Late-life enalapril administration induces nitric oxide-dependent and independent metabolic adaptations in the rat skeletal muscle.

Recently, we showed that administration of the angiotensin-converting enzyme inhibitor enalapril to aged rats attenuated muscle strength decline and mitigated apoptosis in the gastrocnemius muscle. The aim of the present study was to investigate possible mechanisms underlying the muscle-protective effects of enalapril. We also sought to discern the effects of enalapril mediated by nitric oxide (NO) from those independent of this signaling molecule. Eighty-seven male Fischer 344 × Brown Norway rats were randomly assigned to receive enalapril (n = 23), the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; n = 22), enalapril + L-NAME (n = 19), or placebo (n = 23) from 24 to 27 months of age. Experiments were performed on the tibialis anterior muscle. Total NOS activity and the expression of neuronal, endothelial, and inducible NOS isoforms (nNOS, eNOS, and iNOS) were determined to investigate the effects of enalapril on NO signaling. Transcript levels of tumor necrosis factor-alpha (TNF-α) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were assessed to explore actions of enalapril on inflammation and mitochondrial biogenesis, respectively. Protein expression of energy-sensing and insulin signaling mediators, including protein kinase B (Akt-1), phosphorylated Akt-1 (pAkt-1), mammalian target of rapamycin (mTOR), AMP-activated protein kinase subunit alpha (AMPKα), phosphorylated AMPKα (pAMPKα), and the glucose transporter GLUT-4, was also determined. Finally, the generation of hydrogen peroxide (H2O2) was quantified in subsarcolemmal (SSM) and intermyofibrillar (IFM) mitochondria. Enalapril increased total NOS activity, which was prevented by L-NAME co-administration. eNOS protein content was enhanced by enalapril, but not by enalapril + L-NAME. Gene expression of iNOS was down-regulated by enalapril either alone or in combination with L-NAME. In contrast, protein levels of nNOS were unaltered by treatments. The mRNA abundance of TNF-α was reduced by enalapril relative to placebo, with no differences among any other group. PCG-1α gene expression was unaffected by enalapril and lowered by enalapril + L-NAME. No differences in protein expression of Akt-1, pAkt-1, AMPKα, pAMPKα, or GLUT-4 were detected among groups. However, mTOR protein levels were increased by enalapril compared with placebo. Finally, all treatment groups displayed reduced SSM, but not IFM H2O2 production relative to placebo. Our data indicate that enalapril induces a number of metabolic adaptations in aged skeletal muscle. These effects result from the concerted modulation of NO and angiotensin II signaling, rather than from a dichotomous action of enalapril on the two pathways. Muscle protection by enalapril administered late in life appears to be primarily mediated by mitigation of oxidative stress and pro-inflammatory signaling.

The tomato cis-prenyltransferase gene family.

cis-prenyltransferases (CPTs) are predicted to be involved in the synthesis of long-chain polyisoprenoids, all with five or more isoprene (C5) units. Recently, we identified a short-chain CPT, neryl diphosphate synthase (NDPS1), in tomato (Solanum lycopersicum). Here, we searched the tomato genome and identified and characterized its entire CPT gene family, which comprises seven members (SlCPT1-7, with NDPS1 designated as SlCPT1). Six of the SlCPT genes encode proteins with N-terminal targeting sequences, which, when fused to GFP, mediated GFP transport to the plastids of Arabidopsis protoplasts. The SlCPT3-GFP fusion protein was localized to the cytosol. Enzymatic characterization of recombinant SlCPT proteins demonstrated that SlCPT6 produces Z,Z-FPP, and SlCPT2 catalyzes the formation of nerylneryl diphosphate while SlCPT4, SlCPT5 and SlCPT7 synthesize longer-chain products (C25-C55). Although no in vitro activity was demonstrated for SlCPT3, its expression in the Saccharomyces cerevisiae dolichol biosynthesis mutant (rer2) complemented the temperature-sensitive growth defect. Transcripts of SlCPT2, SlCPT4, SlCPT5 and SlCPT7 are present at low levels in multiple tissues, SlCPT6 is exclusively expressed in red fruit and roots, and SlCPT1, SlCPT3 and SlCPT7 are highly expressed in trichomes. RNAi-mediated suppression of NDPS1 led to a large decrease in ß-phellandrene (which is produced from neryl diphosphate), with greater reductions achieved with the general 35S promoter compared to the trichome-specific MKS1 promoter. Phylogenetic analysis revealed CPT gene families in both eudicots and monocots, and showed that all the short-chain CPT genes from tomato (SlCPT1, SlCPT2 and SlCPT6) are closely linked to terpene synthase gene clusters.

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.

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 (R(2) = 0.78; Q(2) = 0.61) as well as gait speed (R(2) = 0.81; Q(2) = 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.

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.

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.

Photosynthetic redox imbalance influences flavonoid biosynthesis in Lemna gibba.

Plants accumulate flavonoids in response to a myriad of environmental challenges, especially when exposed to ultraviolet (UV) radiation or situations causing oxidative stress. However, the origin and nature of the signal triggering their accumulation remain obscure. In this study, a group of flavonoids belonging to the flavone class was identified in Lemna gibba (duckweed). These flavones accumulated upon exposure to UV radiation, low temperature, copper and the photosynthetic electron transport (PET) inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and 1,2-dihydroxyanthraquinone (DHATQ). All of these stressors were also shown to promote PET chain (PETC) reduction; however, in the co-presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or a light regime that oxidized the PETC, flavonoid accumulation ceased. Chloroplast-derived reactive oxygen species (ROS) were not associated with all of the stress conditions that promoted both PETC reduction and flavonoid synthesis, indicating that ROS were not a strict requisite for flavonoid accumulation. Transcripts for the flavonoid biosynthetic genes, chalcone synthase (CHS) and chalcone isomerase, were similarly responsive to the PETC redox state, as were a panel of transcripts revealed by differential display PCR. Collectively, these results provide evidence that PETC redox status is one of the factors affecting flavonoid biosynthesis.

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.

Effects of short-term GH supplementation and treadmill exercise training on physical performance and skeletal muscle apoptosis in old rats.

Growth hormone (GH) supplementation at old age has been shown to improve body composition, although its effect on muscle performance is still debated. On the other hand, resistance training increases muscle mass and strength even when initiated at advanced age. In the present study, we investigated the effects of short-term GH supplementation and exercise training on physical performance and skeletal muscle apoptosis in aged rats. Old (28 mo) male Fischer 344 x Brown Norway rats were randomized to 4 wk of GH supplementation (300 mug subcutaneous, twice daily) or 4 wk of treadmill running or used as sedentary controls. Eight-month-old rats, sedentary or exercised, were used as young controls. Exercise training improved exercise capacity and muscle strength in old animals. In soleus muscle, age and exercise were not associated with significant changes in the extent of apoptosis. However, we detected an age-related increase of cleaved caspase-8 (+98%), cleaved caspase-3 (+136%), and apoptotic DNA fragmentation (+203%) in the extensor digitorum longus muscle of old sedentary rats, which was attenuated by exercise. GH administration neither ameliorated physical performance nor attenuated apoptosis in extensor digitorum longus and was associated with increased apoptosis in soleus muscle (+206% vs. old controls). Our findings indicate that a short-term program of exercise training started at advanced age reverses age-related skeletal muscle apoptosis and represents an effective strategy to improve physical performance. In contrast, short-term administration of GH late in life does not provide any protection against functional decline or muscle aging and may even accelerate apoptosis in slow-twitch muscles, such as the soleus.