Generation of allogenic and xenogeneic functional muscle stem cells for intramuscular transplantation.

Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected mouse DMD-induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD-iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Remarkably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation, and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.

Robust, Precise, and Deep Proteome Profiling Using a Small Mass Range and Narrow Window Data-Independent-Acquisition Scheme.

In recent years, a plethora of different data-independent acquisition methods have been developed for proteomics to cover a wide range of requirements. Current deep proteome profiling methods rely on fractionations, elaborate chromatography, and mass spectrometry setups or display suboptimal quantitative precision. We set out to develop an easy-to-use one shot DIA method that achieves high quantitative precision and high proteome coverage. We achieve this by focusing on a small mass range of 430-670 m/z using small isolation windows without overlap. With this new method, we were able to quantify >9200 protein groups in HEK lysates with an average coefficient of variance of 3.2%. To demonstrate the power of our newly developed narrow mass range method, we applied it to investigate the effect of PGC-1α knockout on the skeletal muscle proteome in mice. Compared to a standard data-dependent acquisition method, we could double proteome coverage and, most importantly, achieve a significantly higher quantitative precision, as compared to a previously proposed DIA method. We believe that our method will be especially helpful in quantifying low abundant proteins in samples with a high dynamic range. All raw and result files are available at massive.ucsd.edu (MSV000092186).

Molecular control of endurance training adaptation in male mouse skeletal muscle.

Skeletal muscle has an enormous plastic potential to adapt to various external and internal perturbations. Although morphological changes in endurance-trained muscles are well described, the molecular underpinnings of training adaptation are poorly understood. We therefore aimed to elucidate the molecular signature of muscles of trained male mice and unravel the training status-dependent responses to an acute bout of exercise. Our results reveal that, even though at baseline an unexpectedly low number of genes define the trained muscle, training status substantially affects the transcriptional response to an acute challenge, both quantitatively and qualitatively, in part associated with epigenetic modifications. Finally, transiently activated factors such as the peroxisome proliferator-activated receptor-γ coactivator 1α are indispensable for normal training adaptation. Together, these results provide a molecular framework of the temporal and training status-dependent exercise response that underpins muscle plasticity in training.

The molecular athlete: exercise physiology from mechanisms to medals.

Human skeletal muscle demonstrates remarkable plasticity, adapting to numerous external stimuli including the habitual level of contractile loading. Accordingly, muscle function and exercise capacity encompass a broad spectrum, from inactive individuals with low levels of endurance and strength to elite athletes who produce prodigious performances underpinned by pleiotropic training-induced muscular adaptations. Our current understanding of the signal integration, interpretation, and output coordination of the cellular and molecular mechanisms that govern muscle plasticity across this continuum is incomplete. As such, training methods and their application to elite athletes largely rely on a "trial-and-error" approach, with the experience and practices of successful coaches and athletes often providing the bases for "post hoc" scientific enquiry and research. This review provides a synopsis of the morphological and functional changes along with the molecular mechanisms underlying exercise adaptation to endurance- and resistance-based training. These traits are placed in the context of innate genetic and interindividual differences in exercise capacity and performance, with special consideration given to aging athletes. Collectively, we provide a comprehensive overview of skeletal muscle plasticity in response to different modes of exercise and how such adaptations translate from "molecules to medals."

Drugs, clocks and exercise in ageing: hype and hope, fact and fiction.

Ageing is a biological process that is linked to a functional decline, ultimately resulting in death. Large interindividual differences exist in terms of life- and healthspan, representing life expectancy and the number of years spent in the absence of major diseases, respectively. The genetic and molecular mechanisms that are involved in the regulation of the ageing process, and those that render age the main risk factor for many diseases are still poorly understood. Nevertheless, a growing number of compounds have been put forward to affect this process. However, for scientists and laypeople alike, it is difficult to separate fact from fiction, and hype from hope. In this review, we discuss the currently pursued pharmacological anti-ageing approaches. These are compared to non-pharmacological interventions, some of which confer powerful effects on health and well-being, in particular an active lifestyle and exercise. Moreover, functional parameters and biological clocks as well as other molecular marks are compared in terms of predictive power of morbidity and mortality. Then, conceptual aspects and roadblocks in the development of anti-ageing drugs are outlined. Finally, an overview on current and future strategies to mitigate age-related pathologies and the extension of life- and healthspan is provided.

Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle.

Preserving skeletal muscle function is essential to maintain life quality at high age. Calorie restriction (CR) potently extends health and lifespan, but is largely unachievable in humans, making "CR mimetics" of great interest. CR targets nutrient-sensing pathways centering on mTORC1. The mTORC1 inhibitor, rapamycin, is considered a potential CR mimetic and is proven to counteract age-related muscle loss. Therefore, we tested whether rapamycin acts via similar mechanisms as CR to slow muscle aging. Here we show that long-term CR and rapamycin unexpectedly display distinct gene expression profiles in geriatric mouse skeletal muscle, despite both benefiting aging muscles. Furthermore, CR improves muscle integrity in mice with nutrient-insensitive, sustained muscle mTORC1 activity and rapamycin provides additive benefits to CR in naturally aging mouse muscles. We conclude that rapamycin and CR exert distinct, compounding effects in aging skeletal muscle, thus opening the possibility of parallel interventions to counteract muscle aging.

Interleukin-6 potentiates endurance training adaptation and improves functional capacity in old mice.

BACKGROUND: Interventions to preserve functional capacities at advanced age are becoming increasingly important. So far, exercise provides the only means to counteract age-related decrements in physical performance and muscle function. Unfortunately, the effectiveness of exercise interventions in elderly populations is hampered by reduced acceptance and compliance as well as disuse complications. We therefore studied whether application of interleukin-6 (IL-6), a pleiotropic myokine that is induced by skeletal muscle activity and exerts broad systemic effects in response to exercise, affects physical performance and muscle function alone or in combination with training in aged mice. METHODS: Sedentary old male mice (Sed+Saline, n = 15) were compared with animals that received recombinant IL-6 (rIL-6) in an exercise-mimicking pulsatile manner (Sed+IL-6, n = 16), were trained with a moderate-intensity, low-volume endurance exercise regimen (Ex+Saline, n = 13), or were exposed to a combination of these two interventions (Ex+IL-6, n = 16) for 12 weeks. Before and at the end of the intervention, mice underwent a battery of tests to quantify endurance performance, muscle contractility in situ, motor coordination, and gait and metabolic parameters. RESULTS: Mice exposed to enhanced levels of IL-6 during endurance exercise bouts showed superior improvements in endurance performance (33% more work and 12% greater peak power compared with baseline), fatigue resistance in situ (P = 0.0014 vs. Sed+Saline; P = 0.0199 vs. Sed+IL-6; and P = 0.0342 vs. Ex+Saline), motor coordination (rotarod performance, P = 0.0428), and gait (gait speed, P = 0.0053) following training. Pulsatile rIL-6 treatment in sedentary mice had only marginal effects on glucose tolerance and some gait parameters. No increase in adverse events or mortality related to rIL-6 treatment was observed. CONCLUSIONS: Administration of rIL-6 paired with treadmill running bouts potentiates the adaptive response to a moderate-intensity low-volume endurance exercise regimen in old mice, while being safe and well tolerated.

PGC-1α regulates myonuclear accretion after moderate endurance training.

