Aged Muscle Research Articles

#2694 URAEMIC CARDIOMYOPATHY AS PREMATURE PRESBYCARDIA

Abstract Background and Aims Presbycardia, the age-related decline in cardiac function, has been shown in the general population by demonstrating gradual decline in peak cardiac performance with advancing age [1]. Chronic kidney disease (CKD) has shown phenotypic similarities with premature aging. Studies have shown accelerated vascular aging and skeletal muscle wasting in CKD. However, it is not known whether CKD patients show age related decline in cardiac function and whether the impairment is more pronounced for the given age (premature presbycardia). In the present study, we set out to test the hypothesis that CKD patients have premature presbycardia by measuring peak cardiac power (CPOpeak) non-invasively across different age groups. Method A cross sectional study (n = 170) of healthy male volunteers (n = 100) and male CKD patients (n = 70) spanning ages 19 to 75 years. CKD patients were grouped into early (CKD 2–3, n = 29) and late CKD (CKD 4–5, n = 41). Patients with diabetes or any known cardiovascular diseases were excluded. CPOpeak was measured using a specialised cardiopulmonary exercise test (CPX) using CO2 rebreathing method [2]. The association between age and CPOpeak was evaluated. The regression lines of CKD patients and healthy volunteers were compared using ANCOVA. P<0.05 is considered significant. Results The mean eGFR of early CKD and late CKD were 57.8±17.2 ml/min and 16.8±5.8 ml/min respectively. The mean CPOpeak of the study groups were 5.35±0.94 W (Control), 4.93±0.71 W (early CKD) and 4.26±0.69 W (late CKD). Fig. 1 shows the association between CPOpeak and age along with the regression equations for the individual study groups. Comparison of the regression lines using ANCOVA did not show any difference in slopes between the groups (P = NS). There was a diminution in CPOpeak of 0.2W per 10 years of advancing age. However, for any given age, the CPOpeak was diminished by 0.38 W in early CKD (P = 0.023) and 0.93 W in late CKD (P<10−3) compared to control. In other words, the cardiac age was older by 19 years in early CKD and by 46.5 years in late CKD compared to healthy volunteers. Conclusion The results demonstrate that CKD is associated with premature presbycardia. This phenomenon may offer explanation for the high prevalence of heart failure in advanced CKD. Further studies exploring the underlying mechanisms of premature aging in CKD may be worthwhile in the future.

Sources and Sinks of Serine in Nutrition, Health, and Disease.

Amino acid dysregulation has emerged as an important driver of disease progression in various contexts. l-Serine lies at a central node of metabolism, linking carbohydrate metabolism, transamination, glycine, and folate-mediated one-carbon metabolism to protein synthesis and various downstream bioenergetic and biosynthetic pathways. l-Serine is produced locally in the brain but is sourced predominantly from glycine and one-carbon metabolism in peripheral tissues via liver and kidney metabolism. Compromised regulation or activity of l-serine synthesis and disposal occurs in the context of genetic diseases as well as chronic disease states, leading to low circulating l-serine levels and pathogenesis in the nervous system, retina, heart, and aging muscle. Dietary interventions in preclinical models modulate sensory neuropathy, retinopathy, tumor growth, and muscle regeneration. A serine tolerance test may provide a quantitative readout of l-serine homeostasis that identifies patients who may be susceptible to neuropathy or responsive to therapy.

Targeting mitochondrial quality control in muscle aging: Natural dietary products as potential interventions

AbstractMitochondria are the main sources of energy production for muscle tissues, including skeletal, cardiac, or smooth muscle, to support their activities. Mitochondrial dysfunction and dysregulation of their quality control systems are significant characteristics in aged muscle. Increasing evidence indicates that natural products, especially phytochemicals, can prevent or improve age‐related muscle disorders. Among them, some dietary components specifically regulate mitochondrial quality control (MQC) to play their efficacy. However, the content of related studies is currently not systematically reviewed. In this review, composition, changes, and associated regulators in the MQC network during muscle aging were summarized by comprehensive literature retrieval. Subsequently, the effects and mechanisms of dietary active ingredients on this control network were described. This review showed that mitochondrial dysfunction is a common feature in different types of muscle aging. And four main ways of MQC are involved in maintaining mitochondrial homeostasis and they form a tight and integral system, for muscle‐healthy aging. The benefits of specific endogenous metabolites and exogenous components on aged muscle health are mediated in the above ways. Dietary phytochemicals including phenols, terpenoids, and alkaloids, unfold their influential roles in delaying muscle aging via promoting mitochondrial biogenesis and mitophagy. These findings suggest that MQC is a promising action site against muscle aging and food or herbal‐derived natural products are representative potential interventions. More clinical studies in the future are worthy of future investigation of these natural products.

