Reduction in Acetylation of Superoxide Dismutase 2 in Skeletal Muscle Improves Exercise Capacity in Mice With Heart Failure

Figures

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  FIGURE 1. Honokiol decreases the acetylation of SOD2 in C2C12 myotubes in a SIRT3‐dependent manner. (A) Experimental protocol for Honokiol treatment of C2C12 myotubes. (B) Representative western blots (left) and summary data (right) of SIRT3 and acetylated lysine levels in whole lysates from C2C12 myotubes treated with vehicle (n = 9) or Honokiol (n = 9). Results were normalized to non‐specific bands of CBB‐stained gel. (C) Representative western blots (top) and summary data (bottom) of acetylated SOD2 and total SOD2 levels in C2C12 myotubes treated with vehicle (n = 5) or Honokiol (n = 5). Acetylated SOD2 was normalized to total SOD2. (D) Experimental protocol for Honokiol treatment of C2C12 myotubes transfected with SIRT3 siRNA. (E) Representative western blot (top) and summary data (bottom) of SIRT3 in C2C12 myotubes transfected with SIRT3 siRNA or scramble. (F) Representative western blots (top) and summary data (bottom) of acetylated SOD2 and SOD2 levels in C2C12 myotubes treated with vehicle (n = 6) or Honokiol (n = 6) with transfection of SIRT3 siRNA. Acetylated SOD2 was normalized to total SOD2. Data are shown as the mean ± SD p values were calculated by the unpaired Student t‐test…

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  FIGURE 2. Honokiol ameliorates the reduced exercise capacity of MI mice. (A) Experimental protocol for Honokiol treatment to MI mice. Summary data of body weight and gastrocnemius weight/body weight (B), work, running distance and running time (C), and lactate production after exercise/work (D) in sham + vehicle (n = 8), sham + Honokiol (n = 7), MI + vehicle (n = 8) and MI + Honokiol mice (n = 6). Data are shown as the mean ± SD p values of the main effect for each factor and interaction effect between two factors were calculated by two‐way ANOVA, with the factors of MI and Honokiol, and if there was an interaction effect between 2 factors, the Tukey post hoc test was performed. MI, myocardial infarction.

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  FIGURE 3. Honokiol treatment improved mitochondrial respiration and the decrease in fibre Type I in the skeletal muscle of MI mice. (A) Summary data of complex I‐linked State 2, complex I‐linked State 3, and complex I + II‐linked State 3 respiration in isolated mitochondria from the gastrocnemius tissues form sham + vehicle (n = 6), MI + vehicle (n = 7) and MI + Honokiol (n = 6). (B) Representative high‐power photomicrographs of gastrocnemius muscle tissue sections stained with MHC immunofluorescence staining (left, blue: Type I fibre, green: Type IIa fibre, red: Type IIb fibre), and summary data (right) of the proportion of Type I fibre to the total fibres in sham + vehicle (n = 5), MI + vehicle (n = 5), and MI + Honokiol mice (n = 5). (C) Summary data of citrate synthase activity in the skeletal muscle of sham + vehicle (n = 5), MI + vehicle (n = 5), and MI + Honokiol mice (n = 5). (D) Representative transmission electron microscopy images of gastrocnemius muscle (left) and summary data of number of mitochondria, mitochondrial cross‐sectional area and mitochondrial circularity (right) in sham + vehicle (n = 3), MI + vehicle (n = 3), and MI + Honokiol mice (n = 3). Six images from 3 mice…

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  FIGURE 4. Honokiol treatment attenuated the acetylation of SOD2 and ROS production in the skeletal muscle of MI mice. (A) Representative western blots (left) and summary data (right) of SIRT3 and acetylated lysine levels in whole lysates from the skeletal muscle of sham + vehicle (n = 6), MI + vehicle (n = 4), and MI + Honokiol mice (n = 5). Results were normalized to non‐specific bands of the CBB‐stained gel. (B) Representative western blots (top) and summary data (bottom) of acetylated SOD2 and total SOD2 levels in the skeletal muscle of sham + vehicle (n = 7), MI + vehicle (n = 5) and MI + Honokiol mice (n = 6). Acetylated SOD2 was normalized to total SOD2. (C) Summary data of H2O2 production from isolated mitochondria in gastrocnemius muscle of sham + vehicle (n = 6), MI + vehicle (n = 6) and MI + Honokiol mice (n = 6). (D) Summary data of aconitase activity in gastrocnemius muscle of sham + vehicle (n = 6), MI + vehicle (n = 6), and MI + Honokiol mice (n = 6). Data are shown as the mean ± SD p values were calculated by one‐way ANOVA followed by the Tukey post hoc test. MI, myocardial infarction; SIRT3, sirtuin 3; CBB, Coomassie Brilliant Blue; SOD2, superoxide dismutase 2.

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  FIGURE 5. Overexpression of SIRT3 in skeletal muscle ameliorates reduced exercise capacity in MI mice. (A) Experimental protocol for AAV9 injection to MI mice. (B) Representative images of GFP expression in MI + AAV9‐Control mice in gastrocnemius muscle (low‐magnification, upper‐left), gastrocnemius muscle (high‐magnification, upper‐right), heart (bottom‐left), and liver (bottom‐right). (C) Representative western blots (left) and summary data (right) of SIRT3 in the gastrocnemius muscle of MI + AAV9‐Control (n = 7) and MI + AAV9‐SIRT3 mice (n = 7). (D) Summary data of work, running distance and running time of MI + AAV9‐Control (n = 7) and MI + AAV9‐SIRT3 mice (n = 7). Data are shown as the mean ± SD p values were calculated by the unpaired Student t‐test. AAV9, adeno‐associated virus serotype 9; SIRT3, sirtuin 3; GFP, green‐fluorescence protein; CBB, Coomassie Brilliant Blue; MI, myocardial infarction.

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  FIGURE 6. Schematic diagram of the potential roles of SIRT 3 and acetylated SOD2 in the skeletal muscle of MI mice and a possible therapeutic target for SIRT3 activator.