The myokine FGF21 associates with enhanced survival in ALS and mitigates stress-induced cytotoxicity.

Amyotrophic lateral sclerosis (ALS) is an age-related and fatal neurodegenerative disease characterized by progressive muscle weakness. There is marked heterogeneity in clinical presentation, progression, and pathophysiology with only modest treatments to slow disease progression. Molecular markers that provide insight into this heterogeneity are crucial for clinical management and identification of new therapeutic targets. In a prior muscle miRNA sequencing investigation, we identified altered FGF pathways in ALS muscle, leading us to investigate FGF21. We analyzed human ALS muscle biopsy samples and found a large increase in FGF21 expression with localization to atrophic myofibers and surrounding endomysium. A concomitant increase in FGF21 was detected in ALS spinal cords which correlated with muscle levels. FGF21 was increased in the SOD1G93A mouse beginning in presymptomatic stages. In parallel, there was dysregulation of the co-receptor, ß-Klotho. Plasma FGF21 levels were increased and high levels correlated with slower disease progression, prolonged survival, and increased body mass index. In NSC-34 motor neurons and C2C12 muscle cells expressing SOD1G93A or exposed to oxidative stress, ectopic FGF21 mitigated loss of cell viability. In summary, FGF21 is a novel biomarker in ALS that correlates with slower disease progression and exerts trophic effects under conditions of cellular stress.

Differentially co-expressed myofibre transcripts associated with abnormal myofibre proportion in chronic obstructive pulmonary disease.

BACKGROUND: Skeletal muscle dysfunction is a common extrapulmonary manifestation of chronic obstructive pulmonary disease (COPD). Alterations in skeletal muscle myosin heavy chain expression, with reduced type I and increased type II myosin heavy chain expression, are associated with COPD severity when studied in largely male cohorts. The objectives of this study were (1) to define an abnormal myofibre proportion phenotype in both males and females with COPD and (2) to identify transcripts and transcriptional networks associated with abnormal myofibre proportion in COPD. METHODS: Forty-six participants with COPD were assessed for body composition, strength, endurance and pulmonary function. Skeletal muscle biopsies from the vastus lateralis were assayed for fibre-type distribution and cross-sectional area via immunofluorescence microscopy and RNA-sequenced to generate transcriptome-wide gene expression data. Sex-stratified k-means clustering of type I and IIx/IIax fibre proportions was used to define abnormal myofibre proportion in participants with COPD and contrasted with previously defined criteria. Single transcripts and weighted co-expression network analysis modules were tested for correlation with the abnormal myofibre proportion phenotype. RESULTS: Abnormal myofibre proportion was defined in males with COPD (n = 29) as <18% type I and/or >22% type IIx/IIax fibres and in females with COPD (n = 17) as <36% type I and/or >12% type IIx/IIax fibres. Half of the participants with COPD were classified as having an abnormal myofibre proportion. Participants with COPD and an abnormal myofibre proportion had lower median handgrip strength (26.1 vs. 34.0 kg, P = 0.022), 6-min walk distance (300 vs. 353 m, P = 0.039) and forced expiratory volume in 1 s-to-forced vital capacity ratio (0.42 vs. 0.48, P = 0.041) compared with participants with COPD and normal myofibre proportions. Twenty-nine transcripts were associated with abnormal myofibre proportions in participants with COPD, with the upregulated NEB, TPM1 and TPM2 genes having the largest fold differences. Co-expression network analysis revealed that two transcript modules were significantly positively associated with the presence of abnormal myofibre proportions. One of these co-expression modules contained genes classically associated with muscle atrophy, as well as transcripts associated with both type I and type II myofibres, and was enriched for genetic loci associated with bone mineral density. CONCLUSIONS: Our findings indicate that there are significant transcriptional alterations associated with abnormal myofibre proportions in participants with COPD. Transcripts canonically associated with both type I and type IIa fibres were enriched in a co-expression network associated with abnormal myofibre proportion, suggesting altered transcriptional regulation across multiple fibre types.

When a calorie is not just a calorie: Diet quality and timing as mediators of metabolism and healthy aging.

