Somatic mutagenesis in satellite cells associates with human skeletal muscle aging.

Human aging is associated with a decline in skeletal muscle (SkM) function and a reduction in the number and activity of satellite cells (SCs), the resident stem cells. To study the connection between SC aging and muscle impairment, we analyze the whole genome of single SC clones of the leg muscle vastus lateralis from healthy individuals of different ages (21-78 years). We find an accumulation rate of 13 somatic mutations per genome per year, consistent with proliferation of SCs in the healthy adult muscle. SkM-expressed genes are protected from mutations, but aging results in an increase in mutations in exons and promoters, targeting genes involved in SC activity and muscle function. In agreement with SC mutations affecting the whole tissue, we detect a missense mutation in a SC propagating to the muscle. Our results suggest somatic mutagenesis in SCs as a driving force in the age-related decline of SkM function.

The Impact of Endurance Training on Human Skeletal Muscle Memory, Global Isoform Expression and Novel Transcripts.

Regularly performed endurance training has many beneficial effects on health and skeletal muscle function, and can be used to prevent and treat common diseases e.g. cardiovascular disease, type II diabetes and obesity. The molecular adaptation mechanisms regulating these effects are incompletely understood. To date, global transcriptome changes in skeletal muscles have been studied at the gene level only. Therefore, global isoform expression changes following exercise training in humans are unknown. Also, the effects of repeated interventions on transcriptional memory or training response have not been studied before. In this study, 23 individuals trained one leg for three months. Nine months later, 12 of the same subjects trained both legs in a second training period. Skeletal muscle biopsies were obtained from both legs before and after both training periods. RNA sequencing analysis of all 119 skeletal muscle biopsies showed that training altered the expression of 3,404 gene isoforms, mainly associated with oxidative ATP production. Fifty-four genes had isoforms that changed in opposite directions. Training altered expression of 34 novel transcripts, all with protein-coding potential. After nine months of detraining, no training-induced transcriptome differences were detected between the previously trained and untrained legs. Although there were several differences in the physiological and transcriptional responses to repeated training, no coherent evidence of an endurance training induced transcriptional skeletal muscle memory was found. This human lifestyle intervention induced differential expression of thousands of isoforms and several transcripts from unannotated regions of the genome. It is likely that the observed isoform expression changes reflect adaptational mechanisms and processes that provide the functional and health benefits of regular physical activity.

Improvement of insulin sensitivity in response to exercise training in type 2 diabetes mellitus is associated with vascular endothelial growth factor A expression.

PURPOSE: Insulin sensitivity changes in response to exercise training demonstrate a large variation. Vascular endothelial growth factor A could promote increased insulin sensitivity through angiogenesis. We investigated associations between changes in expression of key genes and insulin sensitivity, aerobic capacity and glycaemic control following exercise training in diabetes mellitus type 2. METHODS: Subjects with diabetes mellitus type 2 underwent 12 weeks of structured exercise. Euglycaemic clamp, exercise test and HbA1c were performed. Muscle biopsies were obtained for mRNA expression. RESULTS: A total of 16 subjects completed the study. Change in vascular endothelial growth factor A expression was positively associated with an increase in insulin sensitivity (p = 0.004) and with a decrease in HbA1c (p = 0.034). Vascular endothelial growth factor A receptor-1 expression showed similar associations. CONCLUSION: The variation in physical adaptation to exercise training in diabetes mellitus type 2 was associated with changes in expression of vascular endothelial growth factor A in muscle. This difference in induced gene expression could contribute to the variation in exercise training effects on insulin sensitivity. Measures of capillary blood flow need to be assessed in future studies.

PlanHab (Planetary Habitat Simulation): the combined and separate effects of 21 days bed rest and hypoxic confinement on human skeletal muscle miRNA expression.

The study concerns effects of 21 days of sustained bedrest and hypoxia, alone and in combination, on skeletal muscle microRNA (miRNA) expression. It is expected that astronauts undertaking long-duration missions will be exposed not only to microgravity but also to a hypoxic environment. The molecular machinery underlying microgravity-induced alterations in skeletal muscle structure and function is still largely unknown. One possible regulatory mechanism is altered expression of miRNAs, a group of noncoding RNAs which down-regulate many different target genes through increased degradation or translation of their messenger RNA Thirteen healthy men underwent three 21-day interventions, interspersed by 4-month washout periods: horizontal bedrest in normoxia, bedrest in hypoxia, ambulation in hypoxia. The level of hypoxia corresponded to 4000 m altitude. miRNAs from v. lateralis muscle biopsies were analyzed using a microarray covering ≈4000 human miRNAs. Sixteen mature miRNAs were up-regulated and three down-regulated after bedrest. The magnitudes of these changes were small and a large portion of the miRNAs affected by bedrest was also differentially expressed after washout periods. In fact, the number of differentially expressed probe sets over time was substantially larger than what could be detected after bedrest. Still, the majority of the miRNAs (let-7, miR-15, miR-25, miR-199, miR-133) that were differentially expressed following bedrest, belong to miRNA families previously reported in the context of muscle physiology, in particular to respond to changes in mechanical loading. Since only minor changes in miRNA expression could be detected after bedrest, our data indicate miRNA to play only a minor role in the substantial change in muscle phenotype seen with unloading.

