Hazel A. Lees

Skeletal muscle autophagy and apoptosis during aging: effects of calorie restriction and life-long exercise

Sarcopenia, loss of muscle mass and function, is a common feature of aging. Oxidative damage and apoptosis are likely underlying factors. Autophagy, a process for the degradation of cellular constituents, may be a mechanism to combat cell damage and death. We investigated the effect of age on autophagy and apoptosis in plantaris muscle of male Fischer 344 rats that were either fed ad libitum, or mild, life-long calorie restricted (CR) alone or combined with life-long voluntary exercise. Upstream autophagy-regulatory proteins were either upregulated with age (Beclin-1) or unchanged (Atg7 and 9). LC3 gene and protein expression pattern as well as LAMP-2 gene expression, both downstream regulators of autophagy, however, suggested an age-related decline in autophagic degradation. Atg protein expression and LC3 and LAMP-2 gene expression were improved in CR rats with or without exercise. The age-related increase in oxidative damage and apoptosis were attenuated by the treatments. Both, oxidative damage and apoptosis correlated negatively with autophagy. We conclude that mild CR attenuates the age-related impairment of autophagy in rodent skeletal muscle, which might be one of the mechanisms by which CR attenuates age-related cellular damage and cell death in skeletal muscle in vivo.

The Impact of Aging on Mitochondrial Function and Biogenesis Pathways in Skeletal Muscle of Sedentary High- and Low-Functioning Elderly Individuals

Age‐related loss of muscle mass and strength (sarcopenia) leads to a decline in physical function and frailty in the elderly. Among the many proposed underlying causes of sarcopenia, mitochondrial dysfunction is inherent in a variety of aged tissues. The intent of this study was to examine the effect of aging on key groups of regulatory proteins involved in mitochondrial biogenesis and how this relates to physical performance in two groups of sedentary elderly participants, classified as high‐ and low‐functioning based on the Short Physical Performance Battery test. Muscle mass was decreased by 38% and 30% in low‐functioning elderly (LFE) participants when compared to young and high‐functioning elderly participants, respectively, and positively correlated to physical performance. Mitochondrial respiration in permeabilized muscle fibers was reduced (41%) in the LFE group when compared to the young, and this was associated with a 30% decline in cytochrome c oxidase activity. Levels of key metabolic regulators, SIRT3 and PGC‐1α, were significantly reduced (50%) in both groups of elderly participants when compared to young. Similarly, the fusion protein OPA1 was lower in muscle from elderly subjects; however, no changes were detected in Mfn2, Drp1 or Fis1 among the groups. In contrast, protein import machinery components Tom22 and cHsp70 were increased in the LFE group when compared to the young. This study suggests that aging in skeletal muscle is associated with impaired mitochondrial function and altered biogenesis pathways and that this may contribute to muscle atrophy and the decline in muscle performance observed in the elderly population.

Age-related activation of mitochondrial caspase-independent apoptotic signaling in rat gastrocnemius muscle

Mitochondria-mediated apoptosis represents a central process driving age-related muscle loss. However, the temporal relation between mitochondrial apoptotic signaling and sarcopenia as well as the regulation of release of pro-apoptotic factors from the mitochondria has not been elucidated. In this study, we investigated mitochondrial apoptotic signaling in skeletal muscle of rats across a wide age range. We also investigated whether mitochondrial-driven apoptosis was accompanied by changes in the expression of Bcl-2 proteins and components of the mitochondrial permeability transition pore (mPTP). Analyses were performed on gastrocnemius muscle of 8-, 18-, 29- and 37-month-old male Fischer344 x Brown Norway rats (9 per group). Muscle weight declined progressively with advancing age, concomitant with increased apoptotic DNA fragmentation. Cytosolic and nuclear levels of apoptosis inducing factor (AIF) and endonuclease G (EndoG) increased in old and senescent animals. In contrast, cytosolic levels of cytochrome c were unchanged with age. Mitochondrial Bcl-2, Bax and Bid increased dramatically in 37-month-old rats, with no changes in the Bax/Bcl-2 ratio in any of the age groups. Finally, expression of cyclophilin D (CyPD) was enhanced at very old age. Our findings indicate that the mitochondrial caspase-independent apoptotic pathway may play a more prominent role in skeletal muscle loss than caspase-mediated apoptosis.

Sarcopenia of aging: Underlying cellular mechanisms and protection by calorie restriction

Sarcopenia, the loss of muscle mass and function, is a common feature of aging and impacts on individual health and quality of life. Several cellular mechanisms have been involved in the pathogenesis of this syndrome, including mitochondrial dysfunction, altered apoptotic and autophagic signaling, and, more recently, trace metal dyshomeostasis. Calorie restriction (CR) without malnutrition has been shown to ameliorate the age‐related loss of muscle mass in a variety a species. Mechanisms of protection span from preservation of mitochondrial functional and structural integrity to mitochondrial biogenesis, reduction of oxidative stress, and favorable modulation of apoptotic and autophagic signaling pathways. Importantly, preliminary evidence indicates that moderate CR may promote muscle mitochondrial biogenesis in middle‐aged human subjects. Further research is warranted to investigate whether CR may represent a safe and efficient strategy to delay the onset and mitigate the progression of sarcopenia in older adults.