An epidemic of obesity has affected large portions of the world, increasing the risk of developing many different age-associated diseases, including cancer, cardiovascular disease, and diabetes. In contrast with the prevailing notion that "a calorie is just a calorie," there are clear differences, within and between individuals, in the metabolic response to different macronutrient sources. Recent findings challenge this oversimplification; calories from different macronutrient sources or consumed at different times of day have metabolic effects beyond their value as fuel. Here, we summarize discussions conducted at a recent NIH workshop that brought together experts in calorie restriction, macronutrient composition, and time-restricted feeding to discuss how dietary composition and feeding schedule impact whole-body metabolism, longevity, and healthspan. These discussions may provide insights into the long-sought molecular mechanisms engaged by calorie restriction to extend lifespan, lead to novel therapies, and potentially inform the development of a personalized food-as-medicine approach to healthy aging.

Impairments in SHMT2 expression or cellular folate availability reduce oxidative phosphorylation and pyruvate kinase activity.

BACKGROUND: Serine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine-producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the protein's active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cell models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression. In this study, primary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6J dam crossed with a heterozygous Shmt2+/- male to generate Shmt2+/+ (wild-type) or Shmt2+/- (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression (ΔSHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function. RESULTS: Homozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, formate production, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not ΔSHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in ΔSHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2+/- MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss. CONCLUSIONS: The results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, mitochondrial formate production, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.

Genetic variation in genes regulating skeletal muscle regeneration and tissue remodelling associated with weight loss in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally. COPD patients with cachexia or weight loss have increased risk of death independent of body mass index (BMI) and lung function. We tested the hypothesis genetic variation is associated with weight loss in COPD using a genome-wide association study approach. METHODS: Participants with COPD (N = 4308) from three studies (COPDGene, ECLIPSE, and SPIROMICS) were analysed. Discovery analyses were performed in COPDGene with replication in SPIROMICS and ECLIPSE. In COPDGene, weight loss was defined as self-reported unintentional weight loss > 5% in the past year or low BMI (BMI < 20 kg/m2 ). In ECLIPSE and SPIROMICS, weight loss was calculated using available longitudinal visits. Stratified analyses were performed among African American (AA) and Non-Hispanic White (NHW) participants with COPD. Single variant and gene-based analyses were performed adjusting for confounders. Fine mapping was performed using a Bayesian approach integrating genetic association results with linkage disequilibrium and functional annotation. Significant gene networks were identified by integrating genetic regions associated with weight loss with skeletal muscle protein-protein interaction (PPI) data. RESULTS: At the single variant level, only the rs35368512 variant, intergenic to GRXCR1 and LINC02383, was associated with weight loss (odds ratio = 3.6, 95% confidence interval = 2.3-5.6, P = 3.2 × 10-8 ) among AA COPD participants in COPDGene. At the gene level in COPDGene, EFNA2 and BAIAP2 were significantly associated with weight loss in AA and NHW COPD participants, respectively. The EFNA2 association replicated among AA from SPIROMICS (P = 0.0014), whereas the BAIAP2 association replicated in NHW from ECLIPSE (P = 0.025). The EFNA2 gene encodes the membrane-bound protein ephrin-A2 involved in the regulation of developmental processes and adult tissue homeostasis such as skeletal muscle. The BAIAP2 gene encodes the insulin-responsive protein of mass 53 kD (IRSp53), a negative regulator of myogenic differentiation. Integration of the gene-based findings participants with PPI data revealed networks of genes involved in pathways such as Rho and synapse signalling. CONCLUSIONS: The EFNA2 and BAIAP2 genes were significantly associated with weight loss in COPD participants. Collectively, the integrative network analyses indicated genetic variation associated with weight loss in COPD may influence skeletal muscle regeneration and tissue remodelling.

NaCT/SLC13A5 facilitates citrate import and metabolism under nutrient-limited conditions.

