Multi-transcriptome analysis following an acute skeletal muscle growth stimulus yields tools for discerning global and MYC regulatory networks

Myc is a powerful transcription factor implicated in epigenetic reprogramming, cellular plasticity, and rapid growth as well as tumorigenesis. Cancer in skeletal muscle is extremely rare despite marked and sustained Myc induction during loading-induced hypertrophy. Here, we investigated global, actively transcribed, stable, and myonucleus-specific transcriptomes following an acute hypertrophic stimulus in mouse plantaris. With these datasets, we define global and Myc-specific dynamics at the onset of mechanical overload-induced muscle fiber growth. Data collation across analyses reveals an under-appreciated role for the muscle fiber in extracellular matrix remodeling during adaptation, along with the contribution of mRNA stability to epigenetic-related transcript levels in muscle. We also identify Runx1 and Ankrd1 (Marp1) as abundant myonucleus-enriched loading-induced genes. We observed that a strong induction of cell cycle regulators including Myc occurs with mechanical overload in myonuclei. Additionally, in vivo Myc-controlled gene expression in the plantaris was defined using a genetic muscle fiber-specific doxycycline-inducible Myc-overexpression model. We determined Myc is implicated in numerous aspects of gene expression during early-phase muscle fiber growth. Specifically, brief induction of Myc protein in muscle represses Reverbα, Reverbβ, and Myh2 while increasing Rpl3, recapitulating gene expression in myonuclei during acute overload. Experimental, comparative, and in silico analyses place Myc at the center of a stable and actively transcribed, loading-responsive, muscle fiber–localized regulatory hub. Collectively, our experiments are a roadmap for understanding global and Myc-mediated transcriptional networks that regulate rapid remodeling in postmitotic cells. We provide open webtools for exploring the five RNA-seq datasets as a resource to the field

Disrupted circadian oscillations in type 2 diabetes are linked to altered rhythmic mitochondrial metabolism in skeletal muscle

Funding: The authors are supported by grants from the AstraZeneca SciLifeLab Research Programme, Novo Nordisk Foundation (NNF14OC0011493, and NNF17OC0030088), Swedish Diabetes Foundation (DIA2018-357), Swedish Research Council (2015-00165 and 2018-02389), the Knut and Alice Wallenberg Foundation (2018-0094), the Strategic Research Programme in Diabetes at Karolinska Institutet (2009-1068), the Stockholm County Council (SLL20170159), and the Swedish Research Council for Sport Science (P2019-0140). B.M.G. was supported by fellowships from the Novo Nordisk Foundation (NNF19OC0055072), the Wenner-Gren Foundation, an Albert Renold Travel Fellowship from the European Foundation for the Study of Diabetes, and an Eric Reid Fund for Methodology from the Biochemical Society. N.J.P. and L.S.-P. were supported by an Individual Fellowship from the Marie Skłodowska-Curie Actions (European Commission: 704978 and 675610). X.Z. and K.A.E. were supported by NIH R01AR066082. N.J.P. was supported by grants from the Sigurd och Elsa Goljes Minne and Lars Hierta Memorial Foundations (Sweden). We acknowledge the Beta Cell in-vivo Imaging/Extracellular Flux Analysis core facility supported by the Strategic Research Program in Diabetes for the usage of the Seahorse flux analyzer. Additional support was received from the Novo Nordisk Foundation Center for Basic Metabolic Research at the University of Copenhagen (NNF18CC0034900). The Novo Nordisk Foundation Center for Basic Metabolic Research is an independent research center at the University of Copenhagen, partially funded by an unrestricted donation from the Novo Nordisk Foundation. We acknowledge the Single-Cell Omics platform at the Novo Nordisk Foundation Center for Basic Metabolic Research for technical and computational expertise and support. Schematics are created with BioRender.com.Peer reviewedPublisher PD

Skeletal muscle PGC-1α1 reroutes kynurenine metabolism to increase energy efficiency and fatigue-resistance

