SR9009 administered for one day after myocardial ischemia-reperfusion prevents heart failure in mice by targeting the cardiac inflammasome

Figures

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  Fig. 1. Short-term pharmacological targeting of REV-ERB after mI/R protects against HF. a Experimental design, left anterior descending coronary artery ligation for 45 min of ischemia followed by reperfusion and SR9009 treatment (i.p. once daily at ZT06 for up to 5 days), and pathophysiologic assessments up to 8 weeks post-mI/R. b All infarcts were reproducibly similar within the first day post-mI/R, as quantified by area at risk:left ventricle (AAR:LV), infarct area:LV (IN:LV), and infarct area:area at risk (IN:AAR) (n = 5 hearts/group). c mI/R mice treated with SR9009 for just 5 days had reduced heart weight (HW) and HW:body weight (BW) vs. mI/R+vehicle controls, at 8 weeks post-mI/R (see Table 1 for details). *p < 0.01 mI/R+SR9009 vs. mI/R+vehicle. d Functional benefits of short-term SR9009 treatment after mI/R include smaller LV diastolic (LVIDd) and systolic (LVIDs) dimensions and better % ejection fraction (% EF) and % fractional shortening (% FS), as compared with mI/R+vehicle, by echocardiography over 8 weeks post-mI/R (see Table 1 for all echocardiography values). *p < 0.0001 mI/R+SR9009 vs. mI/R+vehicle. e Preserved normal pressure–volume (PV) dynamics in SR9009-treated mI/R…

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  Fig. 2. Short-term pharmacological targeting of REV-ERB benefits outcome post-mI/R by limiting inflammatory responses in infarcted myocardium. a SR9009 reduced cardiac Rev-Erbα and Rev-Erbβ mRNA expression and b protein abundance by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot as quantified in vivo, ZT07, *p < 0.05 (n = 4 hearts/group). c SR9009 treatment for 5 days (green line) post-mI/R reduced Rev-Erbα and Rev-Erbβ mRNA expression in infarcted hearts only during the time of treatment, and d cardiac cytokine mRNA expression was also reduced in infarcted hearts, *p < 0.05 (n = 5 hearts/time point/group), see Supplementary Table 1 for RT-PCR values. e Neutrophil recruitment to the infarcted myocardium was reduced with SR9009 treatment vs. mI/R + vehicle by myeloperoxidase (MPO) assay (*p < 0.01, n = 5 hearts/time point/group). f Fewer leukocytes infiltrated infarcted myocardium following SR9009 treatment, as shown by flow-cytometry staining of CD45+ cells, and g less macrophages (CD11b+F4/80+) infiltrated, including both the (h) M1 (CD206−) and M2 (CD206+) subpopulations as compared with mI/R + vehicle controls, and i the proportion of reparative…

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  Fig. 3. SR9009 reduces the adverse NLRP3 inflammasome, and genetic loss of REV-ERB repressor activity. a SR9009 treatment reduced cardiac Nlrp3 mRNA expression, and b protein abundance as early as 1 day post-mI/R as compared with vehicle-treated controls, *p < 0.01. SR9009 treatment also reduced inflammasome cytokines in the heart, including c Il-1β mRNA expression and d IL-1β protein, and e Il-18 mRNA expression and f IL-18 protein abundance versus controls, ZT07, *p < 0.01 (n = 5 hearts/group). g Representative genotyping of homozygous Nr1d1−/−, heterozygous Nr1d1+/−, and WT Nr1d1+/+ mice (top), and wheel running actigraphy (bottom) of mice under diurnal (12 h light:12 h dark; LD) and circadian (constant darkness; DD) conditions. Nr1d1−/− mice exhibited a shortened free-running circadian period, as anticipated. h Homozygous (Nr1d1−/−) mice lacking REV-ERB were not protected by treatment with the REV-ERB agonist SR9009, with no benefits on cardiac structure and function post-mI/R as compared with WT littermates with a functional target, see Supplementary Table 3 for pathophysiology values. i Treatment with SR9009 also showed no benefits on the hearts of Nr1d1−/− mice for inflammatory…

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  Fig. 4. Cellular benefits of SR9009 treatment post-mI/R. a To investigate the role of immune cells, lethally irradiated WT (CD45.1+) mice were reconstituted with bone marrow cells from WT or Nr1d1−/− donor (CD45.2+) mice, generating bone marrow transplant BMTWT→WT or BMTNr1d1−/−→WT chimeras. b Peripheral blood mononuclear cells (PBMCs) and bone marrow cells had >96% reconstitution with CD45.2+ donor cells, at 8 weeks after BMT by flow cytometry (n ≥ 5/group). BMT mice were subjected to mI/R and treatment with SR9009, and c infarct size by day 2 was significantly reduced, and d there was less activation of cardiac inflammasome and cytokine mRNA expression versus vehicle-treated controls, ZT07, *p < 0.05 (n = 5 hearts/group). e Cardiomyocytes treated with SR9009 showed no difference in hypoxia-induced cell death versus vehicle-treated controls (n = 6/group). f LPS activates the inflammasome in cardiac fibroblasts, and co-treatment with SR9009 decreased levels in the cells, and g supernatant, *p < 0.05 (n = 4/group)

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  Fig. 5. Treatment with SR9009 is time-of-day dependent, and treatment for just 1 day leads to improved outcomes post-mI/R. a Mice subjected to mI/R during their sleep time (ZT01-04) or wake time (ZT13-16) were chronotherapeutically treated with SR9009 at time points corresponding to the peak (ZT06) or trough (ZT18) of cardiac REV-ERB expression (see Supplementary Fig. 4). First or subsequent treatments at sleep time (ZT06) were most effective; see Supplementary Table 6 for all pathophysiology values. b Mice were subjected to mI/R (ZT01-04) and treated with SR9009 (ZT06) for 1, 3, or 5 days. Just 1 day of SR9009 treatment was sufficient to benefit outcomes versus vehicle-treated control; see Table 2 for pathophysiology data. c Proposed mechanism for the beneficial effects of targeting REV-ERB post-mI/R. SR9009 enhances REV-ERB repressor activity leading to reduced NLRP3 inflammasome priming and activation in the infarcted myocardium post reperfusion, leading to more efficient repair processes and protecting against HF