same intervention.</p><p>Echocardiographic parameters in SERCA2a KO and control mice 8 weeks after gene excision and 4 weeks after initiation of sustained TLR9 stimulation.</p

Increased mortality and deteriorated cardiac functions and structures in TLR9 stimulated (CpG B) SERCA2A KO mice 8 weeks after gene excision and 4 weeks after initiation of sustained TLR9 stimulation.

<p>(A) Survival analysis: median 59 days in TLR9 stimulated SERCA2a KO mice vs. 64.5 days in SERCA2a KO. Groups were compared using Log rank (Mantel Cox test, n = 16–19 per group, n = 5 in TLR9 stimulated control group). (B) LV inner diastolic diameter/TL (LVID;d, mm). (C) LV inner systolic diameter/TL (LVID;s, mm). (D) Left atrium/TL (mm). (E) Early ventricular filling velocity (e’) (F) LV fractional shortening (LVFS%) (G) Longitudinal strain (%). LVID (B and C), left atrium (D) and LVFS (F) were determined using echocardiography (n = 6–12 per group), and e’ (E) and longitudinal strain (G) were determined using PC-MRI (n = 5–7 per group). TL, tibia length (mm). Statistics were done using Mann Whitney U- test. Data are mean± SEM. *<i>P</i><0.05, **<i>P</i> <0.01, ***<i>P</i><0.001 vs. SERCA2a KO mice. ^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. control with same intervention.</p

Increased number of MAC-2 positive cells in TLR9 stimulated mouse hearts.

<p>(A) Photos taken with 40x objective (scale bar 50μm). (B) MAC-2 image based quantification of MAC-2 positive cells. Statistics were done using Mann Whitney U- test (n = 5–8 per group). Lines and error bars are mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01 vs. SERCA2a KO mice. ^#<i>P</i><0.05 vs. control with same intervention.</p

Organ weights of mice at 8 weeks after SERCA2A gene excision and 4 weeks after initiation of sustained TLR9 stimulation.

<p>(A) Lung weight (mg)/TL (B) Liver weight (mg)/TL (C) Spleen weight (mg)/TL. TL, tibia length (mm). Statistics were done Mann Whitney U- test (n = 7–12 per group). Data are meanxSEM. *<i>P</i><0.05, **<i>P</i><0.01, **<i>P</i> <0.001 vs. SERCA2a KO. ^#<i>P</i><0.05 vs. control with same intervention.</p

Histology of haematoxylin and eosin stained hearts.

<p>(A) Photos taken with 40x objective (scale bar 50μm). (A-B) <i>Cardiac cell stress and/or death</i>: increased nucleus-to-cytoplasm ratio (light blue arrow), swollen cardiomyocyte cytoplasm (dark blue arrow), vacuolization (red arrow), cardiac cell death (green arrow). (A-C) <i>Interstitial leukocyte infiltration</i> (black arrow). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139715#pone.0139715.s006" target="_blank">S2 Table</a> for details. Distribution between the groups was compared using Chi-square test (n = 5–8 per group). *<i>P</i><0.05, **<i>P</i><0.01 vs. SERCA2a KO mice.</p

Phase contrast magnetic resonance imaging in SERCA2A KO and control mice 8 weeks after gene excision and 4 weeks after initiation of sustained TLR9 stimulation.

<p>Long.strain rate, Longitudinal strain rate; Max.rad velocity, Maximum radial velocity; Min. Rad velocity, Minimum radial velocity; Circ.strain, Circumferential strain. Data are expressed as mean±SD.</p><p>Phase contrast magnetic resonance imaging in SERCA2A KO and control mice 8 weeks after gene excision and 4 weeks after initiation of sustained TLR9 stimulation.</p