The coactivator PGC-1α1 is activated by exercise training in skeletal muscle and promotes fatigue-resistance. In exercised muscle, PGC-1α1 enhances the expression of kynurenine aminotransferases (Kats), which convert kynurenine into kynurenic acid. This reduces kynurenine-associated neurotoxicity and generates glutamate as a byproduct. Here, we show that PGC-1α1 elevates aspartate and glutamate levels and increases the expression of glycolysis and malate-aspartate shuttle (MAS) genes. These interconnected processes improve energy utilization and transfer fuel-derived electrons to mitochondrial respiration. This PGC-1α1-dependent mechanism allows trained muscle to use kynurenine metabolism to increase the bioenergetic efficiency of glucose oxidation. Kat inhibition with carbidopa impairs aspartate biosynthesis, mitochondrial respiration, and reduces exercise performance and muscle force in mice. Our findings show that PGC-1α1 activates the MAS in skeletal muscle, supported by kynurenine catabolism, as part of the adaptations to endurance exercise. This crosstalk between kynurenine metabolism and the MAS may have important physiological and clinical implications

Ca2+ fluxes and insulin action in cardiac and skeletal muscles

Obesity and type 2 diabetes are major and rapidly increasing health problems in society. They are associated with several life-threatening conditions, including heart and renal failure, and damage to the nervous system. An inability of cells to respond normally to insulin, insulin resistance, is a key feature in obesity and type 2 diabetes. Ca2+ is a versatile messenger that regulates diverse cellular functions such as fertilization, electrical signaling, contraction, synaptic transmission, gene transcription, hormonal signaling, metabolism, and cell death. To exert these diverse effects, duration, amplitude and spatial distribution of Ca2+ need to be tightly regulated. The role of Ca2+ in insulin signaling under normal conditions and in association with insulin resistance is uncertain. This thesis focuses on Ca2+ fluxes and insulin action in cardiac and skeletal muscles. In the first two papers we examined the effect of insulin on Ca2+ homeostasis in normal, freshly isolated mouse ventricular cardiomyocytes and how Ca2+ handling was changed in an animal model of obesity and insulin resistance, ob/ob mice. Ob/ob cardiomyocytes showed prolonged electrically evoked Ca2+ transients and impaired mitochondrial Ca2+ handling, which resulted in extra Ca2+ transients that may predispose for arrhythmias in vivo. Moreover, we observed decreased ion fluxes through canonical transient receptor potential 3 (TRPC3) channels, which may affect intracellular Ca2+ homeostasis and hence cellular function. In the following two papers, we investigated the role of Ca2+ in insulin-mediated glucose uptake in adult skeletal muscles. Increased Ca2+ influx in the presence of insulin potentiated glucose uptake in muscles from both normal and ob/ob mice, whereas decreased Ca2+ influx was associated with decreased insulinmediated glucose uptake. In addition, TRPC3 protein expression was knocked down using a novel transfection technique with small interfering RNA coupled to carbon nanotubes, which resulted in large decreases in diacylglycerol-induced Ca2+ influx and insulin-mediated glucose uptake. Insulin-mediated glucose uptake occurs via the glucose transporter 4 (GLUT4) that was found to co-localize with TRPC3 in the t-tubular system, which is considered to be the predominant site of glucose uptake in skeletal muscle. Taken together, these studies shed light on how insulin and Ca2+ interact in signaling in cardiac and skeletal muscles. In the heart, components and channels that alter intracellular Ca2+ handling and might be involved in the development of acute cardiac failure in insulin resistant conditions have been identified. Further, we demonstrate that Ca2+ is important for insulin-mediated glucose uptake. Thus, the present data identify specific sites for therapeutic intervention in the treatment of conditions associated with insulin resistance

