Fig. 4: The infarct-limiting effect of metoprolol is time-of-day dependent.

A Mouse model of myocardial ischemia–reperfusion injury (IRI) induced at different times of the day (ZT 1-5, ZT 8-13, and ZT 18-22) for the estimation of area-at-risk and infarct size (NA/AAR, %) by Evans Blue and TTC staining. WT mice were treated 10 min before reperfusion by i.v. injection of vehicle (0.9% NaCl) or metoprolol (12.5 mg/kg). B Representative images of 1-mm-thick transverse LV slices showing AAR (negative for Evans Blue) and the infarcted area (extent of necrosis; TTC-negative area) after IRI induced at different times of day. C Histological analysis of IS in mice subjected to IRI and randomized to receive vehicle or metoprolol before reperfusion. Vehicle, n = 12 for ZT1; n = 10 for ZT 9-13; n = 8 for ZT 18-22. Metoprolol, n = 10 for ZT 1-5; n = 9 for ZT 9-13; n = 8 for ZT 18-22. D Flow cytometry analysis of neutrophil infiltration of the LV 24 h after induced IRI in LysM-GFP reporter mice at ZT 1-5 (control, n = 5; metoprolol, n = 4) and ZT 18-22 (control, n = 5; metoprolol, n = 6). LysM-GFP⁺Ly6G⁺ cells were counted in dispersed LV cells. Representative plots are shown. E Flow cytometry analysis of co-aggregate formation between neutrophils and platelets (Ly6G⁺CD41⁺ cells) in peripheral blood 24 h after induced IRI in LysM-GFP reporter mice at ZT 1-5 (control, n = 9; metoprolol, n = 6) and ZT 18-22 (n = 5 per condition). Representative plots are shown. F Myocardial IRI model in mice lacking Adrb1 in neutrophils (Mrp8-Cre^-/+ Adrb1^FLOX/FLOX, KO) and littermates (Mrp8-Cre^-/- Adrb1^FLOX/FLOX, WT). G Representative images of Evans Blue and TTC staining in 1 mm-thick transverse LV slices. Histological analysis of (H) AAR (% LV) and (I) IS (NA/AAR, %) in mice lacking Adrb1 (blue) or littermates (red) 24 h after induced IRI at ZT 1-5 (n = 10 per condition) and ZT 18–22 (n = 4 for WT; n = 9 for KO). Squares represent individual mice. (C–E, H, I) Data are presented as mean ± SD and compared by 2-tailed unpaired Student’s t-test or Mann-Whitney test, for normally or non-normally distributed data, respectively. Differences were deemed statistically significant at P < 0.05. AAR, area-at-risk; IS, infarct size (NA/AAR, %); LV left ventricle, NA necrotic area, TTC triphenyl tetrazolium chloride, WT Wild-type, ZT Zeitgeber time. Source data are provided as a Source Data file.