The transcriptional demands of skeletal muscle fibres are high and require hundreds of nuclei (myonuclei) to produce specialised contractile machinery and multiple mitochondria along their length. Each myonucleus spatially regulates gene expression in a finite volume of cytoplasm, termed the myonuclear domain (MND), which positively correlates with fibre cross-sectional area (CSA). Endurance training triggers adaptive responses in skeletal muscle, including myonuclear accretion, decreased MND sizes and increased expression of the transcription co-activator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Previous work has shown that overexpression of PGC-1α in skeletal muscle regulates mitochondrial biogenesis, myonuclear accretion and MND volume. However, whether PGC-1α is critical for these processes in adaptation to endurance training remained unclear. To test this, we evaluated myonuclear distribution and organisation in endurance-trained wild-type mice and mice lacking PGC-1α in skeletal muscle (PGC-1α mKO). Here, we show a differential myonuclear accretion response to endurance training that is governed by PGC-1α and is dependent on muscle fibre size. The positive relationship of MND size and muscle fibre CSA trended towards a stronger correlation in PGC-1a mKO versus control after endurance training, suggesting that myonuclear accretion was slightly affected with increasing fibre CSA in PGC-1α mKO. However, in larger fibres, the relationship between MND and CSA was significantly altered in trained versus sedentary PGC-1α mKO, suggesting that PGC-1α is critical for myonuclear accretion in these fibres. Accordingly, there was a negative correlation between the nuclear number and CSA, suggesting that in larger fibres myonuclear numbers fail to scale with CSA. Our findings suggest that PGC-1α is an important contributor to myonuclear accretion following moderate-intensity endurance training. This may contribute to the adaptive response to endurance training by enabling a sufficient rate of transcription of genes required for mitochondrial biogenesis.

Remodeling of metabolism and inflammation by exercise ameliorates tumor-associated anemia.

A considerable number of patients with cancer suffer from anemia, which has detrimental effects on quality of life and survival. The mechanisms underlying tumor-associated anemia are multifactorial and poorly understood. Therefore, we aimed at systematically assessing the patho-etiology of tumor-associated anemia in mice. We demonstrate that reduced red blood cell (RBC) survival rather than altered erythropoiesis is driving the development of anemia. The tumor-induced inflammatory and metabolic remodeling affect RBC integrity and augment splenic phagocyte activity promoting erythrophagocytosis. Exercise training normalizes these tumor-associated abnormal metabolic profiles and inflammation and thereby ameliorates anemia, in part, by promoting RBC survival. Fatigue was prevented in exercising tumor-bearing mice. Thus, exercise has the unique potential to substantially modulate metabolism and inflammation and thereby counteracts pathological remodeling of these parameters by the tumor microenvironment. Translation of this finding to patients with cancer could have a major impact on quality of life and potentially survival.

Branched-chain amino acid metabolism is regulated by ERRα in primary human myotubes and is further impaired by glucose loading in type 2 diabetes.

AIMS/HYPOTHESIS: Increased levels of branched-chain amino acids (BCAAs) are associated with type 2 diabetes pathogenesis. However, most metabolomic studies are limited to an analysis of plasma metabolites under fasting conditions, rather than the dynamic shift in response to a metabolic challenge. Moreover, metabolomic profiles of peripheral tissues involved in glucose homeostasis are scarce and the transcriptomic regulation of genes involved in BCAA catabolism is partially unknown. This study aimed to identify differences in circulating and skeletal muscle BCAA levels in response to an OGTT in individuals with normal glucose tolerance (NGT) or type 2 diabetes. Additionally, transcription factors involved in the regulation of the BCAA gene set were identified. METHODS: Plasma and vastus lateralis muscle biopsies were obtained from individuals with NGT or type 2 diabetes before and after an OGTT. Plasma and quadriceps muscles were harvested from skeletal muscle-specific Ppargc1a knockout and transgenic mice. BCAA-related metabolites and genes were assessed by LC-MS/MS and quantitative RT-PCR, respectively. Small interfering RNA and adenovirus-mediated overexpression techniques were used in primary human skeletal muscle cells to study the role of PPARGC1A and ESRRA in the expression of the BCAA gene set. Radiolabelled leucine was used to analyse the impact of oestrogen-related receptor α (ERRα) knockdown on leucine oxidation. RESULTS: Impairments in BCAA catabolism in people with type 2 diabetes under fasting conditions were exacerbated after a glucose load. Branched-chain keto acids were reduced 37-56% after an OGTT in the NGT group, whereas no changes were detected in individuals with type 2 diabetes. These changes were concomitant with a stronger correlation with glucose homeostasis biomarkers and downregulated expression of branched-chain amino acid transaminase 2, branched-chain keto acid dehydrogenase complex subunits and 69% of downstream BCAA-related genes in skeletal muscle. In primary human myotubes overexpressing peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α, encoded by PPARGC1A), 61% of the analysed BCAA genes were upregulated, while 67% were downregulated in the quadriceps of skeletal muscle-specific Ppargc1a knockout mice. ESRRA (encoding ERRα) silencing completely abrogated the PGC-1α-induced upregulation of BCAA-related genes in primary human myotubes. CONCLUSIONS/INTERPRETATION: Metabolic inflexibility in type 2 diabetes impacts BCAA homeostasis and attenuates the decrease in circulating and skeletal muscle BCAA-related metabolites after a glucose challenge. Transcriptional regulation of BCAA genes in primary human myotubes via PGC-1α is ERRα-dependent.

