Immunohistochemical analysis of myosin heavy chain (MHC) isoform expression in perinatal and adult rat diaphragm muscles was performed with antibodies which permitted the identification of all known MHC isoforms found in typical rat muscles. Isoform switching, leading to the emergence of the adult phenotype, was more complex than had been previously described. As many as four isoforms could be coexpressed in a single myofiber. Elimination of developmental isoforms did not usually result in the myofiber immediately achieving its adult phenotype. Activation of genes for specific adult isoforms might be delayed to puberty. For example, two of the three fast MHCs, MHC2X and MHC2A appeared perinatally, while MHC2B did not appear until 30 days postnatal. By Day 60 this isoform was present in approximately 27% of the myofibers, but in most myofibers expression of this isoform was transient (i.e., at Day greater than or equal to 115, less than 4% of the myofibers expressed MHC2B). Fibers which contained MHC beta/slow during the late fetal and early neonatal period coexpressed MHCemb. A marked increase in the frequency of fibers containing MHC beta/slow occurred between 4 and 21 days postnatal. These slow fibers arose from a population of myofibers which expressed MHCemb and MHCneo during their development, and they accounted for the majority of slow fibers found in the adult diaphragm. The adult myosin phenotype of the diaphragm myofibers (as determined with immunocytochemistry, and 5% SDS-PAGE) was not achieved until the rat was greater than or equal to 115 days old.

Emergence of mature myosin phenotype in rat diaphragm muscle

Mechanical ventilation (MV) is associated with diaphragmatic oxidative stress that contributes to both diaphragmatic atrophy and contractile dysfunction. However, the pathways responsible for oxidant production in the diaphragm during MV remain unknown. To address this issue, we tested the hypothesis that diaphragmatic nitric oxide synthase (NOS) activity is elevated during MV, resulting in nitration of diaphragmatic proteins. Rats were mechanically ventilated for 18 h, and time-matched, anesthetized but spontaneously breathing animals served as controls. Protein levels of endothelial NOS, inducible NOS, and neuronal NOS were measured in diaphragms from all animals. 3-Nitrotyrosine levels were also measured as an index of protein nitration, and S-nitrosothiol levels were measured as a marker of nitric oxide reactions with molecules containing sulfhydryl groups. Levels of nitrates and nitrites were measured as markers of stable end products of nitric oxide metabolism. Finally, as a marker of oxidative stress, diaphragmatic levels of reduced GSH were also analyzed. MV did not promote an increase in diaphragmatic protein levels of endothelial NOS or neuronal NOS. Moreover, inducible NOS was not detected in the diaphragms of either experimental group. Consistent with these findings, MV did not elevate diaphragmatic 3-nitrotyrosine levels in any subcellular fraction of the diaphragm, including the cytosolic, mitochondrial, membrane, and insoluble protein fractions. Moreover, prolonged MV did not elevate diaphragmatic levels of S-nitrosothiols, nitrate, or nitrite. Finally, prolonged MV significantly reduced diaphragmatic levels of GSH, which is consistent with diaphragmatic oxidative stress. Collectively, these data reveal that MV-induced oxidative stress in the diaphragm is not due to increases in nitric oxide production by NOS.

