Endotoxin Pretreatment Mitigates Myocardial Ischemia-Reperfusion Injury Through Preservation of Mitochondrial Respiration: A Combined Assessment of In Vivo, Ex Vivo, and In Vitro Data

Figures

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  Figure 1. Effect of endotoxin preconditioning on left ventricular ejection fraction and myocardial oxygen consumption after ischemia and reperfusion. (A) Representative echocardiographic images. (B) Pressure–Volume Area (PVA) scatter plots with linear trendlines. (C) Quantitative analysis of Left Ventricular Ejection Fraction (LVEF, %) and (D) PVA (mmHg·µL) in Sham, ischemia/reperfusion (I/R; 30 min ischemia + 2 weeks reperfusion), and I/R with endotoxin preconditioning (I/R + LPS) groups (n = 5 per group). I/R = ischemia/reperfusion; I/R + LPS = ischemia/reperfusion with endotoxin preconditioning; LPS = lipopolysaccharide. Data are presented as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. ns = not significant (p ≥ 0.05); * p < 0.05; ** p < 0.01.

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  Figure 2. Effect of Endotoxin Preconditioning on Myocardial Fibrosis Following Ischemia–Reperfusion. (A) Representative histological images of hematoxylin and eosin (H&E) and Masson’s trichrome-stained left ventricular sections from Sham, ischemia/reperfusion (I/R), and endotoxin preconditioning + ischemia/reperfusion (I/R & LPS) groups (n = 5 per group). Scale bar = 200 μm. (B) Quantification of fibrotic area expressed as a percentage (%) of total myocardial tissue area. Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test for multiple group comparisons. ** p < 0.01, *** p < 0.001 versus I/R group. Abbreviations: I/R, ischemia/reperfusion; LPS, lipopolysaccharide.

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  Figure 3. Effect of endotoxin pretreatment on left ventricular function following ischemia–reperfusion under isolated normothermic perfusion conditions. (A) Representative table summarizing measured cardiac parameters. Quantitative analysis of (B) left ventricular developed pressure (LVDP, mmHg), (C) heart rate (HR, bpm), and (D) coronary flow (CF, mL/min/g) in Sham, ischemia/reperfusion (I/R; 30 min ischemia + 45 min reperfusion), and endotoxin preconditioning + ischemia/reperfusion (I/R & LPS) groups (n = 5 per group). Abbreviations: I/R, ischemia/reperfusion; LPS, lipopolysaccharide. Data are presented as mean ± standard deviation (SD). Statistical comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. ns, not significant (p ≥ 0.05); * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 versus I/R group.

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  Figure 4. Effect of endotoxin preconditioning on cardiomyocyte structure after hypoxia/reoxygenation injury. (A) Representative confocal microscopy images of neonatal primary rat cardiomyocytes in the control, 4 h hypoxia + 2 h reoxygenation (H/R), and endotoxin preconditioning + H/R (H/R & LPS) groups (n = 3 per group). Cells were immunostained for sarcomeric α-actinin (red) and counterstained with DAPI for nuclei (blue). Scale bar: 20 μm. (B) Sarcomere length and (C) cell density quantification. H/R = hypoxia/reoxygenation; H/R & LPS = hypoxia/reoxygenation with endotoxin preconditioning; LPS = lipopolysaccharide. Data are presented as mean ± standard deviation. One-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons. * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 5. Effect of different endotoxin dosages on cardiomyocyte viability following hypoxia–reoxygenation. Representative bar graph showing cell viability of primary neonatal rat cardiomyocytes in control, 4 h hypoxia + 2 h reoxygenation (H/R), and endotoxin-preconditioned H/R groups treated with varying concentrations of lipopolysaccharide (LPS: 0.1 (p = 0.0008), 0.5, 1.0, and 2.0 µg/mL) (n = 3 per group). H/R = hypoxia–reoxygenation, H/R & LPS = hypoxia–reoxygenation with endotoxin preconditioning. Data are presented as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. ** p < 0.01; *** p < 0.001 versus untreated H/R group.

