Mitochondrial aldehyde dehydrogenase rescues against diabetic cardiomyopathy through GSK3β-mediated preservation of mitochondrial integrity and Parkin-mediated mitophagy

Figures

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  Figure 1. WCGNA of GSE4745 dataset constructed in response to the temporal-correlated pathological changes of myocardium in diabetic rattus norvegicus. (A) The heatmap showing the clustering analysis of sample transcript profile and phenotypic traits involving STZ injection days, body weight (BW), ventricular weight (VW), blood glucose (Bglu), ventricular weight/body weight ratio (VW/BW ratio), and body weight/control average (BW/Control). (B) The coexpression gene modules were identified through the hierarchical cluster algorithm and merged by the dynamic tree cut method, in which the cutting height is 0.25. (C) The heatmap showing the Pearson’s correlation analysis among modules and clinical traits. (D) The heatmap showing coexpression correlations among gene modules based on the hierarchical cluster algorithm.

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  Figure 2. The detection of pathway regulation network, gene set variation score, and gene set enrichment. (A and B) KEGG pathway regulation network was constructed based on the Metascape database involving genes in magenta or pink module. (C) GO functional enrichment map showing that adrenergic/calcium/cAMP signaling pathway, AGE–RAGE signaling pathway, and chemical diabetic/carcinogenesis/cardiomyopathy categories were significantly enriched. (D) GSVA of GSE4745 dataset reveals temporal variation of mitophagy and PI3K–Akt activation pathway scores in response to pathological changes in cardiomyopathy following STZ injection. (E and F) GSVA and GSEA of GSE150649 dataset validate mitophagy and PI3K–Akt activation pathway score variation and enrichment in diabetic hearts of newborn rats.

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  Figure 3. Influence of ALDH2 overexpression (ALDH2OE) and knockout on diabetes mellitus (DM)-induced changes in myocardial morphology, oxidative stress, and apoptosis. (A) Representative Lectin (top), Masson trichrome (middle), and DCF (bottom) staining micrographs depicting cardiomyocyte cross-sectional area, interstitial fibrosis, and oxidative stress, respectively. (B–D) Quantitative analysis of cardiomyocyte cross-sectional area (B), interstitial fibrosis (C), and DCF fluorescence intensity (D). (E) Representative TUNEL/DAPI/cardiac troponin T (cTnT) staining micrographs depicting cardiomyocyte apoptosis. (F) Quantitative analysis for the percentage of TUNEL-positive cells. Mean ± SEM, n = 6–8 mice or myocardial sections per group; *P < 0.05 vs. WT group, #P < 0.05 vs. WT-DM group.

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  Figure 4. Influence of ALDH2 overexpression and knockout on diabetes mellitus-induced changes in mitochondrial ultrastructure, protein expression, and function. (A) Representative TEM ultrastructural images. (B) Mitochondrial area (% total area). (C) Mitochondrial circularity index (major axis length/minor axis length). (D) Representative immunoblot and protein level of PGC-1α (GAPDH as loading control). (E) Representative immunoblot and protein level of UCP2 (α-tubulin as loading control). (F) Aconitase activity. (G) Representative JC-1 staining (aggregate, monomer, and merged) micrographs. (H) Quantitative analysis of JC-1 staining for MMP. Mean ± SEM, n = 5 ultrastructural images (B), 42–54 mitochondria (C), or 6–8 mice or myocardial micrographs (D–F and H) per group; *P < 0.05 vs. WT group, #P < 0.05 vs. WT-DM group.

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  Figure 5. ALDH2 overexpression and knockout on diabetes mellitus-induced changes in myocardial ALDH2, apoptosis, and Ca2+ regulatory proteins as well as plasma FFAs. (A) Plasma ALDH2 activity in normal individuals and patients with diabetes mellitus. (B–I) WT, ALDH2OE, and ALDH2−/− mice were challenged with or without STZ. (B) Representative immunoblots depicting ALDH2, SERCA2a, Na+–Ca2+ exchanger (NCX), and phospholamban (PLB) (GAPDH as loading control). (C) ALDH2 activity in the heart. (D) ALDH2 protein levels in hearts. (E) Plasma FFA levels. (F) Myocardial capsase-3 activity. (G–I) Levels of Ca2+ regulatory proteins SERCA2a, NCX, and PLB in hearts. Mean ± SEM, n = 5 patients (A) or 6–8 mice (C–I) per group; *P < 0.05 vs. normal or WT group, #P < 0.05 vs. WT-DM group.

