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

Cristine J Reitz  1 ,

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

Cristine J Reitz et al. Commun Biol. .

Abstract

Reperfusion of patients after myocardial infarction (heart attack) triggers cardiac inflammation that leads to infarct expansion and heart failure (HF). We previously showed that the circadian mechanism is a critical regulator of reperfusion injury. However, whether pharmacological targeting using circadian medicine limits reperfusion injury and protects against HF is unknown. Here, we show that short-term targeting of the circadian driver REV-ERB with SR9009 benefits long-term cardiac repair post-myocardial ischemia reperfusion in mice. Gain and loss of function studies demonstrate specificity of targeting REV-ERB in mice. Treatment for just one day abates the cardiac NLRP3 inflammasome, decreasing immunocyte recruitment, and thereby allowing the vulnerable infarct to heal. Therapy is given in vivo, after reperfusion, and promotes efficient repair. This study presents downregulation of the cardiac inflammasome in fibroblasts as a cellular target of SR9009, inviting more targeted therapeutic investigations in the future.

Keywords: Cardiovascular diseases; Heart failure.

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Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

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 mice, as compared with mI/R+vehicle, representative image (see Table 1 for all hemodynamics values). f Representative hearts showing cardiac morphology in sham (top panel) and mI/R mice. The SR9009-treated mI/R hearts have less hypertrophy (less equidistant sections; middle panels), as compared with mI/R+vehicle mice (more equidistant sections; bottom panels). Heart sections are collected every 300 µm. Sections are stained with Masson’s trichrome. g The LV wall of sections #5 (s5) and #6 (s6) (from Fig. 1f) are shown with increased magnification to highlight comparisons in the infarct region. Top panel, normal LV wall in sham mice. Middle panel, transmural infarct in LV wall of mI/R hearts, as indicated by the blue-staining region. Bottom panel, animals treated short-term with SR9009 after mI/R have reduced infarct expansion by 8 weeks after mI/R. Scale bar (top right of each paired image) = 500 μm. h Quantification demonstrating that there is significantly (*p < 0.001) less infarct volume and infarct expansion in the SR9009-treated hearts by 8 weeks post-mI/R

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 M2 cells over the adverse M1 macrophages was higher in SR9009-treated mice consistent with the improved outcomes (*p < 0.001, n = 5 hearts/time point/group). See Supplementary Fig. 1 for leukocyte gating, and Supplementary Table 2 for leukocyte values. j To assess T cell responses, we first injected fluorescent particles into the anterior free LV wall at the time of surgery, which are taken up by CD11b+ myeloid cells, and trafficked to the mediastinal lymph nodes (Supplementary Fig. 2). SR9009-treated mice had reduced trafficking to the heart-draining mediastinal lymph nodes by day 1, k resulting in less proliferation of Ki67+ lymphatic CD4+ T cells, and l reduced adverse T cell infiltration in the infarcted myocardium by day 3 (*p < 0.001, n ≥ 4 hearts/group)

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 cytokine mRNA expression nor NLRP3 inflammasome expression as compared with WT littermates (ΔΔCT, *p < 0.05, n = 4 hearts/group). j Nr1d1−/− cardiac fibroblasts in culture exhibited increased expression of Nlrp3, Il-1β, and Il-18 (inflammasome) mRNA levels, consistent with a loss of REV-ERB repressor activity, and k LPS stimulated the inflammasome in cardiac fibroblasts isolated from Nr1d1+/+ or Nr1d1−/− mice, and l SR9009 was most effective at reducing the inflammasome activation in the Nr1d1+/+ cells with a functional REV-ERB target (1/CT, *p < 0.05, n = 4/group). See Supplementary Tables 3, 4, 5, and Supplementary Fig. 3 for genetic (Nr1d1, ClockΔ19/Δ19) loss and gain of function studies

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)

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

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