Tackling Brain and Muscle Dysfunction in Acute Respiratory Distress Syndrome Survivors: National Heart, Lung, and Blood Institute Workshop Report.

Acute respiratory distress syndrome (ARDS) is associated with long-term impairments in brain and muscle function that significantly impact the quality of life of those who survive the acute illness. The mechanisms underlying these impairments are not yet well understood, and evidence-based interventions to minimize the burden on patients remain unproven. The National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health assembled a workshop in April 2023 to review the state of the science regarding ARDS-associated brain and muscle dysfunction, to identify gaps in current knowledge, and to determine priorities for future investigation. The workshop included presentations by scientific leaders across the translational science spectrum and was open to the public as well as the scientific community. This report describes the themes discussed at the workshop as well as recommendations to advance the field toward the goal of improving the health and wellbeing of ARDS survivors.

ATF4-dependent increase in mitochondrial-endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle.

Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate interorganellar communication by contributing to the dynamic transfer of Ca2+, lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and correlated with an induction of Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells alters MERC formation by evaluating multiple myocyte systems, including from mice and Drosophila, and in primary myotubes. Our results revealed that OPA1 deficiency induced tighter and more frequent MERCs in concert with a greater abundance of MERC proteins involved in calcium exchange. Additionally, loss of OPA1 increased the expression of activating transcription factor 4 (ATF4), an integrated stress response (ISR) pathway effector. Reducing Atf4 expression prevented the OPA1-loss-induced tightening of MERC structures. OPA1 reduction was associated with decreased mitochondrial and sarcoplasmic reticulum, a specialized form of ER, calcium, which was reversed following ATF4 repression. These data suggest that mitochondrial stress, induced by OPA1 deficiency, regulates skeletal muscle MERC formation in an ATF4-dependent manner.

A Specialized Centrosome-Proteasome Axis Mediates Proteostasis and Influences Cardiac Stress through Txlnb.

Background: Centrosomes localize to perinuclear foci where they serve multifunctional roles, arranging the microtubule organizing center (MTOC) and anchoring ubiquitin-proteasome system (UPS) machinery. In mature cardiomyocytes, centrosomal proteins redistribute into a specialized perinuclear cage-like structure, and a potential centrosome-UPS interface has not been studied. Taxilin-beta (Txlnb), a cardiomyocyte-enriched protein, belongs to a family of centrosome adapter proteins implicated in protein quality control. We hypothesize that Txlnb plays a key role in centrosomal-proteasomal crosstalk in cardiomyocytes. Methods: Integrative bioinformatics assessed centrosomal gene dysregulation in failing hearts. Txlnb gain/loss-of-function studies were conducted in cultured cardiomyocytes and mice. Txlnb's role in cardiac proteotoxicity and hypertrophy was examined using CryAB-R120G mice and transverse aortic constriction (TAC), respectively. Molecular modeling investigated Txlnb structure/function. Results: Human failing hearts show consistent dysregulation of many centrosome-associated genes, alongside UPS-related genes. Txlnb emerged as a candidate regulator of cardiomyocyte proteostasis that localizes to the perinuclear centrosomal compartment. Txlnb's interactome strongly supports its involvement in cytoskeletal, microtubule, and UPS processes, particularly centrosome-related functions. Overexpressing Txlnb in cardiomyocytes reduced ubiquitinated protein accumulation and enhanced proteasome activity during hypertrophy. Txlnb-knockout (KO) mouse hearts exhibit proteasomal insufficiency and altered cardiac growth, evidenced by ubiquitinated protein accumulation, decreased 26Sß5 proteasome activity, and lower mass with age. In Cryab-R120G mice, Txlnb loss worsened heart failure, causing lower ejection fractions. After TAC, Txlnb-KO mice also showed reduced ejection fraction, increased heart mass, and elevated ubiquitinated protein accumulation. Investigations into the molecular mechanisms revealed that Txlnb-KO did not affect proteasomal subunit expression but led to the upregulation of Txlna and several centrosomal proteins (Cep63, Ofd1, and Tubg) suggesting altered centrosomal dynamics. Structural predictions support Txlnb's role as a specialized centrosomal-adapter protein bridging centrosomes with proteasomes, confirmed by microtubule-dependent perinuclear localization. Conclusions: Together, these data provide initial evidence connecting Txlnb to cardiac proteostasis, hinting at the potential importance of functional bridging between specialized centrosomes and UPS in cardiomyocytes.

