Activation of PI3Kγ/Akt pathway increases cardiomyocyte HMGB1 expression in diabetic environment

Figures

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  Figure 1. HG increases cardiomyocyte HMGB1 production in culture Mouse cardiomyocytes were exposed to HG (30 mM). Cardiomyocytes and their supernatants were harvested at the indicated times for the assessment of intracellular (A: Western blot) and extracellular (B: ELISA) HMGB1, respectively. n = 3, *P < 0.05 as compared with time 0.

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  Figure 2. PI3Kγ and Akt are activated in cardiomyocytes under HG condition Cardiomyocytes were treated with HG (30 mM). At the indicated time points, cardiomyocytes were harvested for the assessment (Western blot analysis) of both phosphorylated and total PI3Kγ (A) and Akt (B) proteins. Representative blots are shown above, and densitometric analysis is visible below. n = 3, *P < 0.05 when compared with control (time 0).

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  Figure 3. The PI3Kγ/Akt pathway is associated with the expression of HMGB1 in HG-conditioned cardiomyocytes A and B: Cardiomyocytes were pretreated with either AS605240 (a PI3Kγ inhibitor; 0.25 μM) or PI3K p110γ CRISPR/Cas9 KO plasmid. Subsequently, the cardiomyocytes were conditioned with HG or mannitol for either 30 minutes or 24 hours. The cardiomyocytes were harvested for the assessment of Akt phosphorylation (A) or HMGB1 expression (B). n = 3; *P < 0.05 versus dimethyl sulfoxide (DMSO) or control plasmid; #P < 0.05 versus HG. (C) Upper panel, cardiomyocytes were pretreated with an Akt inhibitor, A6730 (0–30 μM) or DMSO (vehicle) for 1 hour and conditioned with HG for 24 hours. The cardiomyocytes were harvested for the Western blot assessment of HMGB1 production. n = 3; *P < 0.05 versus DMSO; #P < 0.05 versus HG. Lower panel: cardiomyocytes were transfected with the control or Akt1/2/3 siRNA, and they were then conditioned with HG (30 mM) for 24 hours. The cardiomyocytes were harvested for HMGB1 expression. n = 3; *P < 0.05 versus control; #P < 0.05 versus HG.

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  Figure 4. Mitotempo prevents HG-induced PI3Kγ/Akt activation and HMGB1 expression Cardiomyocytes were conditioned with HG or mannitol (Mtl) with or without Mitotempo (25 mM) for 30 minutes or 24 hours. Subsequently, the cardiomyocytes were harvested to detect oxidative stress with a DCFDA microplate assay (A); phosphorylation of PI3Kγ and phosphorylation of Akt (B); and HMGB1 expression (C). n = 3, *P < 0.05 compared to Mtl; #P < 0.05 compared to HG.

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  Figure 5. A schematic representation of a working hypothesis illustrating the signaling pathway involved in the induction of HMGB1 expression in cardiomyocytes conditioned with HG