Citrate lies at a critical node of metabolism, linking tricarboxylic acid metabolism and lipogenesis via acetyl-coenzyme A. Recent studies have observed that deficiency of the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, dysregulates hepatic metabolism and drives pediatric epilepsy. To examine how NaCT contributes to citrate metabolism in cells relevant to the pathophysiology of these diseases, we apply 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cells and primary rat cortical neurons. Exogenous citrate appreciably contributes to intermediary metabolism only under hypoxic conditions. In the absence of glutamine, citrate supplementation increases de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromises citrate uptake and catabolism. Citrate supplementation rescues Huh7 cell viability in response to glutamine deprivation or Zn2+ treatment, and NaCT deficiency mitigates these effects. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient-limited conditions and may facilitate resistance to metal toxicity.

A defined N6-methyladenosine (m6A) profile conferred by METTL3 regulates muscle stem cell/myoblast state transitions.

Muscle-specific adult stem cells (MuSCs) are required for skeletal muscle regeneration. To ensure efficient skeletal muscle regeneration after injury, MuSCs must undergo state transitions as they are activated from quiescence, give rise to a population of proliferating myoblasts, and continue either to terminal differentiation, to repair or replace damaged myofibers, or self-renewal to repopulate the quiescent population. Changes in MuSC/myoblast state are accompanied by dramatic shifts in their transcriptional profile. Previous reports in other adult stem cell systems have identified alterations in the most abundant internal mRNA modification, N6-methyladenosine (m6A), conferred by its active writer, METTL3, to regulate cell state transitions through alterations in the transcriptional profile of these cells. Our objective was to determine if m6A-modification deposition via METTL3 is a regulator of MuSC/myoblast state transitions in vitro and in vivo. Using liquid chromatography/mass spectrometry we identified that global m6A levels increase during the early stages of skeletal muscle regeneration, in vivo, and decline when C2C12 myoblasts transition from proliferation to differentiation, in vitro. Using m6A-specific RNA-sequencing (MeRIP-seq), a distinct profile of m6A-modification was identified, distinguishing proliferating from differentiating C2C12 myoblasts. RNAi studies show that reducing levels of METTL3, the active m6A methyltransferase, reduced global m6A levels and forced C2C12 myoblasts to prematurely differentiate. Reducing levels of METTL3 in primary mouse MuSCs prior to transplantation enhanced their engraftment capacity upon primary transplantation, however their capacity for serial transplantation was lost. In conclusion, METTL3 regulates m6A levels in MuSCs/myoblasts and controls the transition of MuSCs/myoblasts to different cell states. Furthermore, the first transcriptome wide map of m6A-modifications in proliferating and differentiating C2C12 myoblasts is provided and reveals a number of genes that may regulate MuSC/myoblast state transitions which had not been previously identified.

Whole Blueberry and Isolated Polyphenol-Rich Fractions Modulate Specific Gut Microbes in an In Vitro Colon Model and in a Pilot Study in Human Consumers.

Blueberry (BB) consumption is linked to improved health. The bioconversion of the polyphenolic content of BB by fermentative bacteria in the large intestine may be a necessary step for the health benefits attributed to BB consumption. The identification of specific gut microbiota taxa that respond to BB consumption and that mediate the bioconversion of consumed polyphenolic compounds into bioactive forms is required to improve our understanding of how polyphenols impact human health. We tested the ability of polyphenol-rich fractions purified from whole BB-namely, anthocyanins/flavonol glycosides (ANTH/FLAV), proanthocyanidins (PACs), the sugar/acid fraction (S/A), and total polyphenols (TPP)-to modulate the fecal microbiota composition of healthy adults in an in vitro colon system. In a parallel pilot study, we tested the effect of consuming 38 g of freeze-dried BB powder per day for 6 weeks on the fecal microbiota of 17 women in two age groups (i.e., young and older). The BB ingredients had a distinct effect on the fecal microbiota composition in the artificial colon model. The ANTH/FLAV and PAC fractions were more effective in promoting microbiome alpha diversity compared to S/A and TPP, and these effects were attributed to differentially responsive taxa. Dietary enrichment with BB resulted in a moderate increase in the diversity of the microbiota of the older subjects but not in younger subjects, and certain health-relevant taxa were significantly associated with BB consumption. Alterations in the abundance of some gut bacteria correlated not only with BB consumption but also with increased antioxidant activity in blood. Collectively, these pilot data support the notion that BB consumption is associated with gut microbiota changes and health benefits.

