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

Abstract

Responding appropriately to exercise is essential to maintenance of skeletal muscle mass and function at all ages and particularly during aging. Here, a hypothesis is presented that a key component of the inability of skeletal muscle to respond effectively to exercise in aging is a denervation-induced failure of muscle redox signalling. This novel hypothesis proposes that an initial increase in oxidation in muscle mitochondria leads to a paradoxical increase in the reductive state of specific cysteines of signalling proteins in the muscle cytosol that suppresses their ability to respond to normal oxidising redox signals during exercise. The following are presented for consideration:Transient loss of integrity of peripheral motor neurons occurs repeatedly throughout life and is normally rapidly repaired by reinnervation, but this repair process becomes less efficient with aging. Each transient loss of neuromuscular integrity leads to a rapid, large increase in mitochondrial peroxide production in the denervated muscle fibers and in neighbouring muscle fibers. This peroxide may initially act to stimulate axonal sprouting and regeneration, but also stimulates retrograde mitonuclear communication to increase expression of a range of cytoprotective proteins in an attempt to protect the fiber and neighbouring tissues against oxidative damage. The increased peroxide within mitochondria does not lead to an increased cytosolic peroxide, but the increases in adaptive cytoprotective proteins include some located to the muscle cytosol which modify the local cytosol redox environment to induce a more reductive state in key cysteines of specific signalling proteins. Key adaptations of skeletal muscle to exercise involve transient peroxiredoxin oxidation as effectors of redox signalling in the cytosol. This requires sensitive oxidation of key cysteine residues. In aging, the chronic change to a more reductive cytosolic environment prevents the transient oxidation of peroxiredoxin 2 and hence prevents essential adaptations to exercise, thus contributing to loss of muscle mass and function. Experimental approaches suitable for testing the hypothesis are also outlined.

Keywords: Aging; Exercise; Muscle; Redox; Training.

Graphical abstract

Fig. 1

Potential mechanisms underlying generation of ROS (superoxide and H₂O₂) during muscle contractions and activation of key redox-sensitive signalling molecules involved in muscle adaptations to the contractile activity. From Ref. [44].

Fig. 2

A. Images of the peroneal nerve in Thy1-YFP mice to show one of the 3 branches of the peroneal nerve (indicated by * in the figure) transected immediately prior to entry into the Tibialis Anterior (TA) muscle leaving 2 branches intact. This image was taken at 7 days post-transection showing lack of any regrowth of the nerve (a). Intact muscles stained with α-bungarotoxin to visualise the AChR showed variability in NMJ structure (b). At 7 days post-surgery, four regions were identified where: all fibers had lost axonal input but retained AChR (R1), all fibers retained full innervation (R3), or fibers had a mix of innervated and some denervated fibers (R2, R4). Reproduced from Ref. [79]. B.The regions identified in Fig. 2A in longitudinal section were identified on transverse sections of the TA muscle and small bundles of fibers were obtained from each region (b). These fibers were permeablised and state 1 mitochondrial peroxide generation examined at 7 (b) days post-surgery in comparison with sham-operated control muscles; *P < 0.05 compared with fibers from the same region of sham-operated muscles. Reproduced from Ref. [79].

Fig. 3

A.Representative fluorescent images of NMJ's from adult (6–8 months) (A) and old (26 months) (B) Thy1-CFP mice showing the pre-synaptic terminal motor nerves (blue) and motor endplates stained with α-bungarotoxin (red) (scale bar = 50 μm). No significant difference was observed between the cytosolic H₂O₂ content (indicated by the cyto-HyPer2 fluorescence) of transfected AT muscles from adult and old mice when assessing the ratio of emissions at 516 nm after 488 and 405 excitation (488/405 ratio) from individual fibers (C). Mitochondrial peroxide generation monitored by the rate of amplex red oxidation (expressed as H₂O₂ generation) during state 1 respiration (i.e. in the absence of added substrates) from permeablised AT fibers from adult and old mice (D). ***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.)

