Figure legends
Figure 1. Experimental protocols to investigate the effects of pinocembrin on MI/R. (A) Protocol of I/R ex vivo study (1). (B) Protocol of MI/R in vivo study.
Figure 2. Compound pinocembrin significantly improves cardiac contractile recovery, reduces infarct size and and LDH leakage after ischemia-reperfusion (I/R) ex vivo . A-D. Time course of LVDP (A), LVEDP (B), and +dP/dtmax (C) and -dP/dtmax (D); n = 10 each. E. Effect of pinocembrin on the LDH activity in the coronary effluent; n = 10 each. F. Representative images and analysis of the infarct size in isolated I/R (30 min/2 h) hearts; n=7 in control group; n=10 in pinocembrin group. Data represent the mean ± SEM. *P <0.05 versus I/R control group.
Figure 3. Pinocembrin protects from myocardial I/R (MI/R) injury in vivo in C57/BL6 mice. Wild-type (WT) mice were subjected to 30 minutes of the left anterior descending coronary artery (LAD) ligation, followed by 24h of reperfusion, vehicle or pinocembrin intravenously (5 and 10 mg/kg) 5 min before reperfusion. A. Representative 2,3,5-triphenyltetra-zolium chloride (TTC)-stained LV transverse slices (scale bar,1 mm). Areas stained dark blue, white and red represented non-risk, infarcted and ischaemic but non-infarcted zones, respectively. B. Analysis of AAR (calculated as total infarcted plus ischaemic but non-infarcted zones, % total LV). C. Analysis of Myocardial infarct size (%AAR). D-E, Plasma LDH activity and cTnI levels. F-G, Representative images and left ventricular ejection fraction (EF%) and fractional shortening (FS%) by echocardiography. n=9 each. Data represent the mean ± SEM. *P <0.05 versus sham group.#P <0.05 versus I/R control group.
Figure 4. Protective actions of pinocembrin against cardiomyocyte injury responses in vitro. A. Effect of pinocembrin (30 µM, added during reperfusion) on rat cardiomyocyte cTnI release subsequent to H/R. B. Effect of pinocembrin on rat cardiomyocyte LDH release subsequent to H/R. C. Effect of pinocembrin on mouse cardiomyocyte cTnI release subsequent to H/R. D. Effect of pinocembrin on mouse cardiomyocyte LDH release subsequent to H/R. n=5 each. Data represent the mean ± SEM. *P <0.05 versus normoxia with vehicle group.#P <0.05 versus H/R with vehicle group.
Figure 5. Pinocembrin increases glycolysis in primary cardiomyocytes. A. Seahorse XFe24 extracellular flux analyzer traces of a glycolysis stress test in rat cardiomyocytes measured as the ECAR by sequential injection of 20 mM glucose, 1 μM oligomycin, and 100 mM 2-deoxy-d-glucose (2-DG). B. Quantification of glycolysis, glycolytic capacity, and glycolytic reserves in rat cardiomyocytes pretreated with pinocembrin or control. C. Seahorse XFe24 extracellular flux analyzer traces of a glycolysis stress test in mouse cardiomyocytes measured as the ECAR by sequential injection of 20 mM glucose, 1 μM oligomycin, and 100 mM 2-DG. D. Quantification of glycolysis, glycolytic capacity, and glycolytic reserves in mouse cardiomyocytes pretreated with pinocembrin or control. E. Mouse cardiomyocytes were treatment with pinocembrin for 24 h, and then the glycolysis-related genes were detected by qRT-PCR. n = 3/treatment in all Seahorse assays, each in triplicate. Data represent the mean ± SEM. *P < 0.05, significantly different as indicated. NS, no significant difference as indicated.
Figure 6. Pinocembrin protect cardiomyocytes from simulated I/R injury via enhancing glycolysis. A-D Treatment with PFKFB3 inhibitor, 3PO, results in decline of glycolysis and glycolytic capacity in mouse and rat cardiomyocytes. n=3/treatment in all Seahorse assays, each in triplicate. E-F. Effect of PFKFB3 inhibitor, 3PO, on mouse and rat cardiomyocyte cTnI release subsequent to H/R. n=5 each. G-H. Effect of PFKFB3 inhibitor, 3PO, on mouse and rat cardiomyocyte LDH release subsequent to H/R. n=5 each. Data represent the mean ± SEM. *P <0.05 versus H/R with vehicle group.#P <0.05 versus H/R with PB group.
Figure 7. Knock down of PFKFB3 in myocardium using AAV9 impairs pinocembrin induced cardioprotection after MI/R. A. Representative infarct staining from AAV-shNC or AAV-shPFKFB3 mice treated with vehicle or pinocembrin. B-C AAR and infarct sizes from AAV-shNC or AAV-shPFKFB3 mice treated with vehicle or pinocembrin. D-E. Plasma LDH activity and cTnI levels. F. Representative echocardiographic images, assessment of LV EF and FS by echocardiography from AAV-shNC or AAV-shPFKFB3 mice treated with vehicle or pinocembrin. n=8 each. Data represent the mean ± SEM. *P <0.05, significantly different as indicated.