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  Figure 6. Influence of ALDH2 overexpression and knockout on diabetes mellitus-induced changes in autophagy/mitophagy and related regulatory post-insulin receptor signaling. (A) Representative immunoblots depicting autophagy/mitophagy protein markers (GAPDH as loading control). (B) LC3BII-to-LC3BI ratio. (C–F) Protein levels of TOM20 (C), Parkin (D), FUNDC1 (E), and BNIP3 (F). (G) Representative immunoblots depicting total and phosphorylated Akt, GSK3β, PTEN, and Foxo3a (GAPDH as loading control). (H) pAkt-to-Akt ratio. (I) pGSK3β-to-GSK3β ratio. (J) pPTEN-to-PTEN ratio. (K) pFoxo3a-to-Foxo3a ratio. Mean ± SEM, n = 6–8 mice per group; *P < 0.05 vs. WT group, #P < 0.05 vs. WT-DM group.

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  Figure 7. Influence of the newly screened ALDH2 agonist torezolid on STZ- or high glucose (HG)-induced myocardial contractile dysfunction. (A) Molecular docking simulation of the binding between torezolid and ALDH2. (B) ALDH2 activity in cardiomyocytes from WT mice exposed to normal glucose (NG, 5.5 mM) or HG (25.5 mM) in the absence or presence of torezolid (5 μM) or the ALDH2 inhibitor Daidzin (120 μM). (C–H) Echocardiographic properties in control or STZ diabetic mice treated with or without torezolid (10 mg/kg, i.p., twice daily for 4 weeks, initiated concurrently with STZ injection). (C) LVEDD. (D) LVESD. (E) Fractional shortening. (F) Ejection fraction. (G) LV wall thickness. (H) Heart rate. (I–M) In vitro mechanical properties in cardiomyocytes from WT or Parkin knockout (PRKKO) mice exposed to NG or HG in the presence or absence of torezolid for 24 h. (I) PS (normalized to cell length). (J) +dL/dt. (K) –dL/dt. (L) TPS. (M) TR90. Mean ± SEM, n = 5 isolations (B), 6 mice (C–H), or 60 cells (I–M) per group; *P < 0.05 vs. NG or control group, #P < 0.05 vs. DM or HG group.

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  Figure 8. Influence of ALDH2 overexpression or the ALDH2 agonist Alda-1 on HG-induced MMP collapse and cardiomyocyte contractile dysfunction in the absence or presence of various pharmacological inhibitors. (A) Representative flow cytometric histograms of TMRE (1 μl/ml)-loaded H9C2 cells with or without ALDH2 overexpression exposed to NG (5.5 mM) or HG (25.5 mM) for 24 h. (B) Quantitative analysis of TMRE mean intensity for MMP in H9C2 cells. (C–H) Cardiomyocytes from WT mice were exposed to NG or HG for 24 h in the absence or presence of Alda-1 (20 μM), the Akt inhibitor AKTi (1 μM), the GSK3β activator LY294002 (10 μM), or the mitochondrial uncoupler FCCP (1 μM) prior to assessment of mechanical properties. (C) Resting cell length. (D) PS (normalized to cell length). (E) +dL/dt. (F) –dL/dt. (G) TPS. (H) TR90. (I) Scheme depicting the proposed role of ALDH2 in diabetic cardiomyopathy. Diabetes mellitus or hyperglycemia downregulates ALDH2, which promotes activation of PTEN and deactivation of Akt and GSK3β, leading to mitochondrial injury. Meanwhile, diabetes-evoked downregulation of ALDH2 suppresses Foxo3a activation and Parkin-mediated mitophagy, also favoring mitochondrial injury.…