Mitochondrial death effectors: relevance to sarcopenia and disuse muscle atrophy.

Accelerated apoptosis in skeletal muscle is increasingly recognized as a potential mechanism contributing to the development of sarcopenia of aging and disuse muscle atrophy. Given their central role in the regulation of apoptosis, mitochondria are regarded as key players in the pathogenesis of myocyte loss during aging and other atrophying conditions. Oxidative damage to mitochondrial constituents, impaired respiration and altered mitochondrial turnover have been proposed as potential triggering events for mitochondrial apoptotic signaling. In addition, iron accumulation within mitochondria may enhance the susceptibility to apoptosis during the development of sarcopenia and possibly acute muscle atrophy, likely through exacerbation of oxidative stress. Mitochondria can induce myocyte apoptosis via both caspase-dependent and independent pathways, although the apoptogenic mediators involved may be different depending on age, muscle type and specific atrophying conditions. Despite the considerable advances made, additional research is necessary to establish a definite causal link between apoptotic signaling and the development of sarcopenia and acute atrophy. Furthermore, a translational effort is required to determine the role played by apoptosis in the pathogenesis of sarcopenia and disuse-induced muscle loss in human subjects.

Changes in IL-15 expression and death-receptor apoptotic signaling in rat gastrocnemius muscle with aging and life-long calorie restriction.

TNF-alpha-mediated apoptosis is enhanced in aged rodent muscles, suggesting that this pathway may be involved in sarcopenia. Interleukin-15 (IL-15), a muscle-derived anabolic cytokine, mitigates muscle wasting and apoptosis in cachectic rats. This effect is thought to occur through inhibition of TNF-alpha-triggered apoptosis. We investigated IL-15 signaling and the TNF-alpha-mediated pathway of apoptosis in the gastrocnemius muscle of Fischer344xBrown Norway rats across the ages of 8, 18, 29 and 37 months, in relation to life-long calorie restriction (CR, 40% calorie intake reduction). Aging caused loss of muscle mass and increased apoptotic DNA fragmentation, which were mitigated by CR. Protein levels of IL-15 and mRNA abundance of IL-15 receptor a-chain decreased in senescent ad libitum (AL) fed rats, but were maintained in CR rodents. Elevations of TNF-alpha, TNF-receptor 1, cleaved caspase-8 and -3 were observed at advanced age in AL rats. These changes were prevented or mitigated by CR. Our results indicate that aging is associated with decreased IL-15 signaling in rat gastrocnemius muscle, which may contribute to sarcopenia partly through enhanced TNF-alpha-mediated apoptosis. Preservation of IL-15 signaling by CR may therefore represent a further mechanism contributing to the anti-aging effect of this dietary intervention in skeletal muscle.

Myogenic and proteolytic mRNA expression following blood flow restricted exercise.

Aim:  Resistance exercise performed at low loads (20–30% of maximal strength) with blood flow restriction (BFR) acutely increases protein synthesis and induces hypertrophy when performed chronically. We investigated myogenic and proteolytic mRNA expression 8 h following an acute bout of knee extension exercise.

Skeletal muscle apoptotic signaling predicts thigh muscle volume and gait speed in community-dwelling older persons: an exploratory study.

Background Preclinical studies strongly suggest that accelerated apoptosis in skeletal myocytes may be involved in the pathogenesis of sarcopenia. However, evidence in humans is sparse. In the present study, we investigated whether apoptotic signaling in the skeletal muscle was associated with indices of muscle mass and function in older persons. Methodology/Principal Findings Community-dwelling older adults were categorized into high-functioning (HF) or low-functioning (LF) groups according to their short physical performance battery (SPPB) summary score. Participants underwent an isokinetic knee extensor strength test and 3-dimensional magnetic resonance imaging of the thigh. Vastus lateralis muscle samples were obtained by percutaneous needle biopsy and assayed for the expression of a set of apoptotic signaling proteins. Age, sex, number of comorbid conditions and medications as well as knee extensor strength were not different between groups. HF participants displayed greater thigh muscle volume compared with LF persons. Multivariate partial least squares (PLS) regressions showed significant correlations between caspase-dependent apoptotic signaling proteins and the muscular percentage of thigh volume (R2 = 0.78; Q2 = 0.61) as well as gait speed (R2 = 0.81; Q2 = 0.56). Significant variables in the PLS model of percent muscle volume were active caspase-8, cleaved caspase-3, cytosolic cytochrome c and mitochondrial Bak. The regression model of gait speed was mainly described by cleaved caspase-3 and mitochondrial Bax and Bak. PLS predictive apoptotic variables did not differ between functional groups. No correlation was determined between apoptotic signaling proteins and muscle strength or quality (strength per unit volume). Conclusions/Significance Data from this exploratory study show for the first time that apoptotic signaling is correlated with indices of muscle mass and function in a cohort of community-dwelling older persons. Future larger-scale studies are needed to corroborate these preliminary findings and determine if down-regulation of apoptotic signaling in skeletal myocytes will provide improvements in the muscle mass and functional status of older persons.