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.

Effects of different training modalities on phosphate homeostasis and local vitamin D metabolism in rat bone.

OBJECTIVES: Mechanical loading may be an important factor in the regulation of bone derived hormones involved in phosphate homeostasis. This study investigated the effects of peak power and endurance training on expression levels of fibroblast growth factor 23 (FGF23) and 1α-hydroxylase (CYP27b1) in bone. METHODS: Thirty-eight rats were assigned to six weeks of training in four groups: peak power (PT), endurance (ET), PT followed by ET (PET) or no training (control). In cortical bone, FGF23 was quantified using immunohistochemistry. mRNA expression levels of proteins involved in phosphate and vitamin D homeostasis were quantified in cortical bone and kidney. C-terminal FGF23, 25-hydroxyvitamin D3, parathyroid hormone (PTH), calcium and phosphate concentrations were measured in plasma or serum. RESULTS: Neither FGF23 mRNA and protein expression levels in cortical bone nor FGF23 plasma concentrations differed between the groups. In cortical bone, mRNA expression levels of sclerostin (SOST), dental matrix protein 1 (DMP1), phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and matrix extracellular phosphoglycoprotein (MEPE) were lower after PT compared to ET and PET. Expression levels of CYP27b1 and vitamin D receptor (VDR) in tibial bone were decreased after PT compared to ET. In kidney, no differences between groups were observed for mRNA expression levels of CYP27b1, 24-hydroxylase (CYP24), VDR, NaPi-IIa cotransporter (NPT2a) and NaPi-IIc cotransporter (NPT2c). Serum PTH concentrations were higher after PT compared to controls. CONCLUSION: After six weeks, none of the training modalities induced changes in FGF23 expression levels. However, PT might have caused changes in local phosphate regulation within bone compared to ET and PET. CYP27b1 and VDR expression in bone was reduced after PT compared to ET, suggesting high intensity peak power training in this rat model is associated with decreased vitamin D signalling in bone.

Muscle Wasting Diseases: Novel Targets and Treatments.

Adequate skeletal muscle plasticity is an essential element for our well-being, and compromised muscle function can drastically affect quality of life, morbidity, and mortality. Surprisingly, however, skeletal muscle remains one of the most under-medicated organs. Interventions in muscle diseases are scarce, not only in neuromuscular dystrophies, but also in highly prevalent secondary wasting pathologies such as sarcopenia and cachexia. Even in other diseases that exhibit a well-established risk correlation of muscle dysfunction due to a sedentary lifestyle, such as type 2 diabetes or cardiovascular pathologies, current treatments are mostly targeted on non-muscle tissues. In recent years, a renewed focus on skeletal muscle has led to the discovery of various novel drug targets and the design of new pharmacological approaches. This review provides an overview of the current knowledge of the key mechanisms involved in muscle wasting conditions and novel pharmacological avenues that could ameliorate muscle diseases.

Over-expression of a retinol dehydrogenase (SRP35/DHRS7C) in skeletal muscle activates mTORC2, enhances glucose metabolism and muscle performance.

SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C dehydrogenase/ reductase family 7. Here we show that its over-expression in mouse skeletal muscles induces enhanced muscle performance in vivo, which is not related to alterations in excitation-contraction coupling but rather linked to enhanced glucose metabolism. Over-expression of SRP-35 causes increased phosphorylation of AktS473, triggering plasmalemmal targeting of GLUT4 and higher glucose uptake into muscles. SRP-35 signaling involves RARα and RARγ (non-genomic effect), PI3K and mTORC2. We also demonstrate that all-trans retinoic acid, a downstream product of the enzymatic activity of SRP-35, mimics the effect of SRP-35 in skeletal muscle, inducing a synergistic effect with insulin on AKTS473 phosphorylation. These results indicate that SRP-35 affects skeletal muscle metabolism and may represent an important target for the treatment of metabolic diseases.