Long-term detraining reverses the improvement of lifelong exercise on skeletal muscle ferroptosis and inflammation in aging rats: fiber-type dependence of the Keap1/Nrf2 pathway.

We investigated the effects of lifelong aerobic exercise and 8months of detraining after 10months of aerobic training on circulation, skeletal muscle oxidative stress, and inflammation in aging rats. Sprague-Dawley rats were randomly assigned to the control (CON), detraining (DET), and lifelong aerobic training (LAT) groups. The DET and LAT groups began aerobic treadmill exercise at the age of 8months and stopped training at the 18th and 26th month, respectively; all rats were sacrificed when aged 26months. Compared with CON, LAT remarkably decreased serum and aged skeletal muscle 4-hydroxynonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OHdG) levels. Superoxide dismutase 2(SOD2) levels were higher in the LAT group than in the CON group in skeletal muscle. However, DET remarkably decreased SOD2 protein expression and content in the skeletal muscle and increased malondialdehyde (MDA) level compared with LAT. Compared with LAT, DET remarkably downregulated adiponectin and upregulated tumor necrosis factor alpha (TNF-α) expression, while phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and 70-kDa ribosomal protein S6 kinase (P70S6K) protein expression decreased, and that of FoxO1 and muscle atrophy F-box (MAFbX) proteins increased in the quadriceps femoris. Adiponectin and TNF-α expression in the soleus muscle did not change between groups, whereas that of AKT, mammalian target of rapamycin (mTOR), and P70S6K was lower in the soleus in the DET group than in that in the LAT group. Compared with that in the LAT group, sestrin1 (SES1) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression in the DET group was lower, whereas Keap1 mRNA expression was remarkably upregulated in the quadriceps femoris. Interestingly, the protein and mRNA levels of SES1, Nrf2, and Keap1 in soleus muscle did not differ between groups. LAT remarkably upregulated ferritin heavy polypeptide 1(FTH), glutathione peroxidase 4(GPX4), and solute carrier family 7member 11 (SLC7A11) protein expression in the quadriceps femoris and soleus muscles, compared with CON. However, compared with LAT, DET downregulated FTH, GPX4, and SLC7A11 protein expression in the quadriceps femoris and soleus muscles. Long-term detraining during the aging phase reverses the improvement effect of lifelong exercise on oxidative stress, inflammation, ferroptosis, and muscle atrophy in aging skeletal muscle. The quadriceps femoris is more evident than the soleus, which may be related to the different changes in the Keap1/Nrf2 pathway in different skeletal muscles.

A novel mitochondrial complex I ROS inhibitor partially improves muscle regeneration in adult but not old mice

It is unclear whether mitochondrial dysfunction and redox stress contribute to impaired age-related muscle regenerative capacity. Here we characterized a novel compound, BI4500, that inhibits the release of reactive oxygen species (ROS) from the quinone site in mitochondrial complex I (site IQ). We tested the hypothesis that ROS release from site IQ contributes to impaired regenerative capacity in aging muscle. Electron transfer system site-specific ROS production was measured in adult and aged mouse isolated muscle mitochondria and permeabilized gastrocnemius fibers. BI4500 inhibited ROS production from site IQ in a concentration-dependent manner (IC50 = ∼985 nM) by inhibiting ROS release without impairing complex I-linked respiration. In vivo BI4500 treatment decreased ROS production from site IQ. Muscle injury and sham injury were induced using barium chloride or vehicle injection to the tibialis anterior (TA) muscle in adult and aged male mice. On the same day as injury, mice began a daily gavage of 30 mg/kg BI4500 (BI) or placebo (PLA). Muscle regeneration (H&E, Sirius Red, Pax7) was measured at 5 and 35 days after injury. Muscle injury increased centrally nucleated fibers (CNFs) and fibrosis with no treatment or age effect. There was a significant age by treatment interaction for CNFs at 5- and 35-days post injury with significantly more CNFs in BI adults compared to PLA adults. Muscle fiber cross-sectional area (CSA) recovered significantly more in adult BI mice (−89 ± 365 μm2) compared to old PLA (−599 ± 153 μm2) and old BI (−535 ± 222 μm2, mean ± SD). In situ TA force recovery was measured 35 days after injury and was not significantly different by age or treatment. Inhibition of site IQ ROS partially improves muscle regeneration in adult but not old muscle demonstrating a role for CI ROS in the response to muscle injury. Site IQ ROS does not contribute to impaired regenerative capacity in aging.