Skeletal muscle redox signaling in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovitis and the presence of serum autoantibodies. In addition, skeletal muscle weakness is a common comorbidity that contributes to inability to work and reduced quality of life. Loss in muscle mass cannot alone account for the muscle weakness induced by RA, but instead intramuscular dysfunction appears as a critical factor underlying the decreased force generating capacity for patients afflicted by arthritis. Oxidative stress and associated oxidative post-translational modifications have been shown to contribute to RA-induced muscle weakness in animal models of arthritis and patients with RA. However, it is still unclear how and which sources of reactive oxygen and nitrogen species (ROS/RNS) that are involved in the oxidative stress that drives the progression toward decreased muscle function in RA. Nevertheless, mitochondria, NADPH oxidases (NOX), nitric oxide synthases (NOS) and phospholipases (PLA) have all been associated with increased ROS/RNS production in RA-induced muscle weakness. In this review, we aim to cover potential ROS sources and underlying mechanisms of oxidative stress and loss of force production in RA. We also addressed the use of antioxidants and exercise as potential tools to counteract oxidative stress and skeletal muscle weakness

The underlying mechanisms of diabetic myopathy

National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH), R37-AR055099 / CONICYT-Chile, FONDECYT-11150243 / Facultad de Odontologia, Universidad de Chile, FIOUCh-Enlace 001/2015 / Swedish Research Council / Magnus Bergvall Stiftelse / Ake Wiberg Stiftels

Muscle Weakness in Rheumatoid Arthritis: The Role of Ca2+ and Free Radical Signaling

In addition to the primary symptoms arising from inflammatory processes in the joints, muscle weakness is commonly reported by patients with rheumatoid arthritis (RA). Muscle weakness not only reduces the quality of life for the affected patients, but also dramatically increases the burden on society since patients' work ability decreases. A 25–70% reduction in muscular strength has been observed in pateints with RA when compared with age-matched healthy controls. The reduction in muscle strength is often larger than what could be explained by the reduction in muscle size in patients with RA, which indicates that intracellular (intrinsic) muscle dysfunction plays an important role in the underlying mechanism of muscle weakness associated with RA. In this review, we highlight the present understanding of RA-associated muscle weakness with special focus on how enhanced Ca2+ release from the ryanodine receptor and free radicals (reactive oxygen/nitrogen species) contributes to muscle weakness, and recent developments of novel therapeutic interventions

Role of nitration in control of phosphorylase and glycogenolysis in mouse skeletal muscle.

Phosphorylase is one of the most carefully studied proteins in history, but knowledge of its regulation during intense muscle contraction is incomplete. Tyrosine nitration of purified preparations of skeletal muscle phosphorylase results in inactivation of the enzyme and this is prevented by antioxidants. Whether an altered redox state affects phosphorylase activity and glycogenolysis in contracting muscle is not known. Here, we investigate the role of redox state in control of phosphorylase and glycogenolysis in isolated mouse fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscle preparations during repeated contractions. Exposure of crude muscle extracts to H2O2 had little effect on phosphorylase activity. However, exposure of extracts to peroxynitrite (ONOO-), a nitrating/oxidizing agent, resulted in complete inactivation of phosphorylase (half maximal inhibition at ~200 µM ONOO-), which was fully reversed by the presence of an ONOO-scavanger, dithiothreitol (DTT). Incubation of isolated muscles with ONOO- resulted in nitration of phosphorylase and marked inhibition of glycogenolysis during repeated contractions. ONOO- also resulted in large decreases in high-energy phosphates (ATP and phosphocreatine) in the rested state and following repeated contractions. These metabolic changes were associated with decreased force production during repeated contractions (to ~60% of control). In contrast, repeated contractions did not result in nitration of phosphorylase, nor did DTT or the general antioxidant N-acetylcysteine alter glycogenolysis during repeated contractions. These findings demonstrate that ONOO- inhibits phosphorylase and glycogenolysis in living muscle under extreme conditions. However, nitration does not play a significant role in control of phosphorylase and glycogenolysis during repeated contractions