Advanced Glycation End-Products Exacerbate Myocardial Ischemia/Reperfusion Injury by Promoting Mitochondrial Oxidative Damage and PANoptosis in Diabetes Mellitus

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  Fig. 1. Diabetes mellitus exacerbates myocardial ischemia/reperfusion injury. (A) Serum levels of cardiac troponin T (cTnT). (B) Serum levels of lactate dehydrogenase (LDH). (C) Representative images of TTC/Evans Blue staining in diabetic (db/db) and non-diabetic (db/m) mice. Scale bar: 5 mm. (D) Quantitative analysis of the area at risk (AAR) normalized to left ventricular (LV) area. (E) Quantitative analysis of infarct area (IA) normalized to AAR. (F) Representative images of Masson staining. Scale bar: 5 mm. (G) Quantitative analysis of fibrotic area normalized to LV area. (H) Representative M-mode echocardiographic images. Time stamp: 0.1s; Scale bar: 2.5 mm. (I) Left ventricular ejection fraction (LVEF) measured by echocardiography. (J) Fractional shortening (FS) measured by echocardiography. n = 5 in each group. Data were presented as mean ± SD. (A, B, I, G, J) P-value was produced using two-way ANOVA followed by Sidak's multiple comparisons test. (D, E) P-value was calculated by unpaired Student's t-test. A two-tailed P < 0.05 was considered statistically significant.

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  Fig. 2. Advanced glycation end-products promote PANoptosis in cardiomyocytes after myocardial ischemia/reperfusion. (A) Real-time imaging of cardiomyocyte death during hypoxia/reoxygenation (H/R). scale bar: 300 μm. (B) Total number of viable cardiomyocytes under normoxic conditions. (C) Proportion of PI-positive cells after 12 h of hypoxia. (D, E) Proportion of PI-positive cells at 1 and 2 h after reoxygenation, respectively. (F) Cell survival rate expressed as the percentage of viable cells relative to baseline after 4 h of reoxygenation. (G) Western blot analysis of key proteins associated with apoptosis (cleaved-caspase3), pyroptosis (cleaved-caspase1, GSDMD), and necroptosis (p-RIP1, p-RIP3, p-MLKL). (H, I) Immunofluorescence staining of GSDMD, cleaved-caspase3, and p-MLKL in heart tissues from diabetic (db/db) and non-diabetic (db/m) mice 24 h after MI/R. (J, K) Immunofluorescence colocalization of AIM2, ZBP1, and ASC, demonstrating assembly of the AIM2-ZBP1-PANoptosome in heart tissues from diabetic (db/db) and non-diabetic (db/m) mice 24 h after MI/R. (L) Western blot analysis of PANoptosis executors in cardiomyocytes following genetic ablation of AIM2, ZBP1, or both. c-casp3:…

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  Fig. 3. Advanced glycation end-products exacerbate mitochondrial oxidative damage upon hypoxia/reoxygenation stimulation. (A) Representative images of MitoSOX staining showing mitochondrial ROS levels in cardiomyocytes after H/R stimulation. Scale bar: 200 μm. (B) Quantitative analysis of MitoSOX mean fluorescence intensity (MFI) by flow cytometry, n (biological replicates) = 3. (C) Mitochondrial membrane potential assessed by the JC-1 fluorescence ratio (590/530 nm), n (biological replicates) = 5. (D) Cellular ATP production after H/R stimulation, n (biological replicates) = 5. (E) Transmission electron microscopy images of cardiomyocytes cultured with AGEs-containing or control medium; red arrows indicate mitochondria. Scale bar: 1μm/500 nm(enlarged). (F) Representative western blots of cytochrome c in cytosol. (G) Quantitative analysis of cytosolic cytochrome c levels normalized to β-actin, n (biological replicates) = 3. (H) Quantitative analysis of cytosolic mtDNA, n (biological replicates) = 5. (I) Quantitative analysis (mean fluorescence intensity, MFI) of mtROS in H9C2 cardiomyocytes by flow cytometry using MitoSOX staining, following RAGE knockdown, n (biological replicates) = 3.…