Diaphragmatic nitric oxide synthase is not induced during mechanical ventilation

Cellular homeostasis requires tight coordination between nucleus and mitochondria, organelles that each possesses their own genomes. Disrupted mitonuclear communication has been found to be implicated in many aging processes. However, little is known about mitonuclear signaling regulator in sarcopenia which is a major contributor to the risk of poor health-related quality of life, disability, and premature death in older people. High-temperature requirement protein A2 (HtrA2/Omi) is a mitochondrial protease and plays an important role in mitochondrial proteostasis. HtrA2mnd2(-/-) mice harboring protease-deficient HtrA2/Omi Ser276Cys missense mutants exhibit premature aging phenotype. Additionally, HtrA2/Omi has been established as a signaling regulator in nervous system and tumors. We therefore asked whether HtrA2/Omi participates in mitonuclear signaling regulation in muscle degeneration. Using motor functional, histological, and molecular biological methods, we characterized the phenotype of HtrA2mnd2(-/-) muscle. Furthermore, we isolated the gastrocnemius muscle of HtrA2mnd2(-/-) mice and determined expression of genes in mitochondrial unfolded protein response (UPRmt ), mitohormesis, electron transport chain (ETC), and mitochondrial biogenesis. Here, we showed that HtrA2/Omi protease deficiency induced denervation-independent skeletal muscle degeneration with sarcopenia phenotypes. Despite mitochondrial hypofunction, upregulation of UPRmt and mitohormesis-related genes and elevated total reactive oxygen species (ROS) production were not observed in HtrA2mnd2(-/-) mice, contrary to previous assumptions that loss of protease activity of HtrA2/Omi would lead to mitochondrial dysfunction as a result of proteostasis disturbance and ROS burst. Instead, we showed that HtrA2/Omi protease deficiency results in different changes between the expression of nuclear DNA- and mitochondrial DNA-encoded ETC subunits, which is in consistent with their transcription factors, nuclear respiratory factors 1 and 2, and coactivator peroxisome proliferator-activated receptor γ coactivator 1α. These results reveal that loss of HtrA2/Omi protease activity induces mitonuclear imbalance via differential regulation of mitochondrial biogenesis in sarcopenia. The novel mechanistic insights may be of importance in developing new therapeutic strategies for sarcopenia.

Loss of high-temperature requirement protein A2 protease activity induces mitonuclear imbalance via differential regulation of mitochondrial biogenesis in sarcopenia.

The loss of the ability of skeletal muscle to generate force is one of the most appropriate and valid means to quantify muscle damage. Routine measurements of maximal muscle force, however, include many potential sources of error, the most important of which may be a possible lack of central drive to the muscles. The aim of the present study was to determine the reliability of maximal isometric quadriceps muscle force and voluntary activation over a timescale that is typically employed to examine the aetiology of exercise-induced muscle damage. We also attempted to characterise the reliability of several twitch interpolation variables including the size of the interpolated twitch and the state (i.e. unpotentiated vs potentiated) and size of the resting twitch. Over a 7-day period, eight healthy active males performed repeated maximal voluntary isometric contractions (MVC) of the quadriceps (baseline and 2 h, 6 h, 24 h, 48 h, 72 h and 7 days post). Systematic variations in maximal muscle force, voluntary activation, interpolated twitch, unpotentiated twitch and potentiated twitch were not statistically significant (P>0.05) and 95% repeatability coefficients of ±76.03 N, ±4.42%, ± 8.44 N, ±25.92 N and ±43.58 N were observed, respectively. These data indicate that young healthy well-familiarised male subjects can reproduce their perceived maximal efforts both within and between days where activation levels of >90% are routinely achieved. Providing activation remains within these limits in the 7 days following an acute bout of exercise, the researcher would be 95% certain that exercise-induced muscle damage is present in individual subjects (taken from similar subject populations) if MVC force falls outside these limits.

Reliability of maximal muscle force and voluntary activation as a marker of exercise-induced muscle damage

