Impaired mitochondrial dynamics and bioenergetics in diabetic skeletal muscle

Abstract

In most cells, mitochondria are highly dynamic organelles that constantly fuse, divide and move. These processes allow mitochondria to redistribute in a cell and exchange contents among the mitochondrial population, and subsequently repair damaged mitochondria. However, most studies on mitochondrial dynamics have been performed on cultured cell lines and neurons, and little is known about whether mitochondria are dynamic organelles in vivo, especially in the highly specialized and differentiated adult skeletal muscle cells. Using mitochondrial matrix-targeted photoactivatable green fluorescent protein (mtPAGFP) and electroporation methods combined with confocal microscopy, we found that mitochondria are dynamic in skeletal muscle in vivo, which enables mitochondria exchange contents within the whole mitochondrial population through nanotunneling-mediated mitochondrial fusion. Mitochondrial network promotes rapid transfer of mtPAGFP within the cell. More importantly, the dynamic behavior was impaired in high-fat diet (HFD)-induced obese mice, accompanying with disturbed mitochondrial respiratory function and decreased ATP content in skeletal muscle. We further found that proteins controlling mitochondrial fusion MFN1 and MFN2 but not Opa1 were decreased and proteins governing mitochondrial fission Fis1 and Drp1 were increased in skeletal muscle of HFD-induced mice when compared to normal diet-fed mice. Altogether, we conclude that mitochondria are dynamic organelles in vivo in skeletal muscle, and it is essential in maintaining mitochondrial respiration and bioenergetics.

Figure 1. Dynamic mitochondria in skeletal muscle in vivo.

A, Representative confocal image of mtPAGFP expression in skeletal muscle in anesthetic mouse. Numbers 1, 2, 3 represent three different skeletal muscle fibers. Scale bar: 20 μm. B, Magnification of the rectangle region in (A). Scale bar: 5 μm. C, Representative image showed the distribution and morphology of mitochondria in skeletal muscle after photoactivation of mtPAGFP. D, Redistribution of activated mtPAGFP 30 min after photoactivation. Dashed brown rectangle indicated photoactivated region. Scale bar: 5 μm.

Figure 2. Mitochondrial fusion in skeletal muscle via nanotunneling.

A, Confocal images showed the processes of extending of mitochondrial nanotubule and fusion with neighboring non-activated mitochondrion. Arrows: mitochondrial nanotubule. Scale bar: 2 μm. B, Time-course of fluorescence showed the fluorescence change during mitochondrial fusion.

Figure 3. Mitochondrial network in skeletal muscle in vivo.

A and C, Representative images showed the different morphologies of mitochondrial network soon after photoactivation of mtPAGFP in skeletal muscle. Arrows indicated the interconnected mitochondrial network. Dashed brown rectangle indicated photoactivated region. Scale bar: 2 μm. B and D, Time-course of fluorescence showed synchronous changes of fluorescence intensity within the mitochondrial network.

Figure 4. Biochemical characterizations of HFD-induced obese mice.

A, Body weights of both normal diet (ND) and HFD-induced mice at the age of 44 weeks. B, Blood glucose contents both before and after food intake. C, D and E, Levels of serum insulin (C), cholesterol (D) and triglyceride (E) in control and HFD-induced mice. n = 10 mice for each group. *, P<0.05 compared to ND.

Figure 5. Inhibition of mitochondrial dynamics in skeletal muscle of HFD-induced mice.

A and B, Representative confocal images showed the propagation of activated mtPAGFP in normal diet (ND) (A) and HFD-induced mice (B) 30 min after photoactivation. Dashed brown rectangle indicated photoactivated region with a size of 6.0 μm×6.0 μm. C and D, Statistic analysis on propagated distance (C) along longitudinal direction of the cell and decreased fluorescence of mtPAGFP of activated region (D) in (A) and (B). n = 6 mice for each group. *, P<0.05 compared to ND.

Figure 6. Impaired mitochondrial oxygen consumption and ATP production in skeletal muscle of HFD-induced obese mice.

A and B, Relative oxygen consumption rate of state IV (B) and state III (A) in isolated mitochondria challenged by glutamate/malate and ADP. The rates in normal diet (ND) group was normalized to 1.00. n = 6 mice for each group. C, Respiratory control ratios (ratio of oxygen consumption rate of state III/state IV) in ND and HFD-induced mice. n = 10 mice for each group. D, Relative cellular ATP content in skeletal muscle from ND and HFD-induced mice. The ATP content in ND group was normalized to 1.00. n = 9 mice for each group. *, P<0.05 compared to ND.

Figure 7. Expression of proteins regulating mitochondrial fusion and fission.

A, Representative images of protein expression in skeletal muscle detected by western blot. B and C, The statistical results of normalized relative protein regulating mitochondrial fusion and fission/GADPH (B) or cytochrome c oxidase IV (COX IV) (C) ratio in skeletal muscle of HFD-induced mice when compared to control mice. n = 6 mice for each group. *, P<0.05 compared to ND.