Effect of 1-year daily protein supplementation and physical exercise on muscle protein synthesis rate and muscle metabolome in healthy older Danes: a randomized controlled trial

BackgroundThe skeletal muscle mass decreases with age and the responsiveness of aging muscles’ protein synthesis rate (MPS) to protein intake seems to deteriorate.ObjectiveThis study investigated the impact of 12 months of protein supplementation with or without physical exercise training on the basal and postprandial MPS and the skeletal muscle metabolome of healthy older Danes (> 65 years, 29 females/37 males).MethodsSubjects were randomized to follow one of five intervention groups: (1) carbohydrate, (2) collagen protein, (3) whey protein, (4) home-based light resistance training with whey protein, and (5) center-based heavy-load resistance training with whey protein. Before and after the intervention, a tracer infusion trial was conducted to measure basal and postprandial MPS in response to intake of a cocktail consisting of 20 g whey hydrolysate + 10 g glucose. In addition, the skeletal muscle metabolome was measured using gas chromatography–mass spectrometry (GC–MS) at basal state and 4 h after the intake of the cocktail.ResultsOne year of daily protein or carbohydrate supplementation did not alter the basal and protein-stimulated postprandial muscle protein synthesis rate or the muscle metabolome of healthy older Danes. Basal MPS (%/h) at baseline for all subjects were 0.0034 ± 0,011 (mean ± SD). In contrast to previous studies, no difference was observed in basal MPS between males and females (p = 0.75). With the developed untargeted GC–MS methodology, it was possible to detect and tentatively annotate > 70 metabolites from the human skeletal muscle samples.ConclusionOne year of protein supplementation in comparison to an isocaloric-control supplement seems to affect neither the MPS at basal or postprandial state nor the skeletal muscle metabolome.Clinical trial registryNumber: NCT02115698, clinicaltrials.gov/ct2/show/NCT02115698.

Effects of Resistance Exercise Training on Aged Skeletal Muscle: Potential Role of Muscle Stem Cells

PURPOSE: The prevalence of sarcopenia, which can lead to disability, hospitalization, and death, is increasing among older populations. Resistance exercise training (RT) is currently the most effective strategy for combating sarcopenia by stimulating hypertrophy and increasing strength. This review describes the underlying mechanisms of aging skeletal muscle and whether RT attenuates aging-related loss of muscle function and mass.METHODS: We reviewed and summarized previous research using PubMed, Science Direct, and Google Scholar databases.RESULTS: Load-induced muscle growth is a complex phenomenon that depends on various physiological systems and signaling pathways. Muscle growth occurs through signaling events arising from mechanical stress and consequent muscle protein turnover controlled by the balance between protein synthesis and degradation, which is negatively affected by aging. The authors used the myonuclear domains mediated by muscle satellite cells to explain the molecular machinery of exercise-induced muscle growth and recovery in aging muscles.CONCLUSIONS: Despite a blunted molecular response to an exercise bout, aging muscle cells demonstrated remarkable plasticity, with substantial improvements in myofibril size and strength during RT. More studies are necessary to elucidate the specific mechanisms by which RT activates muscle satellite cells and mitogenic and myogenic signaling in aged muscles.

Unraveling the Interplay of Oxidative Stress, Aging, and Skeletal Muscle: Insights and Interventions for Optimal Muscle Function in the Elderly

Purpose: Aging is related to the onset of sarcopenia. Understand how its development occurs by immunological factors by oxidative stress. One of the main effects for a person is the reduction of muscle function. Aerobic and resistance physical exercise can reduce the progression of oxidative stress and, consequently, sarcopenia.
 Materials and Methods: Systematic review with a sample of six scientific papers published in academic journals.
 Discussion and Conclusion: Studies show that exercise then induces a prevention response to free radical damage. Furthermore, physical exercise induces effects on the concentration of superoxide dismutase enzyme (SOD), catalase, adenosine monophosphate (AMP), sirtuin 1 (SIRT-1) and peroxisome proliferator-activated receptor-gamma coactivator (PGC-1a), which are important for better skeletal muscle condition. Understanding how the elderly muscle responds to exercise is important, and it is necessary to understand the mechanisms of reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation and the modulation of the proinflammatory cytokines and the antioxidant system in response to the exercise.