Skeletal muscle adapts to the stress of contractile activity with a change in gene expression to yield a family of highly conserved cytoprotective proteins known as heat shock proteins (HSPs). These proteins function to restore cellular homeostasis and to protect the cell against further insults. The exercise-induced stress response of rodent muscle is now relatively well defined. Comparable data from human studies, however, are extremely limited and the stress response of human skeletal muscle is far from understood. The main aims of this thesis were to characterise the time-course and magnitude of response of the exercise-induced production of the major HSP families in human skeletal muscle. The role of increased muscle and core temperature in contributing to the exercise-induced production of HSPs was also investigated. Finally, the effects of training status on baseline muscle content of the major HSP families and on the magnitude of the exercise-induced stress response was also examined. All of the exercise related studies undertaken in this thesis employed a 45 min running exercise protocol on a motorised treadmill at an intensity corresponding to the lactate threshold. This protocol was characterised as `non-damaging' in nature as it resulted in no overt structural or functional damage to the muscle of young untrained (27 ±5 years), recreationally active (25 ±2 years) or aerobically trained male subjects (27 ±6 years), as evidenced by indirect indicators of muscle damage such as circulating levels of creatine kinase and maximal quadriceps isometric muscle force. The time-course and magnitude of the exercise-induced response of the major HSP families were characterised in an active young (24 ±4 years) male population. Muscle biopsies were obtained from the vastus lateralis muscle immediately prior to and at 24 h, 48 h, 72 h and 7 days post-exercise. Exercise induced significant and individually variable increases in HSP70, HSC70 and HSP60 content with peak increases typically occurring at 48 h post-exercise. In contrast, exercise did not induce significant increases in either HSP27, aB-crystallin, manganese superoxide dismutase (MnSOD) protein content or the activity of superoxide dismutase (SOD) and catalase. When examining baseline protein levels, HSC70, HSP27 and aB-crystallin appeared consistently expressed between subjects whereas HSP70 and MnSOD displayed marked individual variation of up to 3 and 1.5 fold, respectively. These data demonstrate a differential effect of aerobic exercise on specific HSPs. Data also demonstrate an individual variation in both basal HSP levels and in the magnitude of the stress response to acute exercise, which may be related to individual differences in training status. The role of increased muscle and core temperature in contributing to the exercise induced production of HSPs were subsequently investigated. Active young males (23 A: 3 years) underwent a passive heating protocol of 1h duration during which the temperature of the core and vastus lateralis muscle were increased to similar levels as that occurring during exercise. One limb was immersed in a tank containing warm water whilst the contra-lateral limb remained outside the tank and was not exposed to heat stress. Muscle biopsies were obtained from the vastus lateralis of both legs immediately prior to and at 48 h and 7 days post-heating. The heating protocol induced significant increases in rectal and muscle temperature of the heated leg whilst muscle temperature of the non-heated limb showed no significant change following heating. The heating protocol failed to induce significant increases in muscle content of HSP70, HSC70, HSP60, HSP27, aB-crystallin, MnSOD protein content or the activity of SOD and catalase in either the heated or non-heated leg. Data demonstrate that increases in both systemic and local muscle temperature per se appear not to be mediating the exercise-induced production of HSPs and suggest that non-heat-stress factors associated with muscle contractile activity are of more importance in mediating this response. The influences of aerobic training status on the basal levels of HSPs and on the magnitude of the exercise-induced stress response were also investigated. Muscle biopsies were obtained from the vastus lateralis of young trained (28 ±6 years) and untrained (29 ±6 years) male subjects immediately prior to and at 48 h and 7 days post-exercise. When comparing muscles at rest, trained subjects had significantly higher levels of aB-crystallin, HSP60 and MnSOD compared with untrained subjects. Trained subjects also had a tendency for higher levels of HSP70, HSC70 and total SOD activity compared with untrained subjects. In contrast to the active population examined earlier, neither the trained nor untrained subjects exhibited a stress response to exercise. The absence of a stress response in trained subjects is likely due to the increase in baseline defences and the customary nature of the exercise protocol. The absence of a stress response in untrained subjects may be due to the failure of the exercise protocol to elicit a proposed critical threshold intensity that is required to induce increases in muscle HSP content. This thesis has provided novel data for the literature and has significantly advanced our understanding of the exercise-induced stress response of human skeletal muscle. Future research should examine the effects of exercise intensity on muscle HSP production and investigate the role of reactive oxygen species in contributing to the response. The wider implications of the exercise-induced production of HSPs, such as their potential cytoprotective properties against related and non-related stressors, should also be examined. Key Words: molecular chaperones, oxidative stress, hyperthermia, stress proteins

The exercise-induced expression of heat shock proteins in human skeletal muscle : the role of evaluated muscle and core temperature and the influence of training status.

Mechanisms of Muscle Damage with Injectable Products

Characterisation of nf‐kb activation in regenerating fibres of old mice

atrophy muscle against immobilization-induced muscle Mitochondrial-targeted antioxidants protect skeletal

prolonged cycling exercise time to fatigue and potassium regulation during -acetylcysteine infusion on N Effects of intravenous

Effect of denervation on redox‐regulation in skeletal muscle

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