Osteosarcopenia in Reproductive-Aged Women with Polycystic Ovary Syndrome: A Multicenter Case-Control Study.

CONTEXT: Osteosarcopenia (loss of skeletal muscle and bone mass and/or function usually associated with aging) shares pathophysiological mechanisms with polycystic ovary syndrome (PCOS). However, the relationship between osteosarcopenia and PCOS remains unclear. OBJECTIVE: We evaluated skeletal muscle index% (SMI% = [appendicular muscle mass/weight (kg)] × 100) and bone mineral density (BMD) in PCOS (hyperandrogenism + oligoamenorrhea), and contrasted these musculoskeletal markers against 3 reproductive phenotypes (i): HA (hyperandrogenism + eumenorrhea) (ii); OA (normoandrogenic + oligoamenorrhea) and (iii), controls (normoandrogenic + eumenorrhea). Endocrine predictors of SMI% and BMD were evaluated across the groups. DESIGN, SETTING, AND PARTICIPANTS: Multicenter case-control study of 203 women (18-48 years old) in New York State. RESULTS: PCOS group exhibited reduced SMI% (mean [95% confidence interval (CI)]; 26.2% [25.1,27.3] vs 28.8% [27.7,29.8]), lower-extremity SMI% (57.6% [56.7,60.0] vs 62.5% [60.3,64.6]), and BMD (1.11 [1.08,1.14] vs 1.17 [1.14,1.20] g/cm2) compared to controls. PCOS group also had decreased upper (0.72 [0.70,0.74] vs 0.77 [0.75,0.79] g/cm2) and lower (1.13 [1.10,1.16] vs 1.19 [1.16,1.22] g/cm2) limb BMD compared to HA. Matsuda index was lower in PCOS vs controls and positively associated with SMI% in all groups (all Ps ≤ 0.05). Only controls showed associations between insulin-like growth factor (IGF) 1 and upper (r = 0.84) and lower (r = 0.72) limb BMD (all Ps < 0.01). Unlike in PCOS, IGF-binding protein 2 was associated with SMI% in controls (r = 0.45) and HA (r = 0.67), and with upper limb BMD (r = 0.98) in HA (all Ps < 0.05). CONCLUSIONS: Women with PCOS exhibit early signs of osteosarcopenia when compared to controls likely attributed to disrupted insulin function. Understanding the degree of musculoskeletal deterioration in PCOS is critical for implementing targeted interventions that prevent and delay osteosarcopenia in this clinical population.

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

Peptide YY (PYY) Is Expressed in Human Skeletal Muscle Tissue and Expanding Human Muscle Progenitor Cells.

Peptide YY (PYY) is considered a gut peptide with roles in post-prandial appetite and glucose regulation. Circulating PYY protein levels increase during aerobic exercise. Furthermore, people who have greater increases in muscle progenitor cells (hMPCs), the adult stem cell population responsible for skeletal muscle (SkM) repair, after resistance training have higher PYY transcript levels in SkM prior to training. Currently, examination of PYY expression patterns in SkM and/or hMPCs is lacking. Our objective was to identify the expression patterns of PYY in SkM and hMPCs. PYY and the associated Y receptors were analyzed in SkM biopsy tissue and cultured hMPCs from young and old human participants. Additional experiments to assess the role and regulation of PYY in hMPCs were performed. In SkM, PYY and one of the three Y receptors (Y1r) were detectable, but expression patterns were not affected by age. In expanding hMPCs, PYY and all three Y receptor (Y1r, Y2r, and Y5r) proteins were expressed in a temporal fashion with young hMPCs having greater levels of Y receptors at various time points. Exogenous PYY did not affect hMPC population expansion. hMPC PYY levels increased following the metabolic stimulus, 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), but were not affected by the inflammatory stimulus, tumor necrosis factor alpha (TNFα). In conclusion, PYY and Y receptor expression are not impacted by age in SkM tissue but are reduced in old vs. young expanding hMPCs. Furthermore, endogenous PYY production is stimulated by low energy states and thus may be integral for skeletal muscle and hMPC responses to metabolic stimuli.

