Fig 1.
DKO mice weigh less but consume similar amount of food when compared to WT controls.
A) DKO mice weigh significantly less than WT (p = 0.0298). B) Food consumption between WT and DKO mice is not significantly different.
Fig 2.
Whole body energy expenditure of WT and DKO mice.
A) Rate of oxygen consumption in DKO mice is not significantly different from WT at both night and B) day. Respiratory exchange ratio (F) is similar in WT and DKO mice both at C) night and D) day. Activity counts measured in WT and DKO mice during E) night and F) day. Activity counts are significantly reduced (p = 0.0060) in DKO mice at night compared to WT. Oxygen consumption per unit activity is significantly higher in the DKO mice both during G) night (p = 0.0023) and H) day (p = 0.0463) p<0.05 = significant. * = p<0.05, ** = p<.01.
Fig 3.
Increased oxygen consumption relative to integral force.
A) The fatigue profile of WT and DKO EDL during the 10 minutes fatigue shows that DKO EDL generates lesser force and fatigues less. B) The % of initial force after the 10 minute fatigue is higher in DKO EDL indicating less fatigue (p = 0.0019). C) The quantified force time integral over the entire 10 minutes fatigue protocol is significantly reduced in the DKO EDL compared to WT (p = 0.0097). D) Oxygen consumption over 10 minutes fatigue is not significantly different in WT and DKO EDL muscle. E). Oxygen consumed per unit integral force produced is significantly higher in DKO EDL compared to WT (p = 0.0110). p< 0.05 is significant. * = p<0.05, ** = p<0.01.
Fig 4.
Increased potentiation of force by pyruvate in DKO EDL.
A). Effect of substrate on force production showing an increase in force production using pyruvate as a substrate at lower frequencies in DKO mice. B) B) Specific force produced by WT EDL is significantly higher (p<0.05) than DKO EDL when glucose is used as a substrate at 50 Hz. In the presence of pyruvate the specific force produced by WT EDL is not significantly different from DKO EDL at 50Hz. There is a significant increase in force production in DKO EDL when pyruvate is used as a substrate compared to glucose (p<0.05). However, the force produced by WT EDL in the presence of pyruvate is not significantly different from the force produced in presence of glucose. C) % Increase in force using pyruvate as a substrate relative to glucose is significantly higher in DKO EDL compared to WT at 30Hz (p = 0.0033) and D) 50Hz (p = 0.0011). p< 0.05 is significant. * = p<0.05, ** = p<0.01.
Fig 5.
Increased expression of glycolytic enzymes in DKO muscles.
A) Western blots depicting higher levels of glycolytic enzymes DKO EDL compared to WT. B) HK1 protein level normalized to GAPDH is significantly higher in DKO EDL compared to WT (p = 0.0063) C) PK M2 protein level normalized to GAPDH is significantly higher in DKO EDL compared to WT (p = 0.0001). D) Western blot showing key metabolic regulators are unchanged in DKO EDL compared to WT. E) Western blots showing glycolytic enzymes protein levels are higher in DKO diaphragm. F) HK1 protein level normalized to GAPDH is significantly higher in DKO diaphragm compared to WT (p = 0.0004) and G) PK M2 protein level normalized to GAPDH is significantly higher in DKO diaphragm compared to WT (p = 0.0066). H). Glycolytic enzymes protein expression levels in ventricle. p< 0.05 is significant. * = p<0.05, ** = p<0.01 and *** = p<0.001. HK 1- Hexokinase 1, HK 2- Hexokinase 2, PK M1- Pyruvate kinase M1, PK M2- Pyruvate kinase M2, CS- Citrate synthetase, TFAM- Mitochondrial transcript factor A, LDH- Lactate dehydrogenase.
Fig 6.
Increased expression of mitochondrial fusion and fission regulators in DKO muscles.
A) Western blots depicting mitochondria fission (Drp 1) and fusion (Mfn 2) regulators in WT and DKO EDL. B) Mfn 2 protein level normalized to GAPDH is significantly higher in DKO EDL compared to WT (p = 0.0073). C) Drp 1 protein level normalized to GAPDH is significantly higher in DKO EDL compared to WT (p = 0.0089). D) Western blots depicting mitochondria fission (Drp 1) and fusion (Mfn 2) regulators in WT and DKO diaphragm. E) Mfn 2 protein level normalized to GAPDH is significantly higher in DKO diaphragm compared to WT (p = 0.0074). F) Drp 1 protein level normalized to GAPDH is significantly higher in DKO diaphragm compared to WT (p = 0.0007). Western blots depicting similar mitochondrial electron transport chain complex protein levels in G) EDL and H) diaphragm of WT and DKO mice. p< 0.05 is significant. * = p<0.05, ** = p<0.01. Mfn 2- Mitofusin 2, Drp 1—Dynamin related protein 1, CV ATP5A- Complex V F1-F0 ATP synthase subunit, CIII UQCRC2- Complex III ubiquinol-cytochrome c reductase subunit, CIV MTCO1- Complex IV Cytochrome C Oxidase core subunit, CII SDHB- complex II succinate dehydrogenase subunit.
Fig 7.
Transmission electron microscopic images show mitochondrial localization is altered in DKO EDL muscle.
A) WT EDL B) DKO EDL at 14000X magnification. C) WT and D) DKO at 34000x magnification. The arrows point to the localization of mitochondria (M) which are at the I band on either side of the Z disc in WT, but this tight localization is reduced in the DKO EDL.