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  Figure 6. Effect of endotoxin preconditioning on hypoxia levels in neonatal rat cardiomyocytes after hypoxia–reoxygenation. (A) Representative confocal microscopy images showing HIF-1α (green) and nuclei (DAPI, blue) staining. (B) Quantification of HIF-1α fluorescence intensity. (C) Representative Western blot images of HIF-1α expression. (D) Densitometric analysis of Western blot data. Experimental groups: normoxic control, 4 h hypoxia + 2 h reoxygenation (H/R), and endotoxin preconditioning + H/R (H/R & LPS) (n = 3 per group). Scale bar = 20 μm. H/R = hypoxia/reoxygenation, H/R & LPS = hypoxia/reoxygenation with endotoxin preconditioning, LPS = lipopolysaccharide. Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s post hoc test was used for statistical comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 7. Effect of endotoxin preconditioning on oxidative stress and mitochondrial respiration following ischemia and reoxygenation in neonatal rat cardiomyocytes. (A) Representative fluorescence microscopy images stained with MitoSOX™ Green. (B) Mitochondrial oxygen consumption rate (OCR) plots. Statistical data showing (C) basal mitochondrial respiration, (D) maximal mitochondrial respiration, (E) spare respiratory capacity, (F) adenosine triphosphate (ATP) production, (G) glycolysis, and (H) mitochondrial superoxide (MitoSOX™ Green) intensity. Groups include control, 4 h of hypoxia + 2 h of reoxygenation (H/R), and endotoxin preconditioning + 4 h of hypoxia + 2 h of reoxygenation (H/R & LPS) (n = 3 per group). Scale bar: 50 μm. Data are presented as mean ± standard deviation. One-way ANOVA followed by Tukey’s post hoc test was used for statistical comparisons. ns = not significant (p ≥ 0.05); * p < 0.05; ** p < 0.01; *** p < 0.001. Abbreviations: H/R = hypoxia/reoxygenation; LPS = lipopolysaccharide.

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  Figure 8. Effect of endotoxin preconditioning on cellular apoptosis after ischemia and reoxygenation. (A) Representative flow cytometry images and (B) statistical data depicting the percentage of apoptotic cells in primary neonatal rat cardiomyocytes from control, 4 h of hypoxia + 2 h of reoxygenation (H/R), and endotoxin preconditioning + 4 h of hypoxia + 2 h of reoxygenation (H/R & LPS) groups (n = 3 per group). H/R = hypoxia/reoxygenation, H/R & LPS = hypoxia/reoxygenation with endotoxin preconditioning, LPS = Lipopolysaccharide. Data are presented as mean ± standard deviation. Statistical comparisons among multiple groups were performed using one-way ANOVA followed by Tukey’s post hoc test. ** p < 0.01.

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  Figure 9. Experimental design and timeline for in vivo, ex vivo, and in vitro studies. (A) In vivo: Ischemia–reperfusion injury (IRI) was induced by temporary ligation of the left anterior descending (LAD) coronary artery for 30 min, followed by ligature removal to initiate reperfusion. ECG recordings were obtained before, during, and after LAD ligation (Supplementary Material, Figure S1). (B) Ex vivo: Global ischemia was induced by closing the aortic perfusion cannula for 30 min, followed by reopening to restore reperfusion. Left ventricular developed pressure (LVDP) was continuously monitored before, during, and after ischemia. (C) In vitro: Hypoxia was induced for 4 h, followed by 2 h of reoxygenation before performing cell-based assays. Abbreviations: LPS, lipopolysaccharide; PV loop, pressure–volume loop; LVDP, left ventricular developed pressure (mmHg); IC, immunocytochemistry; WB, Western blot; XFp, Seahorse XFp Mito Stress Test; MitoSOX, MitoSOX™ Green mitochondrial superoxide indicator.