Fig. 4

A. Percentage of Prx1, 2 and 3 found in the oxidised (dimerised) form on non-denaturing gels from FDB muscle fibers from adult (dark blue bars) and old (light blue bars) mice. The graphs show mean ± SEM. *P < 0.05. Redrawn from Stretton et al., [102]. B.Change in the Cys Redox ratio in specific cysteines of peptides found in the gastrocnemius muscles from old and adult mice. Data are expressed as the relative change with increasing age in the reduced/oxidised form (i.e. a ratio greater than one indicates an increased proportion in the reduced form in old mice). Redrawn from Ref. [103]. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

A. Relative abundance of key H₂O₂ -metabolizing enzymes in single type II skeletal muscle fibers. Redrawn from data published by Murgia et al. [108]. B. Selectivity of hydrogen peroxide for peroxiredoxins, protein tyrosine phosphatases and GSH at estimated cellular concentrations, reproduced from Winterbourn [110]. Calculations were performed using competition kinetic analysis. The ratio (r) of the amounts reacting with two substrates is derived from the second-order rate equation and is given by the expression r = k₁ [substrate 1]/k₂ [substrate 2]. With n substrates, the proportion of an oxidant reacting with substrate 1 is given by k₁ [substrate 1]/Σk_n [substrate n]. Concentrations and rate constants: GSH, 2 mM and 0.89 M⁻¹ s⁻¹; human peroxiredoxin 2 (Prx2), 20 μM and 2 × 10⁷ M⁻¹ s⁻¹; protein tyrosine phosphatase 1B (PTP1B), 0.1 μM and 20 M⁻¹ s⁻¹; cell cycle–activating phosphatase Cdc25B, 0.1 μM and 160 M⁻¹ s⁻¹.

Fig. 6

A. Proportion of Prx1, 2 and 3 in the oxidised (dimerised) form on denaturing gels from isolated fibers from the FDB of adult and old WT mice subjected to an electrical stimulation protocol for up to 15 min. Fibers were processed at the stated time points by incubation in 75 mM MMTS for 10 min and then lysed in the presence of MMTS. Lysates were subjected to Western blot analysis using non-reducing gels and the Prx antibodies. Results are presented as the percentage of the total Prx signal found in the dimerised form. Graphs show mean ± SEM. *P < 0.05, †P < 0.05 compared to adult baseline values, ƚ P < 0.05 compared to old baseline values. B. Published data from our group [63] showing the increase in Trx and TrxR in muscle from old compared with adult mice, *P < 0.05, **P < 0.001 compared with adult.

Fig. 7

A. Schematic representation of how peroxides generated in muscle mitochondria in response to denervation may suppress redox signalling of adaptations to contractions. It was originally envisaged that peroxides generated in mitochondria as a result of denervation might directly act on NADPH oxidase mediated redox signalling pathways, but compartmentalisation prevents such direct interference. It is proposed that the increased mitochondrial stress triggers reverse mitonuclear communication that leads to upregulation of regulatory pathways, such as increased Trx, GPx1, catalase [60] and Prx [63], that causes an increased reductive environment and suppression of redox-sensitive contraction-induced adaptations. B. Hypothetical representation of the redox status of critical cysteines involved in signalling responses to contractions in proteins such as the “peroxidatic” cysteine in Prx2. In adult mice at rest the redox balance reflects baseline generation of H₂O₂ and other oxidising agents and the local amount and activity of reductants and regulatory proteins. During exercise activation of NADPH oxidase leads to a shift to a more oxidised status which is sufficient to lead to oxidation of further signalling proteins potentially through a redox relay system. In old mice at rest the redox status of the critical cysteine is shifted to a more reduced state due to the adaptive upregulation of proteins, such as Trx1 which reduce the critical cysteine. During exercise in old mice the oxidation stimulus achieved by activation of NADPH oxidase is therefore insufficient to modify the cysteine sufficiently to activate the signalling pathways.

Fig. 8

It is envisaged that small episodes of denervation, such as loss of single terminal axons occur frequently throughout life and that this is rapidly repaired by axonal sprouting and outgrowth. Each cycle of denervation and re-innervation leads to a transient increase in mitochondrial peroxide generation in the denervated and neighbouring muscle fibers which cause minor oxidative damage and induction of adaptative responses to prevent further oxidative damage. The repetition of this process many times over a lifetime eventually leads to the cumulative increase in oxidative damage seen in muscle tissue of older animals and man, and also to a cumulative adaptive increase in regulatory proteins designed to prevent oxidative damage, including those in the cytosol that cause a greater reduction in the redox status of critical cysteines (as in Fig. 7B). These changes thus lead to the attenuated redox responses to exercise seen in muscle from old mice and humans which compromises the ability to maintain muscle mass and function in old age.