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  Fig. 4. Mitochondrial Oxidative Damage mediates AGEs-induced PANoptosis. (A) Western blot analysis of PANoptosis executors (cleaved caspase-3, GSDMD, and p-MLKL) in H9C2 cardiomyocytes treated with the mitochondrial oxidative stress inducer antimycin A or AGEs. (B) Quantitative analysis of MitoSOX mean fluorescence intensity (MFI), n (biological replicates) = 3. (C) Representative confocal microscopy images of JC-1 staining (scale bar: 100 μm). (D) ATP production in cardiomyocytes after H/R stimulation, n (biological replicates) = 5. (E) mtDNA levels in cytosol of cardiomyocytes after H/R stimulation, n (biological replicates) = 5. (F) Real-time monitoring of cardiomyocyte death during H/R (scale bar: 300 μm). (G) Western blot analysis of apoptosis marker cleaved caspase-3. (H) Western blot analysis of pyroptosis markers cleaved caspase-1 and GSDMD. (I) Western blot analysis of necroptosis markers p-RIP1, p-RIP3, and p-MLKL. c-casp3: cleaved caspase-3. c-casp1: cleaved caspase-1. Data were presented as mean ± SD. (B, D, E) P-value was produced using one-way ANOVA followed by Tukey's multiple comparisons test. A two-tailed P < 0.05 was considered statistically significant.

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  Fig. 5. Treatment with pyridoxamine and empagliflozin attenuates AGE-induced mitochondrial damage and PANoptosis. (A) Representative Western blot analysis of AGE levels in heart tissues from non-diabetic (db/m), diabetic (db/db) mice receiving different treatments. (B) Quantitative analysis of AGE expression normalized to β-actin, n (biological replicates) = 3. (C) Western blot analysis of RAGE, Nrf2, SOD2 in heart tissues from non-diabetic (db/m), diabetic (db/db) mice receiving different treatments. (D) Transmission electron microscopy images of myocardial ultrastructure in diabetic mice treated with saline or PyriEMPA. M: mitochondria; red arrows indicate sarcomeric damage. Scale bar: 2μm/1μm/500 nm. (E) Immunofluorescence colocalization of AIM2, ZBP1, and ASC, indicating AIM2-ZBP1-PANoptosome assembly. Scale bar: 50 μm. (F) Immunofluorescence staining of GSDMD, cleaved caspase-3, and p-MLKL in heart sections from saline- or PyriEMPA-treated diabetic mice. Scale bar: 50 μm. (G) Western blot analysis of AIM2-ZBP1 PANoptome and PANoptosis executors. c-casp3: cleaved caspase-3. c-casp1: cleaved caspase-1. Data were presented as mean ± SD. (B) P-value was generated using one-way ANOVA…

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  Fig. 6. Combination therapy with pyridoxamine and empagliflozin improve MI/RI outcomes in diabetic mice. (A) Serum levels of cardiac troponin T (cTnT). (B) Serum levels of lactate dehydrogenase (LDH). (C) Representative images of TTC/Evans Blue staining in diabetic (db/db) and non-diabetic (db/m) mice. Scale bar: 5 mm. (D) Quantitative analysis of the area at risk (AAR) normalized to left ventricular (LV) area. (E) Quantitative analysis of infarct area (IA) normalized to AAR. (F) Representative images of Masson staining. Scale bar: 500μm/300 μm. (G) Quantitative analysis of fibrotic area normalized to LV area. (H) Representative M-mode echocardiographic images. Time stamp: 0.1s; Scale bar: 5 mm. (I) Left ventricular ejection fraction (LVEF) measured by echocardiography. n (the number of animals used) = 5 in each group. Data were presented as mean ± SD. (A, B, I) P-value was calculated using one-way ANOVA followed by Tukey's multiple comparisons test. (D, E, G) P-value was calculated using two-way ANOVA followed by Sidak's multiple comparisons test. A two-tailed P < 0.05 was considered statistically significant.