Expansion capacity of human muscle progenitor cells differs by age, sex, and metabolic fuel preference.

Activation of satellite cells and expansion of the muscle progenitor cell (MPC) population are essential to generate a sufficient number of cells to repair damaged skeletal muscle. Proliferating MPCs have high energetic and biosynthetic material requirements, and the ability to utilize oxidative phosphorylation (OXPHOS) and/or glycolysis may affect expansion capacity of MPCs. In the present study, we investigated the effect of donor age and sex on human (h)MPC expansion capacity and metabolic fuel preference. hMPCs from young and old male and female donors were grown for 408 h (17 days). Percent confluence, live nuclei count, and dead cell count were measured every 24 h. Metabolic phenotype was assessed by glucose uptake, expression of genes related to glycolysis and OXPHOS, and the Seahorse XF24 Phenotype Test Kit during the exponential phase of growth. hMPCs from old male donors had impaired expansion capacity secondary to heightened cell death early in expansion compared with hMPCs from young male donors, an effect not observed in female hMPCs. Age-related differences in metabolism were also sex dependent; markers of OXPHOS were altered in old (vs. young) male hMPCs, whereas markers of metabolism were largely unaffected by age in female hMPCs. For the first time, we identify sex-specific differences in cell death and OXPHOS that contribute to impaired expansion capacity of hMPC cell populations with age.

Amino acids in healthy aging skeletal muscle.

Life expectancy in the U.S. and globally continues to increase. Despite increased life expectancy quality of life is not enhanced, and older adults often experience chronic age-related disease and functional disability, including frailty. Additionally, changes in body composition such as the involuntary loss of skeletal muscle mass (i.e. sarcopenia) and subsequent increases in adipose tissue can augment disease and disability in this population. Furthermore, increased oxidative stress and decreased antioxidant concentrations may also lead to metabolic dysfunction in older adults. Specific amino acids, including leucine, cysteine and its derivative taurine, and arginine can play various roles in healthy aging, especially in regards to skeletal muscle health. Leucine and arginine play important roles in muscle protein synthesis and cell growth while cysteine and arginine play important roles in quenching oxidative stress. Evidence suggests that supplemental doses of each of these amino acids may improve the aging phenotype. However, additional research is required to establish the doses required to achieve positive outcomes in humans.

Histone Methylation Dynamics and Gene Regulation Occur through the Sensing of One-Carbon Metabolism.

S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) link one-carbon metabolism to methylation status. However, it is unknown whether regulation of SAM and SAH by nutrient availability can be directly sensed to alter the kinetics of key histone methylation marks. We provide evidence that the status of methionine metabolism is sufficient to determine levels of histone methylation by modulating SAM and SAH. This dynamic interaction led to rapid changes in H3K4me3, altered gene transcription, provided feedback regulation to one-carbon metabolism, and could be fully recovered upon restoration of methionine. Modulation of methionine in diet led to changes in metabolism and histone methylation in the liver. In humans, methionine variability in fasting serum was commensurate with concentrations needed for these dynamics and could be partly explained by diet. Together these findings demonstrate that flux through methionine metabolism and the sensing of methionine availability may allow direct communication to the chromatin state in cells.

Increased expression of atrogenes and TWEAK family members after severe burn injury in nonburned human skeletal muscle.

