Denervation Causes Fiber Atrophy and Myosin Heavy Chain Co-Expression in Senescent Skeletal Muscle

Although denervation has long been implicated in aging muscle, the degree to which it is causes the fiber atrophy seen in aging muscle is unknown. To address this question, we quantified motoneuron soma counts in the lumbar spinal cord using choline acetyl transferase immunhistochemistry and quantified the size of denervated versus innervated muscle fibers in the gastrocnemius muscle using the in situ expression of the denervation-specific sodium channel, Nav1.5, in young adult (YA) and senescent (SEN) rats. To gain insights into the mechanisms driving myofiber atrophy, we also examined the myofiber expression of the two primary ubiquitin ligases necessary for muscle atrophy (MAFbx, MuRF1). MN soma number in lumbar spinal cord declined 27% between YA (638±34 MNs×mm−1) and SEN (469±13 MNs×mm−1). Nav1.5 positive fibers (1548±70 μm2) were 35% smaller than Nav1.5 negative fibers (2367±78 μm2; P<0.05) in SEN muscle, whereas Nav1.5 negative fibers in SEN were only 7% smaller than fibers in YA (2553±33 μm2; P<0.05) where no Nav1.5 labeling was seen, suggesting denervation is the primary cause of aging myofiber atrophy. Nav1.5 positive fibers had higher levels of MAFbx and MuRF1 (P<0.05), consistent with involvement of the proteasome proteolytic pathway in the atrophy of denervated muscle fibers in aging muscle. In summary, our study provides the first quantitative assessment of the contribution of denervation to myofiber atrophy in aging muscle, suggesting it explains the majority of the atrophy we observed. This striking result suggests a renewed focus should be placed on denervation in seeking understanding of the causes of and treatments for aging muscle atrophy

Denervation Causes Fiber Atrophy and Myosin Heavy Chain Co-Expression in Senescent Skeletal Muscle

Although denervation has long been implicated in aging muscle, the degree to which it is causes the fiber atrophy seen in aging muscle is unknown. To address this question, we quantified motoneuron soma counts in the lumbar spinal cord using choline acetyl transferase immunhistochemistry and quantified the size of denervated versus innervated muscle fibers in the gastrocnemius muscle using the in situ expression of the denervation-specific sodium channel, Nav1.5, in young adult (YA) and senescent (SEN) rats. To gain insights into the mechanisms driving myofiber atrophy, we also examined the myofiber expression of the two primary ubiquitin ligases necessary for muscle atrophy (MAFbx, MuRF1). MN soma number in lumbar spinal cord declined 27% between YA (638±34 MNs×mm−1) and SEN (469±13 MNs×mm−1). Nav1.5 positive fibers (1548±70 μm2) were 35% smaller than Nav1.5 negative fibers (2367±78 μm2; P<0.05) in SEN muscle, whereas Nav1.5 negative fibers in SEN were only 7% smaller than fibers in YA (2553±33 μm2; P<0.05) where no Nav1.5 labeling was seen, suggesting denervation is the primary cause of aging myofiber atrophy. Nav1.5 positive fibers had higher levels of MAFbx and MuRF1 (P<0.05), consistent with involvement of the proteasome proteolytic pathway in the atrophy of denervated muscle fibers in aging muscle. In summary, our study provides the first quantitative assessment of the contribution of denervation to myofiber atrophy in aging muscle, suggesting it explains the majority of the atrophy we observed. This striking result suggests a renewed focus should be placed on denervation in seeking understanding of the causes of and treatments for aging muscle atrophy

Spinal cord motoneuron counts.

<p>A: Photomicrograph of choline acetyl transferase labeled cross-sections of the spinal cord from a young adult rat and B: senescent rat (scale bar = 400 µm). Insets for both panels are higher power images showing motoneuron soma (arrows) (scale bar = 40 µm). C: mean motoneuron counts for young adult (YA) and senescent (SEN) rats. *P<0.05 versus YA.</p

Alterations in myofiber Nav_1.5 expression with aging.

<p>Photomicrographs of serial sections of the red region of gastrocnemius muscle labeled for Nav_1.5 (A,C) and MHC slow (B,D) in young adult (A,B) and senescent (C,D) rats. In senescent muscle we saw three classes of Nav_1.5 labelling; negative (N), ringed (displaying circumferential labelling, open arrowhead) and cytoplasm (showing cytoplasmic staining, shaded arrowhead). Scale bar is 50 µm.</p

Fiber type changes with aging.

<p>Photomicrographs of serial sections labeled for myosin heavy chain (MHC) slow (A,C) and MHC fast (B,D) within the red region of gastrocnemius muscle from a young adult (A,B) and senescent (C,D) rat. In these images 1 denotes a MHCs fiber, 2 denotes a MHCf fiber, and 3 denotes a fiber co-expressing both MHCs and MHCf. Scale bar is 50 µm.</p

Schematic representation of atrophy in MHC slow versus MHC fast myofibers following denervation in aging muscle.

<p>Thickness of arrow indicates relative proportion of fibers taking a given path. Briefly, upon denervation, MHC slow fibers exhibit increased MAFbx expression, followed by the majority of denervated fibers beginning to co-express MHC fast, coincident with initiation of atrophy. Upon denervation of MHC fast fibers, MAFbx is unchanged but atrophy is already seen, followed by a majority of denervated fibers beginning to co-express MHC slow. MuRF1 increases with prolonged duration of denervation in all MHC categories and is associated with further atrophy in MHC fast and MHC co-expressing myofibers.</p

In Situ myofiber ubiquitin ligase expression with aging.

<p>Serial images of the red region of gastrocnemius muscle labeled for MHC slow (A,B), MAFbx (C,D) and MuRF1 (E,F) in young adult (YA) (A,C,E) and senescent (SEN) (B,D,F) rats. Fibers sharing the same number are the same fiber in serial sections. Scale bar is 100 µm.</p

Protective role of Parkin in skeletal muscle contractile and mitochondrial function

International audienceParkin is an E3 ubiquitin ligase encoded by the Park2 gene. Parkin has been implicated in the regulation of mitophagy, a quality control process in which defective mitochondria are sequestered in autophagosomes and delivered to lysosomes for degradation. Although Parkin has been mainly studied for its implication in neuronal degeneration in Parkinson disease, its role in other tissues remains largely unknown. In the present study, we investigated the skeletal muscles of Park2 knockout (Park2-/- ) mice to test the hypothesis that Parkin plays a physiological role in mitochondrial quality control in normal skeletal muscle, a tissue highly reliant on mitochondrial content and function. We first show that the tibialis anterior (TA) of Park2-/- mice display a slight but significant decrease in its specific force. Park2-/- muscles also show a trend for type IIB fibre hypertrophy without alteration in muscle fibre type proportion. Compared to Park2+/+ muscles, the mitochondrial function of Park2-/- skeletal muscles was significantly impaired, as indicated by the significant decrease in ADP-stimulated mitochondrial respiratory rates, uncoupling, reduced activities of respiratory chain complexes containing mitochondrial DNA (mtDNA)-encoded subunits and increased susceptibility to opening of the permeability transition pore. Muscles of Park2-/- mice also displayed a decrease in the content of the mitochondrial pro-fusion protein Mfn2 and an increase in the pro-fission protein Drp1 suggesting an increase in mitochondrial fragmentation. Finally, Park2 ablation resulted in an increase in basal autophagic flux in skeletal muscles. Overall, the results of the present study demonstrate that Parkin plays a protective role in the maintenance of normal mitochondrial and contractile functions in normal skeletal muscles