Ginsenoside Rh4 delays skeletal muscle aging through SIRT1 pathway

BackgroundThe aging of skeletal muscle is the leading cause of physical disability in older adults, currently effective treatment methods are lacking. Ginsenoside Rh4, an active component extracted from ginseng, possesses beneficial anti-inflammatory and anti-oxidative effects. PurposeThe aim of this study was to elucidate the antioxidant effect of ginsenoside Rh4 on aging skeletal muscle and its molecular mechanism of anti-aging of skeletal muscle. Study designIn this study, we employed a D-galactose-induced model of skeletal muscle aging to investigate whether ginsenoside Rh4 can delay the process of skeletal muscle senescence. MethodsThe effects of ginsenoside Rh4 on oxidative damage and inflammation in aging skeletal muscle were analyzed using immunofluorescence, immunohistochemistry, ELISA kits, H&E staining, flow cytometry, and protein immunoblotting. The changes of ginsenoside Rh4 on mitochondrial morphology were observed by transmission electron microscopy, and ELISA kits and protein immunoblotting analyzed the effects of ginsenoside Rh4 on mitochondrial homeostasis in skeletal muscle cells. The influence of ginsenoside Rh4 on the SIRT1 signaling pathway in aging skeletal muscle were investigated by protein immunoblotting, immunofluorescence, and β-galactosidase staining. ResultsOur results showed that Rh4 improved the morphology of muscle fibers and produced an anti-inflammatory response. Furthermore, in vitro experiments indicated that ginsenosides reduced the production of senescent cells, while Rh4 effectively alleviated oxidative damage in skeletal muscle and restored mitochondrial balance. Transcriptome analysis and molecular docking showed that Rh4 improved mitochondrial homeostasis and delayed skeletal muscle aging by regulating the PGC-1α-TFAM and HIF-1α-c-Myc pathways via targeting SIRT1. ConclusionGinsenoside Rh4 improves oxidative stress and inflammation in skeletal muscle by activating SIRT1, deacetylating Nrf2, regulating PGC-1α-TFAM and HIF-1α-c-Myc pathways, and enhancing mitochondrial homeostasis, thus achieving the effect of delaying skeletal muscle aging.

Adiposity is associated with widespread transcriptional changes and downregulation of longevity pathways in aged skeletal muscle.

Amongst healthy older people, a number of correlates of impaired skeletal muscle mass and function have been defined. Although the prevalence of obesity is increasing markedly in this age group, information is sparse about the particular impacts of obesity on ageing skeletal muscle or the molecular mechanisms that underlie this and associated disease risk. Here, we examined genome-wide transcriptional changes using RNA sequencing in muscle biopsies from 40 older community-dwelling men from the Hertfordshire Sarcopenia Study with regard to obesity (body mass index [BMI] >30kg/m2 , n=7), overweight (BMI 25-30, n=19), normal weight (BMI<25, n=14), and per cent and total fat mass. In addition, we used EPIC DNA methylation array data to investigate correlations between DNA methylation and gene expression in aged skeletal muscle tissue and investigated the relationship between genes within altered regulatory pathways and muscle histological parameters. Individuals with obesity demonstrated a prominent modified transcriptional signature in muscle tissue, with a total of 542 differentially expressed genes associated with obesity (false discovery rate ≤0.05), of which 425 genes were upregulated when compared with normal weight. Upregulated genes were enriched in immune response (P=3.18×10-41 ) and inflammation (leucocyte activation, P=1.47×10-41 ; tumour necrosis factor, P=2.75×10-15 ) signalling pathways and downregulated genes enriched in longevity (P=1.5×10-3 ) and AMP-activated protein kinase (AMPK) (P=4.5×10-3 ) signalling pathways. Furthermore, differentially expressed genes in both longevity and AMPK signalling pathways were associated with a change in DNA methylation, with a total of 256 and 360 significant cytosine-phosphate-guanine-gene correlations identified, respectively. Similar changes in the muscle transcriptome were observed with respect to per cent fat mass and total fat mass. Obesity was further associated with a significant increase in type II fast-fibre area (P=0.026), of which key regulatory genes within both longevity and AMPK pathways were significantly associated. We provide for the first time a global transcriptomic profile of skeletal muscle in older people with and without obesity, demonstrating modulation of key genes and pathways implicated in the regulation of muscle function, changes in DNA methylation associated with such pathways and associations between genes within the modified pathways implicated in muscle regulation and changes in muscle fibre type.

Loss of transcriptional heterogeneity in aged human muscle stem cells.

Age-related loss of muscle mass and function negatively impacts healthspan and lifespan. Satellite cells function as muscle stem cells in muscle maintenance and regeneration by self-renewal, activation, proliferation and differentiation. These processes are perturbed in aging at the stem cell population level, contributing to muscle loss. However, how representation of subpopulations within the human satellite cell pool change during aging remains poorly understood. We previously reported a comprehensive baseline of human satellite cell (Hu-MuSCs) transcriptional activity in muscle homeostasis describing functional heterogenous human satellite cell subpopulations such as CAV1+ Hu-MUSCs. Here, we sequenced additional satellite cells from new healthy donors and performed extended transcriptomic analyses with regard to aging. We found an age-related loss of global transcriptomic heterogeneity and identified new markers (CAV1, CXCL14, GPX3) along with previously described ones (FN1, ITGB1, SPRY1) that are altered during aging in human satellite cells. These findings describe new transcriptomic changes that occur during aging in human satellite cells and provide a foundation for understanding functional impact.