Severe burn induces rapid skeletal muscle proteolysis after the injury, which persists for up to 1 year and results in skeletal muscle atrophy despite dietary and rehabilitative interventions. The purpose of this research was to determine acute changes in gene expression of skeletal muscle mass regulators postburn injury. Specimens were obtained for biopsy from the vastus lateralis of a nonburned leg of eight burned subjects (6M, 2F: 34.8 ± 2.7 years: 29.9 ± 3.1% TBSA burn) at 5.1 ± 1.1 days postburn injury and from matched controls. mRNA expression of cytokines and receptors in the tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) families, and the ubiquitin proteasome E3 ligases, atrogin-1 and MuRF-1, was determined. TNF receptor 1A was over 3.5-fold higher in burn. Expression of TNF-like weak inducer of apoptosis and its receptor were over 1.6 and 6.0-fold higher in burn. IL-6, IL-6 receptor, and glycoprotein 130 were elevated in burned subjects with IL-6 receptor over 13-fold higher. The level of suppressor of cytokine signaling-3 was also increased nearly 6-fold in burn. Atrogin-1 and MuRF-1 were more than 4- and 3-fold higher in burn. These results demonstrate for the first time that severe burn in humans has a remarkable impact on gene expression in skeletal muscle of a nonburned limb of genes that promote inflammation and proteolysis. Because these changes likely contribute to the acute skeletal muscle atrophy in areas not directly affected by the burn, in the future it will be important to determine the responsible systemic cues.

Heightened muscle inflammation susceptibility may impair regenerative capacity in aging humans.

The regenerative response of skeletal muscle to mechanically induced damage is impaired with age. Previous work in our laboratory suggests this may result from higher proinflammatory signaling in aging muscle at rest and/or a greater inflammatory response to damage. We, therefore, assessed skeletal muscle proinflammatory signaling at rest and 24 h after unaccustomed, loaded knee extension contractions that induced modest muscle damage (72% increase in serum creatine kinase) in a cohort of 87 adults across three age groups (AGE40, AGE61, and AGE76). Vastus lateralis muscle gene expression and protein cell signaling of the IL-6 and TNF-α pathways were determined by quantitative PCR and immunoblot analysis. For in vitro studies, cell signaling and fusion capacities were compared among primary myoblasts from young (AGE28) and old (AGE64) donors treated with TNF-α. Muscle expression was higher (1.5- to 2.1-fold) in AGE76 and AGE61 relative to AGE40 for several genes involved in IL-6, TNF-α, and TNF-like weak inducer of apoptosis signaling. Indexes of activation for the proinflammatory transcription factors signal transducer and activator of transcription-3 and NF-κB were highest in AGE76. Resistance loading reduced gene expression of IL-6 receptor, muscle RING finger 1, and atrogin-1, and increased TNF-like weak inducer of apoptosis receptor expression. Donor myoblasts from AGE64 showed impaired differentiation and fusion in standard media and greater NF-κB activation in response to TNF-α treatment (compared with AGE28). We show for the first time that human aging is associated with muscle inflammation susceptibility (i.e., higher basal state of proinflammatory signaling) that is present in both tissue and isolated myogenic cells and likely contributes to the impaired regenerative capacity of skeletal muscle in the older population.

Simvastatin impairs ADP-stimulated respiration and increases mitochondrial oxidative stress in primary human skeletal myotubes.

Statins, the widely prescribed cholesterol-lowering drugs for the treatment of cardiovascular disease, cause adverse skeletal muscle side effects ranging from fatigue to fatal rhabdomyolysis. The purpose of this study was to determine the effects of simvastatin on mitochondrial respiration, oxidative stress, and cell death in differentiated primary human skeletal muscle cells (i.e., myotubes). Simvastatin induced a dose-dependent decrease in viability of proliferating and differentiating primary human muscle precursor cells, and a similar dose-dependent effect was noted in differentiated myoblasts and myotubes. Additionally, there were decreases in myotube number and size following 48 h of simvastatin treatment (5 µM). In permeabilized myotubes, maximal ADP-stimulated oxygen consumption, supported by palmitoylcarnitine+malate (PCM, complex I and II substrates) and glutamate+malate (GM, complex I substrates), was 32-37% lower (P<0.05) in simvastatin-treated (5 µM) vs control myotubes, providing evidence of impaired respiration at complex I. Mitochondrial superoxide and hydrogen peroxide generation were significantly greater in the simvastatin-treated human skeletal myotube cultures compared to control. In addition, simvastatin markedly increased protein levels of Bax (proapoptotic, +53%) and Bcl-2 (antiapoptotic, +100%, P<0.05), mitochondrial PTP opening (+44%, P<0.05), and TUNEL-positive nuclei in human skeletal myotubes, demonstrating up-regulation of mitochondrial-mediated myonuclear apoptotic mechanisms. These data demonstrate that simvastatin induces myotube atrophy and cell loss associated with impaired ADP-stimulated maximal mitochondrial respiratory capacity, mitochondrial oxidative stress, and apoptosis in primary human skeletal myotubes, suggesting that mitochondrial dysfunction may underlie human statin-induced myopathy.

