HyPer2 imaging reveals temporal and heterogeneous hydrogen peroxide changes in denervated and aged skeletal muscle fibers in vivo

Abstract

To determine the role of denervation and motor unit turnover in the age-related increase in skeletal muscle oxidative stress, the hydrogen peroxide (H2O2) specific, genetically-encoded, fluorescent cyto-HyPer2 probe was expressed in mouse anterior tibialis (AT) muscle and compared with ex vivo measurements of mitochondrial oxidant generation. Crush of the peroneal nerve induced increased mitochondrial peroxide generation, measured in permeabilised AT fibers ex vivo and intra vital confocal microscopy of cyto-HyPer2 fluorescence showed increased cytosolic H2O2 in a sub-set (~24%) of individual fibers associated with onset of fiber atrophy. In comparison, mitochondrial peroxide generation was also increased in resting muscle from old (26 month) mice compared with adult (6–8 month) mice, but no age effect on fiber cytosolic H2O2in vivo was seen. Thus ageing is associated with an increased ability of muscle fibers to maintain cytosolic redox homeostasis in the presence of denervation-induced increase in mitochondrial peroxide generation.

Deletion of Sod1 in motor neurons exacerbates age-related changes in axons and NMJs associated with premature muscle atrophy in aging mice

“…Fragmentation of the NMJs was quantified by counting the number of AchR clusters that were clearly separate and these were categorised as previously (Staunton et al, 2019). An intact NMJ was recorded as “none” (i.e.…”

Skeletal muscle transcriptomics identifies common pathways in nerve crush injury and ageing

“…Mice were anaesthetised using isoflurane, the hind limb was shaved, antiseptic skin cleanser (Videne) applied and buprenorphine given (100 μl, 0.3 mg/ml). All animals were maintained under gaseous anaesthesia throughout and all surgery carried out as per Staunton et al [ 99 ]. A small incision (5 mm) was made on the outer side of the limb and the peroneal nerve exposed.…”

“…These factors are believed to cause “self-repair” and facilitate re-growth of the damaged axons and aid recruitment of the re-growing axon to the denervated muscle fibre. This re-growth is evident from intra-vital imaging of the NMJ in adult mice following nerve crush [ 99 ], but the response in aged mice and the mechanisms behind the responses are yet to be defined. Data indicate that lack of neuromuscular transmission is the prime stimulus for release of sprouting factors and these factors are anterograde signals, from Schwann cells or muscle fibres as early as 3 days post injury that ultimately leads to sprouting [ 28 , 98 ].…”

On the mechanisms underlying attenuated redox responses to exercise in older individuals: A hypothesis

“…Data from muscles of old mice show a discrepancy between mitochondrial peroxide generation (which was substantially increased in muscle of old compared with adult mice) and cytosolic H 2 O 2 content where no increase was seen in individual muscle fibres from old compared with adult mice ( Fig. 3 ) [ 94 ]. Thus, these data show clear compartmentalisation of ROS activities with preservation of the muscle cytosolic H 2 O 2 content in the presence of substantial increases in mitochondrial peroxide generation in aging.…”

“…***p < 0.001 Reproduced from Ref. [ 94 ]. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)…”