Muscle Injuries Induce a Prostacyclin-PPARγ/PGC1a-FAO Spike That Boosts Regeneration.

It is well-known that muscle regeneration declines with aging, and aged muscles undergo degenerative atrophy or sarcopenia. While exercise and acute injury are both known to induce muscle regeneration, the molecular signals that help trigger muscle regeneration have remained unclear. Here, mass spectrometry imaging (MSI) is used to show that injured muscles induce a specific subset of prostanoids during regeneration, including PGG1, PGD2, and the prostacyclin PGI2. The spike in prostacyclin promotes skeletal muscle regeneration via myoblasts, and declines with aging. Mechanistically, the prostacyclin spike promotes a spike in PPARγ/PGC1a signaling, which induces a spike in fatty acid oxidation (FAO) to control myogenesis. LC-MS/MS and MSI further confirm that an early FAO spike is associated with normal regeneration, but muscle FAO became dysregulated during aging. Functional experiments demonstrate that the prostacyclin-PPARγ/PGC1a-FAO spike is necessary and sufficient to promote both young and aged muscle regeneration, and that prostacyclin can synergize with PPARγ/PGC1a-FAO signaling to restore aged muscles' regeneration and physical function. Given that the post-injury prostacyclin-PPARγ-FAO spike can be modulated pharmacologically and via post-exercise nutrition, this work has implications for how prostacyclin-PPARγ-FAO might be fine-tuned to promote regeneration and treat muscle diseases of aging.

GCPII Inhibition Delays Age-related Loss of Muscle Mass and Function in Mice

Sarcopenia, the age-related loss of skeletal muscle mass and function, is a significant problem that reduces the quality of life in the elderly. Despite the growing prevalence of sarcopenia, there is still a lack of effective therapies for this disease. Recent research has focused on the role of the enzyme glutamate carboxypeptidase II (GCPII) in regulating muscle function and neuromuscular junction (NMJ) integrity. We have previously shown that inhibition of GCPII with the potent inhibitor 2-(Phosphonomethyl)-pentanedioic acid (2-PMPA) can significantly delay muscle function loss and NMJ denervation in an animal model of ALS. Given that sarcopenia shares similarities with ALS in terms of NMJ deterioration, we aimed to investigate whether 2-PMPA treatment could also preserve muscle function during aging. We measured the activity of GCPII in the gastrocnemius muscles of 4-month-old and 20-month-old mice, and found that GCPII activity was significantly increased in the 20-month-old mice (3188±182 fmol/mg/h) compared to 4-month-old mice (2009±317 fmol/mg/h). Then we treated 15-month-old C57BL6/J mice with 100mg/kg of 2-PMPA IP daily for 5 months and monitored muscle function. After 5 months of 2-PMPA treatment, we observed preservation of gastrocnemius muscle mass (164.8±5.6 vs. 143.6±6.4 mg, 2-PMPA vs. vehicle treated group) and a significant delay in age-related loss in grip strength (-4.75±3.22% vs. -20.98±3.73%, 2-PMPA vs. vehicle). We also found that 2-PMPA treatment increased the mean single-fiber cross-sectional area (759.4±5.2 vs. 722.4±5.1 μm 2 , 2-PMPA vs. vehicle), delayed the decline of nerve signals to the muscles as observed by compound muscle action potential (CMAP) amplitude decline (-7.37±6.16% vs. -26.05±3.71%, 2-PMPA vs. vehicle), and improved ambulation in old mice (3941±153 vs. 3216±188 beam brakes, 2-PMPA vs. vehicle) as observed by open-field test. Immunofluorescent staining showed that GCPII expression in aged muscle is associated with infiltrating macrophages, indicating inflammation as one potential target for the role of 2-PMPA in preserving muscle function during aging. Overall, our current results support that excessive GCPII activity is associated with muscle aging and GCPII inhibition is a potential therapeutic strategy for sarcopenia. This study highlights the potential of GCPII inhibition as a new therapeutic approach to preserving muscle function in the elderly. This work was supported by NIH R01 AG078181-01 (to BSS), R01 AG068130 (to BSS), R01NS093416 (to BSS), and R25GM109441 (Hopkins PREP support for TRJ). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Adult progenitor rejuvenation with embryonic factors