Uncovering the mechanisms of MuRF1-induced ubiquitylation and revealing similarities with MuRF2 and MuRF3.

MuRF1 (Muscle-specific RING finger protein 1; gene name TRIM63) is a ubiquitin E3 ligase, associated with the progression of muscle atrophy. As a RING (Really Interesting New Gene) type E3 ligase, its unique activity of ubiquitylation is driven by a specific interaction with a UBE2 (ubiquitin conjugating enzyme). Our understanding of MuRF1 function remains unclear as candidate UBE2s have not been fully elucidated. In the present study, we screened human ubiquitin dependent UBE2s in vitro and found that MuRF1 engages in ubiquitylation with UBE2D, UBE2E, UBE2N/V families and UBE2W. MuRF1 can cause mono-ubiquitylation, K48- and K63-linked polyubiquitin chains in a UBE2 dependent manner. Moreover, we identified a two-step UBE2 dependent mechanism whereby MuRF1 is monoubiquitylated by UBE2W which acts as an anchor for UBE2N/V to generate polyubiquitin chains. With the in vitro ubiquitylation assay, we also found that MuRF2 and MuRF3 not only share the same UBE2 partners as MuRF1 but can also directly ubiquitylate the same substrates: Titin (A168-A170), Desmin, and MYLPF (Myosin Light Chain, Phosphorylatable, Fast Skeletal Muscle; also called Myosin Light Regulatory Chain 2). In summary, our work presents new insights into the mechanisms that underpin MuRF1 activity and reveals overlap in MuRF-induced ubiquitylation which could explain their partial redundancy in vivo.

A critical discussion on the relationship between E3 ubiquitin ligases, protein degradation, and skeletal muscle wasting: it's not that simple.

Ubiquitination is an important post-translational modification (PTM) for protein substrates, whereby ubiquitin is added to proteins through the coordinated activity of activating (E1), ubiquitin-conjugating (E2), and ubiquitin ligase (E3) enzymes. The E3s provide key functions in the recognition of specific protein substrates to be ubiquitinated and aid in determining their proteolytic or nonproteolytic fates, which has led to their study as indicators of altered cellular processes. MuRF1 and MAFbx/Atrogin-1 were two of the first E3 ubiquitin ligases identified as being upregulated in a range of different skeletal muscle atrophy models. Since their discovery, the expression of these E3 ubiquitin ligases has often been studied as a surrogate measure of changes to bulk protein degradation rates. However, emerging evidence has highlighted the dynamic and complex regulation of the ubiquitin proteasome system (UPS) in skeletal muscle and demonstrated that protein ubiquitination is not necessarily equivalent to protein degradation. These observations highlight the potential challenges of quantifying E3 ubiquitin ligases as markers of protein degradation rates or ubiquitin proteasome system (UPS) activation. This perspective examines the usefulness of monitoring E3 ubiquitin ligases for determining specific or bulk protein degradation rates in the settings of skeletal muscle atrophy. Specific questions that remain unanswered within the skeletal muscle atrophy field are also identified, to encourage the pursuit of new research that will be critical in moving forward our understanding of the molecular mechanisms that govern protein function and degradation in muscle.

Impaired proteostatic mechanisms other than decreased protein synthesis limit old skeletal muscle recovery after disuse atrophy.