An Exploratory Analysis of the Effects of a Weight Loss Plus Exercise Program on Cellular Quality Control Mechanisms in Older Overweight Women

Obese older adults are particularly susceptible to sarcopenia and have a higher prevalence of disability than their peers of normal weight. Interventions to improve body composition in late life are crucial to maintaining independence. The main mechanisms underlying sarcopenia have not been determined conclusively, but chronic inflammation, apoptosis, and impaired mitochondrial function are believed to play important roles. It has yet to be determined whether impaired cellular quality control mechanisms contribute to this process. The objective of this study was to assess the effects of a 6-month weight loss program combined with moderate-intensity exercise on the cellular quality control mechanisms autophagy and ubiquitin-proteasome, as well as on inflammation, apoptosis, and mitochondrial function, in the skeletal muscle of older obese women. The intervention resulted in significant weight loss (8.0 ± 3.9 % vs. 0.4 ± 3.1% of baseline weight, p = 0.002) and improvements in walking speed (reduced time to walk 400 meters, - 20.4 ± 16% vs. - 2.5 ± 12%, p = 0.03). In the intervention group, we observed a three-fold increase in messenger RNA (mRNA) levels of the autophagy regulators LC3B, Atg7, and lysosome-associated membrane protein-2 (LAMP-2) compared to controls. Changes in mRNA levels of FoxO3A and its targets MuRF1, MAFBx, and BNIP3 were on average seven-fold higher in the intervention group compared to controls, but these differences were not statistically significant. Tumor necrosis factor-α (TNF-α) mRNA levels were elevated after the intervention, but we did not detect significant changes in the downstream apoptosis markers caspase 8 and 3. Mitochondrial biogenesis markers (PGC1α and TFAm) were increased by the intervention, but this was not accompanied by significant changes in mitochondrial complex content and activity. In conclusion, although exploratory in nature, this study is among the first to report the stimulation of cellular quality control mechanisms elicited by a weight loss and exercise program in older obese women.

Long-term perturbation of muscle iron homeostasis following hindlimb suspension in old rats is associated with high levels of oxidative stress and impaired recovery from atrophy

In the present study, we investigated the effects of 7 and 14 days of re-loading following 14-day muscle unweighting (hindlimb suspension, HS) on iron transport, non-heme iron levels and oxidative damage in the gastrocnemius muscle of young (6 months) and old (32 months) male Fischer 344×Brown Norway rats. Our results demonstrated that old rats had lower muscle mass, higher levels of total non-heme iron and oxidative damage in skeletal muscle in comparison with young rats. Non-heme iron concentrations and total non-heme iron amounts were 3.4- and 2.3-fold higher in aged rats as compared with their young counterparts, respectively. Seven and 14 days of re-loading was associated with higher muscle weights in young animals as compared with age-matched HS rats, but there was no difference in muscle weights among aged HS, 7 and 14 days of re-loading rats, indicating that aged rats may have a lower adaptability to muscle disuse and a lower capacity to recover from muscle atrophy. Protein levels of cellular iron transporters, such as divalent metal transport-1 (DMT1), transferrin receptor-1 (TfR1), Zip14, and ferroportin (FPN), and their mRNA abundance were determined. TfR1 protein and mRNA levels were significantly lower in aged muscle. Seven and 14 days of re-loading were associated with higher TfR1 mRNA and protein levels in young animals in comparison with their age-matched HS counterparts, but there was no difference between cohorts in aged animals, suggesting adaptive responses in the old to cope with iron deregulation. The extremely low expression of FPN in skeletal muscle might lead to inefficient iron export in the presence of iron overload and play a critical role in age-related iron accumulation in skeletal muscle. Moreover, oxidative stress was much greater in the muscles of the older animals measured as 4-hydroxy-2-nonhenal (HNE)-modified proteins and 8-oxo-7,8-dihydroguanosine levels. These markers remained fairly constant with either HS or re-loading in young rats. In old rats, HNE-modified proteins and 8-oxo-7,8-dihydroguanosine levels were markedly higher in HS and were lower after 7 days of recovery. However, no difference was observed following 14 days of recovery between control and re-loading animals. In conclusion, advanced age is associated with disruption of muscle iron metabolism which is further perturbed by disuse and persists over a longer time period.