During ageing, adult stem cells' regenerative properties decline, as they undergo replicative senescence and lose both their proliferative and differentiation capacities. In contrast, embryonic and foetal progenitors typically possess heightened proliferative capacities and manifest a more robust regenerative response upon injury and transplantation, despite undergoing many rounds of mitosis. How embryonic and foetal progenitors delay senescence and maintain their proliferative and differentiation capacities after numerous rounds of mitosis, remains unknown. It is also unclear if defined embryonic factors can rejuvenate adult progenitors to confer extended proliferative and differentiation capacities, without reprogramming their lineage-specific fates or inducing oncogenic transformation. Here, we report that a minimal combination of LIN28A, TERT, and sh-p53 (LTS), all of which are tightly regulated and play important roles during embryonic development, can delay senescence in adult muscle progenitors. LTS muscle progenitors showed an extended proliferative capacity, maintained a normal karyotype, underwent myogenesis normally, and did not manifest tumorigenesis nor aberrations in lineage differentiation, even in late passages. LTS treatment promoted self-renewal and rescued the pro-senescence phenotype of aged cachexia patients' muscle progenitors, and promoted their engraftment for skeletal muscle regeneration in vivo. When we examined the mechanistic basis for LIN28A's role in the LTS minimum combo, let-7 microRNA suppression could not fully explain how LIN28A promoted muscle progenitor self-renewal. Instead, LIN28A was promoting the translation of oxidative phosphorylation mRNAs in adult muscle progenitors to optimize mitochondrial reactive oxygen species (mtROS) and mitohormetic signalling. Optimized mtROS induced a variety of mitohormetic stress responses, including the hypoxic response for metabolic damage, the unfolded protein response for protein damage, and the p53 response for DNA damage. Perturbation of mtROS levels specifically abrogated the LIN28A-driven hypoxic response in Hypoxia Inducible Factor-1α (HIF1α) and glycolysis, and thus LTS progenitor self-renewal, without affecting normal or TS progenitors. Our findings connect embryonically regulated factors to mitohormesis and progenitor rejuvenation, with implications for ageing-related muscle degeneration.

Impaired proteostatic mechanisms other than protein synthesis limit aged skeletal muscle recovery after disuse atrophy

Older individuals undergo more frequent hospitalizations and periods of bedrest compared to younger persons, causing rapid loss of muscle mass and strength. We and others have shown that old muscle fails to completely regrow following disuse, despite having similar levels of muscle loss during disuse and equal or higher myofibrillar protein synthesis during recovery. Therefore, we hypothesized that a failure of proteostatic mechanisms other than protein synthesis contributes to the inability of old muscle to fully recover after a period of disuse atrophy. Methods: Adult (10 month) and old (30 month) male F344BN rats were hindlimb unloaded for 14 days and then reloaded for up to 60 days. We used a combination of deuterium oxide (D2O) labeling, proteomics, and biochemical analyses to study proteostasis during recovery. Results: There was a failure of old muscle to maintain proteostasis as evident by a greater accumulation of insoluble protein aggregates ( 1.02 ±0.06 vs. 1.22 ±0.06, p&lt;0.05), insoluble collagen ( 4.0 ±1.1 vs. 9.2 ±0.9, p&lt;0.01) and higher amount of advanced glycation end products (AGEs) ( 1.0 ±0.06 vs. 1.5 ±0.08, p&lt;0.001) compared to adult. Old muscle had higher rates of myofibrillar protein synthesis than adult muscle ( 0.029 k/day ±0.002 vs. 0.039 k/day ±0.002, p&lt;0.0001), but also higher rates of breakdown (0.017 1/t ±0.002 vs. 0.028 1/t ±0.004, p&lt;0.05). Differences in the synthesis of collagen were less apparent between adult and old muscle, implying that there was a failure of collagen breakdown in old muscle leading to greater accumulation. Lastly, pathway analysis of proteomic data showed that there were differences in the proteins that accumulated in aggregates between adult and old muscle, and that the synthesis of proteins that regulate proteostasis were different with age. Conclusions: Together, these data indicate that in old muscle altered proteostatic mechanisms other than myofibrillar protein synthesis led to failure to recover from atrophy. Interventions should therefore be targeted at these novel mechanisms instead of protein synthesis alone. NIA Training Grant: 5T32AG052363-04, APS Postdoctoral Fellowship MML, F31AT01147201 ZRH, R01(NCCIH AT009268) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Senomorphic properties of miR-181b in aged human aortic smooth muscle cells