BACKGROUND: Skeletal muscle mass and strength diminish during periods of disuse but recover upon return to weight bearing in healthy adults but are incomplete in old muscle. Efforts to improve muscle recovery in older individuals commonly aim at increasing myofibrillar protein synthesis via mammalian target of rapamycin (mTOR) stimulation despite evidence demonstrating that old muscle has chronically elevated levels of mammalian target of rapamycin complex 1 (mTORC1) activity. We hypothesized that protein synthesis is higher in old muscle than adult muscle, which contributes to a proteostatic stress that impairs recovery. METHODS: We unloaded hindlimbs of adult (10-month) and old (28-month) F344BN rats for 14 days to induce atrophy, followed by reloading up to 60 days with deuterium oxide (D2 O) labelling to study muscle regrowth and proteostasis. RESULTS: We found that old muscle has limited recovery of muscle mass during reloading despite having higher translational capacity and myofibrillar protein synthesis (0.029 k/day ± 0.002 vs. 0.039 k/day ± 0.002, P < 0.0001) than adult muscle. We showed that collagen protein synthesis was not different (0.005 k (1/day) ± 0.0005 vs. 0.004 k (1/day) ± 0.0005, P = 0.15) in old compared to adult, but old muscle had higher collagen concentration (4.5 µg/mg ± 1.2 vs. 9.8 µg/mg ± 0.96, P < 0.01), implying that collagen breakdown was slower in old muscle than adult muscle. This finding was supported by old muscle having more insoluble collagen (4.0 ± 1.1 vs. 9.2 ± 0.9, P < 0.01) and an accumulation of advanced glycation end products (1.0 ± 0.06 vs. 1.5 ± 0.08, P < 0.001) than adult muscle during reloading. Limited recovery of muscle mass during reloading is in part due to higher protein degradation (0.017 1/t ± 0.002 vs. 0.028 1/t ± 0.004, P < 0.05) and/or compromised proteostasis as evidenced by accumulation of ubiquitinated insoluble proteins (1.02 ± 0.06 vs. 1.22 ± 0.06, P < 0.05). Last, we showed that synthesis of individual proteins related to protein folding/refolding, protein degradation and neural-related biological processes was higher in old muscle during reloading than adult muscle. CONCLUSIONS: Our data suggest that the failure of old muscle to recover after disuse is not due to limitations in the ability to synthesize myofibrillar proteins but because of other impaired proteostatic mechanisms (e.g., protein folding and degradation). These data provide novel information on individual proteins that accumulate in protein aggregates after disuse and certain biological processes such as protein folding and degradation that likely play a role in impaired recovery. Therefore, interventions to enhance regrowth of old muscle after disuse should be directed towards the identified impaired proteostatic mechanisms and not aimed at increasing protein synthesis.

Mechanisms of Skeletal Muscle Atrophy and Molecular Circuitry of Stem Cell Fate in Skeletal Muscle Regeneration and Aging.

Skeletal muscle is a complex and highly adaptable tissue. With aging, there is a progressive loss of muscle mass and function, known as sarcopenia, and a reduced capacity for regeneration and repair following injury. A review of the literature shows that the primary mechanisms underlying the age-related loss of muscle mass and the attenuated growth response are multi-factorial and related to alterations in multiple processes, including proteostasis, mitochondrial function, extracellular matrix remodeling, and neuromuscular junction function. Multiple factors influence the rate of sarcopenia, including acute illness and trauma, followed by incomplete recovery and repair. Regeneration and repair of damaged skeletal muscle involve an orchestrated cross-talk between multiple cell populations, including satellite cells, immune cells, and fibro-adipogenic precursor cells. Proof-of-concept studies in mice have demonstrated that reprogramming of this disrupted orchestration, resulting in the normalization of muscle function, may be possible using small molecules that target muscle macrophages. During aging, as well as in muscular dystrophies, disruptions in multiple signaling pathways and in the cross-talk between different cell populations contribute to the failure to properly repair and maintain muscle mass and function.

Sexual dimorphism and the multi-omic response to exercise training in rat subcutaneous white adipose tissue.

Subcutaneous white adipose tissue (scWAT) is a dynamic storage and secretory organ that regulates systemic homeostasis, yet the impact of endurance exercise training and sex on its molecular landscape has not been fully established. Utilizing an integrative multi-omics approach with data generated by the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we identified profound sexual dimorphism in the dynamic response of rat scWAT to endurance exercise training. Despite similar cardiorespiratory improvements, only male rats reduced whole-body adiposity, scWAT adipocyte size, and total scWAT triglyceride abundance with training. Multi-omic analyses of adipose tissue integrated with phenotypic measures identified sex-specific training responses including enrichment of mTOR signaling in females, while males displayed enhanced mitochondrial ribosome biogenesis and oxidative metabolism. Overall, this study reinforces our understanding that sex impacts scWAT biology and provides a rich resource to interrogate responses of scWAT to endurance training.

Multi-omic identification of key transcriptional regulatory programs during endurance exercise training.