Plasticity of the Muscle Stem Cell Microenvironment.

Satellite cells (SCs) are adult muscle stem cells capable of repairing damaged and creating new muscle tissue throughout life. Their functionality is tightly controlled by a microenvironment composed of a wide variety of factors, such as numerous secreted molecules and different cell types, including blood vessels, oxygen, hormones, motor neurons, immune cells, cytokines, fibroblasts, growth factors, myofibers, myofiber metabolism, the extracellular matrix and tissue stiffness. This complex niche controls SC biology-quiescence, activation, proliferation, differentiation or renewal and return to quiescence. In this review, we attempt to give a brief overview of the most important players in the niche and their mutual interaction with SCs. We address the importance of the niche to SC behavior under physiological and pathological conditions, and finally survey the significance of an artificial niche both for basic and translational research purposes.

Paracrine cross-talk between skeletal muscle and macrophages in exercise by PGC-1α-controlled BNP.

Activation of resident and infiltrating immune cells is a central event in training adaptation and other contexts of skeletal muscle repair and regeneration. A precise orchestration of inflammatory events in muscle fibers and immune cells is required after recurrent contraction-relaxation cycles. However, the mechanistic aspects of this important regulation remain largely unknown. We now demonstrate that besides a dominant role in controlling cellular metabolism, the peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) also has a profound effect on cytokine expression in muscle tissue. Muscle PGC-1α expression results in activation of tissue-resident macrophages, at least in part mediated by PGC-1α-dependent B-type natriuretic peptide (BNP) production and secretion. Positive effects of exercise in metabolic diseases and other pathologies associated with chronic inflammation could accordingly involve the PGC-1α-BNP axis and thereby provide novel targets for therapeutic approaches.

Muscle PGC-1α modulates satellite cell number and proliferation by remodeling the stem cell niche.

BACKGROUND: The myogenic capacity of satellite cells (SCs), adult muscle stem cells, is influenced by aging, exercise, and other factors. In skeletal muscle, the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a key regulator of oxidative metabolism and endurance training adaptation. However, a link between PGC-1α and SC behavior remains unexplored. METHODS: We have now studied SC function in a PGC-1α fiber-specific gain-of-function animal model. RESULTS: In surprising contrast to bona fide exercise, muscle-specific PGC-1α transgenic mice have lower SC numbers. Nevertheless, SCs from these mice have a higher propensity for activation and proliferation. Intriguingly, muscle PGC-1α triggers a remodeling of the SC niche by altering the extracellular matrix composition, including the levels of fibronectin, which affects the proliferative output of SCs. CONCLUSIONS: Taken together, PGC-1α indirectly affects SC plasticity in skeletal muscle and thereby might contribute to improved SC activation in exercise.

PGC-1α modulates necrosis, inflammatory response, and fibrotic tissue formation in injured skeletal muscle.

BACKGROUND: Skeletal muscle tissue has an enormous regenerative capacity that is instrumental for a successful defense against muscle injury and wasting. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) exerts therapeutic effects in several muscle pathologies, but its role in damage-induced muscle regeneration is unclear. METHODS: Using muscle-specific gain- and loss-of-function models for PGC-1α in combination with the myotoxic agent cardiotoxin (CTX), we explored the role of this transcriptional coactivator in muscle damage and inflammation. RESULTS: Interestingly, we observed PGC-1α-dependent effects at the early stages of regeneration, in particular regarding macrophage accumulation and polarization from the pro-inflammatory M1 to the anti-inflammatory M2 type, a faster resolution of necrosis and protection against the development of fibrosis after multiple CTX-induced injuries. CONCLUSIONS: PGC-1α exerts beneficial effects on muscle inflammation that might contribute to the therapeutic effects of elevated muscle PGC-1α in different models of muscle wasting.