Introduction: Age associated large artery stiffness (LAS) is an independent predictor of adverse cardiovascular health outcomes in humans and has also been shown to be associated with the development of heart failure, kidney disease, and even dementia. Furthermore, the development of cellular senescence in the aorta, the permanent arrest of the cell cycle and development of pro-inflammatory, pro-fibrotic senescence associated secretory phenotype (SASP), has also been shown to contribute to age associated LAS. MicroRNAs (miR) are small, non-coding RNAs that down-regulate protein expression in a semi-specific manner. miR-181b has been shown to be downregulated with aging, targets many of the secreted factors associated with the aortic SASP and has previously been shown to have a beneficial effect on vascular health. With smooth muscle cells being the predominant cell type in the large arteries, we wanted to address the hypothesis that miR-181b has the potential to act as a senomorphic in human aortic smooth muscle cells (HASMCs) and promote beneficial changes in individual cells that influence LAS. Methods: To test our hypothesis, we obtained young and old HASMCs and transfected them with miR-181b or a scrambled miR control utilizing a lipofectamine transfection protocol. Total RNA was isolated from the HASMCs and quantitative PCR was performed to assess changes of the SASP profile. Cell proliferation was measured using electric cell-substrate impedance sensing (ECIS) technology. Lastly, individual HASMC stiffness measurements were made using twisting magnetic microscopy. Results: Transfection of HASMCs with miR-181b resulted in reduced expression of markers of cell cycle arrest (p16 and p21) and the SASP profile (IL-6, TNF-alpha, MCP1, TGF-b1, IL-1alpha, and MMP2) when compared to scrambled control (p&lt;0.05). HASMC proliferation was decreased in old HASMCs compared to young (p&lt;0.05) and was increased with miR-181b transfection when compared to scramble control (p&lt;0.05). Lastly, individual HASMC stiffness was lower in young cells compared to old (0.4382 ± 0.29 vs. 0.9959 ± 0.72 Pa/nm, n=120, p&lt;0.001). Cell stiffness was decreased in old HASMCs when treated with miR-181b when compared with control (0.7856 ± 0.144 vs. 1.18 ± 0.288 Pa/nm, n=76, p&lt;0.001), but there was no difference in young HASMCs. Conclusions: miR-181b appears to act as a senomorphic agent when transfected in old HASMCs. This is evidenced by noticeable changes in the expression of markers of cellular senescence which corresponded with positive changes in cellular characteristics that are associated with LAS, namely the improvement in cellular proliferation and reduction of individual cell stiffness in the old HASMC groups. Together, these results suggest the modulation of miR-181b may be a potential therapeutic target to improve age associated LAS through an effect on cellular senescence. K08 AG070281 (ET), Western Institute for Veterans Research (ET), U-CARES (ET), R01 AG048366 (LAL), R01 AG050238 (AJD), R01 AG060395 (AJD) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Processing body accumulation in aged muscle stem cells contributes to impaired self-renewal

Stem cell exhaustion is a pillar of aging hallmarks, mediated by an inability of the body’s stem cells to self-renew. In skeletal muscle, impaired self-renewal of muscle stem cells (MuSCs) manifests as a progressive decline in muscle regenerative capacities, leading to functional dependence in the aged. Self-renewal depends on preventing degradation of factors mediating pluripotency during division, thereby averting differentiation, however, mechanisms underlying the inability of MuSCs to self-renew in the aged are unknown. Here, we show processing bodies (p-bodies) accumulate in MuSCs isolated from old mice. As p-bodies canonically represent cellular depots for RNA degradation, our findings suggest age-related changes to RNA degradation may contribute to impaired self-renewal of old MuSCs. To investigate the consequence of p-body enrichment in old MuSCs on self-renewal, we utilized a computational-based lineage analysis termed Dynamo following single cell RNA-sequencing of MuSCs isolated from young and old mice. Dynamo incorporates the velocity of RNA to generate transcriptional vector fields, allowing for inference of lineage specification. Moreover, through characterization of each cell’s transcriptome, this framework also allows for investigation of changes to lineage specification as a result of genetic perturbation. We found that manipulation of scaffold proteins mediating p-body formation resulted in a pronounced effect on lineage specification. Specifically, our results showed that upregulation of LSM14a mRNA processing body assembly factor ( LSM14a) in young MuSCs resulted in a loss of cycling-related gene expression, and subsequently promoted differentiation. Moreover, we found that downregulation of LSM14a in old MuSCs oriented transcriptional vector fields towards a self-renewing phenotype and away from differentiation, mediated by increased expression of cycling-related genes and decreases in differentiation-related genes. Together, the accumulation of p-bodies in old MuSCs coupled with the ability of p-bodies to facilitate self-renewal, suggests that targeted interventions focusing on p-body assembly may be therapeutic in restoring old MuSC self-renewal, and thereby regenerative capacities in the aged. This study was supported by NIA R01 AG061005 (FA), NIA R01 AG052978 (FA), and NIEHS R01 ES025529 (FA). Postdoctoral fellowship funding provided by P30AG024827 (ZH). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex-specific alteration in human muscle transcriptome with age.

