AEOL-induced NRF2 activation and DWORF overexpression mitigate myocardial I/R injury

Figures

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  Fig. 1. AEOL-10150 reduces ROS production upon reoxygenation/reperfusion. A ROS production assessed with the mitochondrial superoxide indicator MitoSOX Red in hiPSCMs subjected to simulated ischemia (1% O2, no glucose) for 45 min followed by 24 h reoxygenation (I/R model). Cells were treated with AEOL (50 µM) or vehicle (saline solution; 0.9% NaCl) at the onset of reoxygenation. B ROS production in hiPSCMs treated with dimethyl succinate (Suc) and oligomycin (Oligo) for 2 h. C Inhibitory effect of AEOL on ROS generation assessed 30 min after mI/R. Mice undergoing the mI/R procedure were treated with a subcutaneous injection of AEOL (25 mg/kg) or vehicle (DPBS) 15 min after reperfusion onset, and ROS were measured from the oxidation of MitoB injected 4 h before the procedure. Data are from n = 5 independent assays per group for in vitro procedures and n = 7 mice per group for in vivo procedures. Quantitative data are presented as mean ± standard error of the mean (SEM). ***p < 0.001; *p < 0.05, determined by one-way ANOVA followed by post hoc Bonferroni correction. BS, blood samples; CS, cardiac samples; I/R, ischemia–reperfusion; Oligo, oligomycin; Suc, succinate; wks, weeks. Other…

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  Fig. 2. AEOL treatment reduces in the reactive oxygen species (ROS) surge triggered by reperfusion and protects against acute ischemia–reperfusion (I/R) injury. A-C, hiPSCMs subjected to ischemia(1% O2) for 45 min followed by 24 h reoxygenation (I/R model) were treated with AEOL (50 µM) and assayed for (A) mitochondrial permeability transition pore opening, B mitochondrial potential (Δψm), and (C) cell viability. D, Representative images and quantification of TUNEL staining in LV sections from mice subjected to mI/R and recovered for 24 h. Mice were treated as indicated with 25 mg/kg AEOL or vehicle (0.9% NaCl) 15 min after starting reperfusion. Scale bar, 50 µm. E Experimental design for determination of myocardial infarct size in mice subjected to mI/R and recovered for 8 weeks, with serial daily treatment with AEOL or vehicle (0.9% NaCl) starting 5 min, 15 min, or 3 days after reperfusion onset. Representative photographs show TTC staining 8 weeks post-reperfusion. F–H Effect of AEOL administration 5 min (F), 15 min (G), or 3 days (H) after reperfusion onset on infarct size determined at 8-weeks post-mI/R. Quantifications were obtained from n = 5 independent assays per group for in…

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  Fig. 3. AEOL treatment in acute-phase mI/R injury improves cardiac function. A, B Heart weight (HW) and HW/body-weight (BW) ratio in mice subjected to mI/R, treated with AEOL or vehicle (0.9% NaCl) 15 min after reperfusion onset, and recovered for 8 weeks. C Evolution of echocardiography measures of cardiac function. Functional benefits of short-term AEOL treatment vs vehicle after mI/R include smaller LV internal diameters at end diastole (LVIDd) and end systole (LVIDs) and better preserved % ejection fraction (% EF) and % fractional shortening (% FS) (full echocardiography data are presented in Table 3). Data were obtained from n = 7 mice/group, and are presented as mean ± SEM. ***p < 0.001; **p < 0.01 or & p < 0.01; &&& p < 0.001 mI/R + AEOL vs mI/R + vehicle, determined by one-way ANOVA followed by post hoc Bonferroni correction. BW, body weight; HW, heart weight. Other abbreviations are defined in the abbreviations list

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  Fig. 4. AEOL increases nuclear translocation of NRF2 and the transcriptional induction of downstream ARE-regulated genes in the infarct border zone. Mice subjected to mI/R were treated with AEOL or vehicle (0.9% NaCl) 15 min after reperfusion onset and sacrificed at 24 h post-mI/R. A,B Cytosolic and nuclear expression of NRF2 in the infarct border zone. C KEAP1 protein expression. D p62 protein expression levels. E LC3 II/LC3 I ratio. F HO-1 protein expression. Data are from n = 7 mice/group and are presented as mean ± SEM. ***p < 0.001; determined by one-way ANOVA followed by post hoc Bonferroni correction. GAPDH, glyceraldehyde phosphate dehydrogenase; H3, histone H3; HO-1, heme oxygenase 1; KEAP1, kelch-like ECH-associated protein 1; LC3, microtubule-associated protein 1 A/1B-light chain 3; NRF2, nuclear factor erythroid 2-related factor 2; p62, sequestosome 1. Other abbreviations are defined in the abbreviations list

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  Fig. 5. AEOL treatment ameliorates mI/R injury through NRF2. A-D Wild type and Nrf2-KO mice subjected to mI/R were treated with AEOL or vehicle (0.9% NaCl) 15 min after reperfusion onset and sacrificed at 8 weeks post-mI/R. A Infarct size calculated as the percentage of LV volume and expressed as fold of control. B Representative LV sections from wild type mice obtained 8 weeks post-mI/R. Collagen in the scar is blue and myocytes are red. Scale bar, 0.5 cm. C Area-at-risk (AAR) calculated as the percentage of LV volume and expressed as fold of control. D Plasma sST2 in mice at 24 h post-mI/R. E hiPSCM culture supernatant content of c-TnT, LDH, and sST2 at 24 h after simulated I/R. Quantifications were obtained from n = 7 mice per group for in vivo assays and n = 5 independent assays per group for in vitro procedures. Quantitative data are presented as mean ± SEM. ***p < 0.001 determined by one-way ANOVA followed by post hoc Bonferroni correction. AAR, area-at-risk; Scr, scramble; KO, knockout. Other abbreviations are defined in the abbreviations list

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  Fig. 6. AEOL-induced DWORF upregulation displaces PLN from SERCA2a. A DWORF accumulation in the infarct border zone of mice 24 h after mI/R. Mice were treated with AEOL or vehicle (0.9% NaCl) 15 min after reperfusion onset. B DWORF accumulation in extracts of hiPSCMs subjected to the simulated I/R protocol (45 min ischemia followed by reoxygenation and maintenance for 24 h). Cells were transfected 48 h before simulated I/R with scrambled (Scr) or siNRF2 siRNA, as indicated, and treated with AEOL or vehicle (0.9% NaCl) at the onset of reoxygenation. C-F Co-immunoprecipitation analysis in hiPSCMs transfected as indicated with Scr, siNRF2, or siDWORF siRNA before simulated I/R. Cells were treated at the onset of reoxygenation with AEOL and DWORF, as indicated, and analyzed at 24 h after simulated I/R. C, D Co-immunoprecipitation and densitometry analysis of co-immunoprecipitated SERCA2a and PLN proteins. E, F Co-immunoprecipitation and densitometry analysis of co-immunoprecipitated SERCA2a and DWORF proteins. A representative input immunoblot is shown above panels C-F. Data were obtained from a sample size of n = 7 mice per group for in vivo assays and n = 5 independent assays per group for…

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  Fig. 7. AEOL treatment enhances SERCA2a activity by upregulating DWORF expression. Maximal SERCA2a activity in the infarct border zone of wild type and Nrf2-KO mice subjected to mI/R, treated as indicated with AEOL and DWORF 15 min after reperfusion onset, and sacrificed at 24 h post-mI/R. Data were obtained from n = 7 mice/group and are presented as mean values ± SEM. ***p < 0.001, determined by one-way ANOVA followed by post hoc Bonferroni correction. Abbreviations are defined in the abbreviations list