The skeletal muscle transcript profile reflects accommodative responses to inadequate protein intake in younger and older males.

Inadequate protein intake initiates adverse changes in skeletal muscle function and structure (i.e., an accommodative response). mRNA level changes due to short-term inadequate dietary protein might be an early indication of subsequent accommodation. The aims of this study were to assess the effects of dietary protein and the diet-by-age interaction on the skeletal muscle transcriptome. Twelve younger (21-43 y) and 10 older (63-79 y) men completed three controlled feeding trials with protein intakes of 0.50 (LPro: lower protein), 0.75 (MPro: medium protein) and 1.00 g protein·kg body weight⁻¹·day⁻¹ (HPro: higher protein). A fasting state biopsy was taken on Day 12 of each trial. Global changes in transcript levels were assessed with Affymetrix genechips and expression patterns determined using self-organizing maps. Nine hundred fifty-eight transcripts were differentially expressed (P<.05) by diet and 853 had a diet-by-age interaction (P<.05). The results for diet alone revealed that LPro was associated with up-regulation of transcripts related to ubiquitin-dependent protein catabolism and muscle contraction and LPro and MPro resulted in up-regulation of transcripts related to apoptosis and down-regulation of transcripts related to cell differentiation, muscle and organ development, extracellular space and responses to stimuli and stress. The diet-by-age effect on protein modification transcripts was consistent with the older males being less responsive to anabolic stimuli (lower protein synthesis at HPro) and more responsive to a catabolic state (protein breakdown at LPro). Changes in skeletal muscle mRNA levels in younger and older males to protein intake near or below the recommended dietary allowance are indicative of an early accommodative response.

Differential genomic responses in old vs. young humans despite similar levels of modest muscle damage after resistance loading.

Across numerous model systems, aging skeletal muscle demonstrates an impaired regenerative response when exposed to the same stimulus as young muscle. To better understand the impact of aging in a human model, we compared changes to the skeletal muscle transcriptome induced by unaccustomed high-intensity resistance loading (RL) sufficient to cause moderate muscle damage in young (37 yr) vs. older (73 yr) adults. Serum creatine kinase was elevated 46% 24 h after RL in all subjects with no age differences, indicating similar degrees of myofiber membrane wounding by age. Despite this similarity, from genomic microarrays 318 unique transcripts were differentially expressed after RL in old vs. only 87 in young subjects. Follow-up pathways analysis and functional annotation revealed among old subjects upregulation of transcripts related to stress and cellular compromise, inflammation and immune responses, necrosis, and protein degradation and changes in expression (up- and downregulation) of transcripts related to skeletal and muscular development, cell growth and proliferation, protein synthesis, fibrosis and connective tissue function, myoblast-myotube fusion and cell-cell adhesion, and structural integrity. Overall the transcript-level changes indicative of undue inflammatory and stress responses in these older adults were not mirrored in young subjects. Follow-up immunoblotting revealed higher protein expression among old subjects for NF-kappaB, heat shock protein (HSP)70, and IL-6 signaling [total and phosphorylated signal transducer and activator of transcription (STAT)3 at Tyr705]. Together, these novel findings suggest that young and old adults are equally susceptible to RL-mediated damage, yet the muscles of older adults are much more sensitive to this modest degree of damage-launching a robust transcriptome-level response that may begin to reveal key differences in the regenerative capacity of skeletal muscle with advancing age.