Mst1 Knockout Alleviates Mitochondrial Fission and Mitigates Left Ventricular Remodeling in the Development of Diabetic Cardiomyopathy

Figures

• 

  Figure 1. Mst1 inhibition alleviates cardiac dysfunction and cardiomyocyte apoptosis in diabetic mice. (A) Representative images of echocardiography. (B) Measurements of LVEF (%). (C) Measurements of LVFS (%). (D) Measurements of LVEDD (mm). (E) Measurements of LVESD (mm). (F) Hemodynamic evaluation of +LV dp/dt max. (G) Hemodynamic evaluation of -LV dp/dt max. The columns and error bars represent the means and SEM. *P < 0.05 vs. WT; #P < 0.05 vs. DM; $P < 0.05 vs. DM + Mst1−/−. (H) Representative images of TUNEL staining in primary cardiomyocytes (scale bar: 40 mm). (I) Quantitative analysis of the apoptotic index (percentage of TUNEL-positive nuclei, %). (J) Representative western blots showing expression of cleaved-caspase 3, caspase 3 and β-actin (loading control) proteins. (K) Quantitative analysis of the ratio of cleaved-caspase 3/caspase 3. The columns and error bars represent the means and SEM. *P < 0.05 vs. Con; #P < 0.05 vs. HG; $P < 0.05 vs. HG + Ad-sh-Mst1; and P < 0.05 vs. HG + Ad-sh-LacZ; and +P < 0.05 vs. HG + Ad-Mst1.

• 

  Figure 2. Mst1 participates in the regulation of mitochondrial dynamics. (A) Representative images of typical damaged and different sized mitochondria (Scale bars: upper panel 2 μm, lower panel 500 nm). (B) Quantitative analysis of the mean mitochondrial size. (C) Quantitative analysis of the number of mitochondria per μm2. The columns and error bars represent the means and SEM. *P < 0.05 vs. WT; #P < 0.05 vs. DM; $P < 0.05 vs. DM +Mst1−/−. (D) Representative images of mitochondria (MitoTracker Red) (Scale bars: upper panel 20 μm, lower panel 5 μm). (E) Quantitative analysis of the mean length of mitochondria rods/branches. (F) Quantitative analysis of the number of branches. (G) Quantitative analysis of the mitochondrial footprint. The columns and error bars represent the means and SEM. *P < 0.05 vs. Con; #P < 0.05 vs. HG; $P < 0.05 vs. HG + Ad-sh-Mst1; and P < 0.05 vs. HG + Ad-sh-LacZ; and +P < 0.05 vs. HG + Ad-Mst1. (H) mtDNA copy number was assessed by complex IV segment. (I) The transcript level of mtDNA was assessed by NADH dehydrogenase subunit 1 (ND1). The columns and error bars represent the means and SEM. *P < 0.05 vs. WT; #P < 0.05 vs. DM; $P < 0.05 vs. DM +Mst1−/−.

• 

  Figure 3. Mst1 knockout decreases Drp1 expression, inhibits Drp1S616 phosphorylation and promotes Drp1S637 phosphorylation in DCM. (A) Protein expression with representative gel blots of t-Drp1, p-Drp1S616, p-Drp1S637, Mff, Mid51, Mid49, Fis1, and β-actin (loading control for Drp1, Mff, Mid51, Mid49 and Fis1). (B) Relative level of t-Drp1. (C) Relative level of p-Drp1S616. (D) Relative level of p-Drp1S637. (E) Relative level of Mff. (F) Relative level of Mid51. (G) Relative level of Mid49. (H) Relative level of Fis1. (I) Protein expression with representative gel blots of Mfn2, Mfn1, Opa1, and β-actin (loading control for Mfn2, Mfn1, Opa1). (J) Relative level of Mfn2. (K) Relative level of Mfn1. (L) Relative level of Opa1. The columns and error bars represent the means and SEM. *P < 0.05 vs. WT; #P < 0.05 vs. DM; and $P < 0.05 vs. HG +Mst1−/−.

• 

  Figure 4. Drp1 knockdown abolishes the effects of Mst1 on mitochondrial fission. (A) Representative images of mitochondria (MitoTracker Red) (Scale bars: upper panel 20 μm, lower panel 5 μm). (B) Quantitative analysis of the mean length of mitochondria rods/branches. (C) Quantitative analysis of the number of branches. (D) Quantitative analysis of the mitochondrial footprint. (E) Representative images of typical damaged and different sized mitochondria (Scale bars: upper panel 1 μm, lower panel 500 nm). (F) Quantitative analysis of the mean mitochondrial size. (G) Quantitative analysis of the number of mitochondria per μm2. The columns and error bars represent the means and SEM. *P < 0.05 vs. Con; #P < 0.05 vs. HG; $P < 0.05 vs. HG + Ad-Mst1; and P < 0.05 vs. HG + Ad-LacZ; +P < 0.05 vs. HG + Ad-sh- Mst1; and %P < 0.05 vs. HG + Ad-sh- LacZ.

• 

  Figure 5. Mst1 knockdown inhibits Drp1 recruitment to the mitochondria. (A) Representative colocalization images of Drp1 (Green) and mitochondria (MitoTracker Red) (Scale bars: upper four panels 20 μm, lower panel 5 μm). (B) Quantitative analysis of Drp1 puncta per cell. (C) Quantitative analysis of Drp1/ MitoTracker Red colocalization. (D) Quantitative analysis of colocalized Drp1 Puncta per cell. (E) Protein expression with representative gel blots of total Drp1, mitochondrial Drp1 and cytoplasmic Drp1. (F) Relative level of total Drp1 (G) Relative level of Drp1 in mitochondria. (H) Relative level of Drp1 in cytoplasm. The columns and error bars represent the means and SEM. *P < 0.05 vs. Con; #P < 0.05 vs. HG; $P < 0.05 vs. HG + Ad-sh-Mst1; and P < 0.05 vs. HG + Ad-sh-LacZ; and +P < 0.05 vs. HG + Ad-Mst1.

• 

  Figure 6. Drp1 knockdown abolishes the effects of Mst1 on mitochondrial membrane potential and mitochondrial dysfunction. (A) Representative images of JC-1 fluorescence imaging (Scale bar: 20 μm). (B) The ratio of aggregated (Red)/monomeric (Green) JC-1. (C) Cellular ATP content. (D) Citrate synthase (CS) activity. (E) Analysis of Complex I activity (ELISA assay) in primary cardiomyocytes. (F) Analysis of Complex II activity (ELISA assay) in primary cardiomyocytes. (G) Analysis of Complex V activity (ELISA assay) in primary cardiomyocytes. The columns and error bars represent the means and SEM. *P < 0.05 vs. Con; #P < 0.05 vs. HG; $P < 0.05 vs. HG + Ad- Mst1; and P < 0.05 vs. HG + Ad-LacZ; +P < 0.05 vs. HG + Ad-sh- Mst1; and %P < 0.05 vs. HG + Ad-sh- LacZ.