Transcription factors (TFs) play a key role in regulating gene expression and responses to stimuli. We conducted an integrated analysis of chromatin accessibility, DNA methylation, and RNA expression across eight rat tissues following endurance exercise training (EET) to map epigenomic changes to transcriptional changes and determine key TFs involved. We uncovered tissue-specific changes and TF motif enrichment across all omic layers, differentially accessible regions (DARs), differentially methylated regions (DMRs), and differentially expressed genes (DEGs). We discovered distinct routes of EET-induced regulation through either epigenomic alterations providing better access for TFs to affect target genes, or via changes in TF expression or activity enabling target gene response. We identified TF motifs enriched among correlated epigenomic and transcriptomic alterations, DEGs correlated with exercise-related phenotypic changes, and EET-induced activity changes of TFs enriched for DEGs among their gene targets. This analysis elucidates the unique transcriptional regulatory mechanisms mediating diverse organ effects of EET.

The mitochondrial multi-omic response to exercise training across tissues.

Mitochondria are adaptable organelles with diverse cellular functions critical to whole-body metabolic homeostasis. While chronic endurance exercise training is known to alter mitochondrial activity, these adaptations have not yet been systematically characterized. Here, the Molecular Transducers of Physical Activity Consortium (MoTrPAC) mapped the longitudinal, multi-omic changes in mitochondrial analytes across 19 tissues in male and female rats endurance trained for 1, 2, 4 or 8 weeks. Training elicited substantial changes in the adrenal gland, brown adipose, colon, heart and skeletal muscle, while we detected mild responses in the brain, lung, small intestine and testes. The colon response was characterized by non-linear dynamics that resulted in upregulation of mitochondrial function that was more prominent in females. Brown adipose and adrenal tissues were characterized by substantial downregulation of mitochondrial pathways. Training induced a previously unrecognized robust upregulation of mitochondrial protein abundance and acetylation in the liver, and a concomitant shift in lipid metabolism. The striated muscles demonstrated a highly coordinated response to increase oxidative capacity, with the majority of changes occurring in protein abundance and post-translational modifications. We identified exercise upregulated networks that are downregulated in human type 2 diabetes and liver cirrhosis. In both cases HSD17B10, a central dehydrogenase in multiple metabolic pathways and mitochondrial tRNA maturation, was the main hub. In summary, we provide a multi-omic, cross-tissue atlas of the mitochondrial response to training and identify candidates for prevention of disease-associated mitochondrial dysfunction.

The role of mTORC1 in the regulation of skeletal muscle mass.

Skeletal muscle mass is a very plastic characteristic of skeletal muscle and is regulated by signaling pathways that control the balance between anabolic and catabolic processes. The serine/threonine kinase mechanistic/mammalian target of rapamycin (mTOR) has been shown to be critically important in the regulation of skeletal muscle mass through its regulation of protein synthesis and degradation pathways. In this commentary, recent advances in the understanding of the role of mTORC1 in the regulation of muscle mass under conditions that induce hypertrophy and atrophy will be highlighted.

Effects of Combined Inorganic Nitrate and Nitrite Supplementation on Cardiorespiratory Fitness and Skeletal Muscle Oxidative Capacity in Type 2 Diabetes: A Pilot Randomized Controlled Trial.