Sarcopenia is a medical condition that progressively develops with age and results in reduced skeletal muscle mass, alteration in muscle composition, and decreased muscle strength. Several clinical studies suggested that sarcopenia disproportionally affects males and females with age. Despite this knowledge, the molecular mechanism governing the pathophysiology is not well understood in a sex-specific manner. In this study, we utilized human gastrocnemius muscles from males and females to identify differentially regulated genes with age. We found 269 genes with at least a twofold expression difference in the aged muscle transcriptome. Among the female muscle samples, there were 239 differentially regulated genes, and the novel protein-coding genes include KIF20A, PIMREG, MTRNR2L6, TRPV6, EFNA2, RNF24, and SFN. In aged male skeletal muscle, there were 166 differentially regulated genes, and the novel-protein coding genes are CENPK, CDKN2A, BHLHA15, and EPHA. Gene Ontology (GO) enrichment revealed glucose catabolism, NAD metabolic processes, and muscle fiber transition pathways that are involved in aged female skeletal muscle, whereas replicative senescence, cytochrome C release, and muscle composition pathways are disrupted in aged male skeletal muscle. Targeting these novels, differentially regulated genes, and signaling pathways could serve as sex-specific therapeutic targets to combat the age-related onset of sarcopenia and promote healthy aging.

Indicators of increased ER stress and UPR in aged D2-mdx and human dystrophic skeletal muscles.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease that results in muscle wasting, wheelchair dependence, and eventual death due to cardiac and respiratory complications. In addition to muscle fragility, dystrophin deficiency also results in multiple secondary dysfunctions, which may lead to the accumulation of unfolded proteins causing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The purpose of this investigation was to understand how ER stress and the UPR are modified in muscle from D2-mdx mice, an emerging DMD model, and from humans with DMD. We hypothesized that markers of ER stress and the UPR are upregulated in D2-mdx and human dystrophic muscles compared to their healthy counterparts. Immunoblotting in diaphragms from 11-month-old D2-mdx and DBA mice indicated increased ER stress and UPR in dystrophic diaphragms compared to healthy, including increased relative abundance of ER stress chaperone CHOP, canonical ER stress transducers ATF6 and pIRE1α S724, and transcription factors that regulate the UPR such as ATF4, XBP1s, and peIF2α S51. The publicly available Affymetrix dataset (GSE38417) was used to analyze the expression of ER stress and UPR-related transcripts and processes. Fifty-eight upregulated genes related to ER stress and the UPR in human dystrophic muscles suggest pathway activation. Further, based on analyses using iRegulon, putative transcription factors that regulate this upregulation profile were identified, including ATF6, XBP1, ATF4, CREB3L2, and EIF2AK3. This study adds to and extends the emerging knowledge of ER stress and the UPR in dystrophin deficiency and identifies transcriptional regulators that may be responsible for these changes and be of therapeutic interest.

Antibody toolkit to investigate eEF1A methylation dynamics in mRNA translation elongation

Protein synthesis is a fundamental step in gene expression, with modulation of mRNA translation at the elongation step emerging as an important regulatory node in shaping cellular proteomes. In this context, five distinct lysine methylation events on eukaryotic elongation factor 1A (eEF1A), a fundamental nonribosomal elongation factor, are proposed to influence mRNA translation elongation dynamics. However, a lack of affinity tools has hindered progress in fully understanding how eEF1A lysine methylation impacts protein synthesis. Here we develop and characterize a suite of selective antibodies to investigate eEF1A methylation and provide evidence that methylation levels decline in aged tissue. Determination of the methyl state and stoichiometry on eEF1A in various cell lines by mass spectrometry shows modest cell-to-cell variability. We also find by Western blot analysis that knockdown of individual eEF1A-specific lysine methyltransferases leads to depletion of the cognate lysine methylation event and indicates active crosstalk between different sites. Further, we find that the antibodies are specific in immunohistochemistry applications. Finally, application of the antibody toolkit suggests that several eEF1A methylation events decrease in aged muscle tissue. Together, our study provides a roadmap for leveraging methyl state and sequence-selective antibody reagents to accelerate discovery of eEF1A methylation-related functions and suggests a role for eEF1A methylation, via protein synthesis regulation, in aging biology.