Nitric oxide (NO) stimulates mitochondrial biogenesis in skeletal muscle. However, NO metabolism is disrupted in individuals with type 2 diabetes mellitus (T2DM) potentially contributing to their decreased cardiorespiratory fitness (i.e., VO2max) and skeletal muscle oxidative capacity. We used a randomized, double-blind, placebo-controlled, 8-week trial with beetroot juice containing nitrate (NO3−) and nitrite (NO2−) (250 mg and 20 mg/day) to test potential benefits on VO2max and skeletal muscle oxidative capacity in T2DM. T2DM (N = 36, Age = 59 ± 9 years; BMI = 31.9 ± 5.0 kg/m2) and age- and BMI-matched non-diabetic controls (N = 15, Age = 60 ± 9 years; BMI = 29.5 ± 4.6 kg/m2) were studied. Mitochondrial respiratory capacity was assessed in muscle biopsies from a subgroup of T2DM and controls (N = 19 and N = 10, respectively). At baseline, T2DM had higher plasma NO3− (100%; p < 0.001) and lower plasma NO2− levels (−46.8%; p < 0.0001) than controls. VO2max was lower in T2DM (−26.4%; p < 0.001), as was maximal carbohydrate- and fatty acid-supported oxygen consumption in permeabilized muscle fibers (−26.1% and −25.5%, respectively; p < 0.05). NO3−/NO2− supplementation increased VO2max (5.3%; p < 0.01). Further, circulating NO2−, but not NO3−, positively correlated with VO2max after supplementation (R2= 0.40; p < 0.05). Within the NO3−/NO2− group, 42% of subjects presented improvements in both carbohydrate- and fatty acid-supported oxygen consumption in skeletal muscle (vs. 0% in placebo; p < 0.05). VO2max improvements in these individuals tended to be larger than in the rest of the NO3−/NO2− group (1.21 ± 0.51 mL/(kg*min) vs. 0.31 ± 0.10 mL/(kg*min); p = 0.09). NO3−/NO2− supplementation increases VO2max in T2DM individuals and improvements in skeletal muscle oxidative capacity appear to occur in those with more pronounced increases in VO2max.

Ubiquitin Ligases in Longevity and Aging Skeletal Muscle.

The development and prevalence of diseases associated with aging presents a global health burden on society. One hallmark of aging is the loss of proteostasis which is caused in part by alterations to the ubiquitin-proteasome system (UPS) and lysosome-autophagy system leading to impaired function and maintenance of mass in tissues such as skeletal muscle. In the instance of skeletal muscle, the impairment of function occurs early in the aging process and is dependent on proteostatic mechanisms. The UPS plays a pivotal role in degradation of misfolded and aggregated proteins. For the purpose of this review, we will discuss the role of the UPS system in the context of age-related loss of muscle mass and function. We highlight the significant role that E3 ubiquitin ligases play in the turnover of key components (e.g., mitochondria and neuromuscular junction) essential to skeletal muscle function and the influence of aging. In addition, we will briefly discuss the contribution of the UPS system to lifespan. By understanding the UPS system as part of the proteostasis network in age-related diseases and disorders such as sarcopenia, new discoveries can be made and new interventions can be developed which will preserve muscle function and maintain quality of life with advancing age.

SnapShot: Skeletal muscle atrophy.

Skeletal muscle size is highly plastic and sensitive to a variety of stimuli. Muscle atrophy occurs as the result of changes in multiple signaling pathways that regulate both protein synthesis and degradation. The signaling pathways that are activated or inhibited depend on the specific stimuli that are altered. To view this SnapShot, open of download the PDF.

Proceedings from the Albert Charitable Trust Inaugural Workshop on 'Understanding the Acute Effects of Exercise on the Brain'.

An inaugural workshop supported by "The Leo and Anne Albert Charitable Trust," was held October 4-7, 2019 in Scottsdale, Arizona, to focus on the effects of exercise on the brain and to discuss how physical activity may prevent or delay the onset of aging-related neurodegenerative conditions. The Scientific Program Committee (led by Dr. Jeff Burns) assembled translational, clinical, and basic scientists who research various aspects of the effects of exercise on the body and brain, with the overall goal of gaining a better understanding as to how to delay or prevent neurodegenerative diseases. In particular, research topics included the links between cardiorespiratory fitness, the cerebrovasculature, energy metabolism, peripheral organs, and cognitive function, which are all highly relevant to understanding the effects of acute and chronic exercise on the brain. The Albert Trust workshop participants addressed these and related topics, as well as how other lifestyle interventions, such as diet, affect age-related cognitive decline associated with Alzheimer's and other neurodegenerative diseases. This report provides a synopsis of the presentations and discussions by the participants, and a delineation of the next steps towards advancing our understanding of the effects of exercise on the aging brain.

Activation of Crtc2/Creb1 in skeletal muscle enhances weight loss during intermittent fasting.

The Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1-mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle-specific overexpression of Crtc2 (Crtc2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, maintenance of higher energy expenditure during the fast and reduced binge-eating during the feeding period. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2-driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.

Identification of the MuRF1 Skeletal Muscle Ubiquitylome Through Quantitative Proteomics.

MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine whether MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.