Bone marrow derived cells in adult skeletal muscle tissue in humans.

BACKGROUND: During the past decade, several animal studies have demonstrated that in addition to local cells, cells from the bone marrow (BM) possess the ability to contribute to regeneration of injured skeletal muscle tissue. In addition, in mice, regular physical activity has been displayed to be a sufficient stimulus for BM-derived cell contribution to the muscle, indicating that this is part of the ongoing physiological remodeling of skeletal muscle. However, whether BM-derived cells participate in human skeletal muscle remodeling is not known. To this end, we analyzed the incorporation of BM-derived cells in healthy human skeletal muscle in women transplanted with male BM. METHODS: Skeletal muscle biopsies were obtained from the m. vastus lateralis of women transplanted with male donor hematopoietic stem cells 6 to 12 years earlier. Healthy women served as controls. Immunohistochemical staining for skeletal muscle fibers, satellite cells (SCs) or endothelial cells (ECs) combined with fluorescent in situ hybridization (FISH) of X and Y chromosomes was used to identify cells of BM origin within the biopsies. Three dimensional confocal imaging was performed to demonstrate colocalization of Y chromosome and DAPI within muscle fibers. To further investigate whether BM-derived cells incorporate into the SC niche, myoblasts were extracted from the biopsies from the transplanted women, cultured, and analyzed using XY FISH and immunocytochemistry. RESULTS: Three dimensional confocal imaging indisputably demonstrated colocalization of Y chromosome and DAPI within muscle fibers. Some Y chromosomes were found within centrally located nuclei. No Y chromosomes were detected in CD56+ SCs in the tissue sections nor in the myoblasts cultured from the extracted SCs. Y chromosome+ ECs were found in all sections from the transplanted subjects. No Y chromosomes were found in the skeletal muscle biopsies obtained from healthy control women. CONCLUSIONS: We demonstrate that BM-derived cells contribute to skeletal muscle fibers and ECs. Our results support that BM contribution to skeletal muscle occurs via direct fusion to muscle fibers, and that the contributing cells derive from the hematopoietic lineage. Thus, the present findings encourage further studies of the importance of this process for the physiological adaptation occurring throughout life.

The truncated splice variants, NT-PGC-1α and PGC-1α4, increase with both endurance and resistance exercise in human skeletal muscle.

Recently, a truncated peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) splice variant, PGC-1α4, that originates from the alternative promoter was shown to be induced by resistance exercise and to elicit muscle hypertrophy without coactivation of "classical" PGC-1α targets involved in mitochondrial biogenesis and angiogenesis. In order to test if distinct physiological adaptations are characterized by divergent induction of PGC-1α splice variants, we investigated the expression of truncated and nontruncated PGC-1α splice variants and PGC-1α transcripts originating from the alternative and the proximal promoter, in human skeletal muscle in response to endurance and resistance exercise. Both total PGC-1α and truncated PGC-1α mRNA expression were increased 2 h after endurance (P < 0.01) and resistance exercise (P < 0.01), with greater increases after endurance exercise (P < 0.05). Expression of nontruncated PGC-1α increased significantly in both exercise groups (P < 0.01 for both groups) without any significant differences between the groups. Both endurance and resistance exercise induced truncated as well as nontruncated PGC-1α transcripts from both the alternative and the proximal promoter. Further challenging the hypothesis that induction of distinct PGC-1α splice variants controls exercise adaptation, both nontruncated and truncated PGC-1α transcripts were induced in AICAR-treated human myotubes (P < 0.05). Thus, contrary to our hypothesis, resistance exercise did not specifically induce the truncated forms of PGC-1α. Induction of truncated PGC-1α splice variants does not appear to underlie distinct adaptations to resistance versus endurance exercise. Further studies on the existence of numerous splice variants originating from different promoters are needed.

Ischemia-reperfusion injury and pregnancy initiate time-dependent and robust signs of up-regulation of cardiac progenitor cells.

To explore how cardiac regeneration and cell turnover adapts to disease, different forms of stress were studied for their effects on the cardiac progenitor cell markers c-Kit and Isl1, the early cardiomyocyte marker Nkx2.5, and mast cells. Adult female rats were examined during pregnancy, after myocardial infarction and ischemia-reperfusion injury with/out insulin like growth factor-1(IGF-1) and hepatocyte growth factor (HGF). Different cardiac sub-domains were analyzed at one and two weeks post-intervention, both at the mRNA and protein levels. While pregnancy and myocardial infarction up-regulated Nkx2.5 and c-Kit (adjusted for mast cell activation), ischemia-reperfusion injury induced the strongest up-regulation which occurred globally throughout the entire heart and not just around the site of injury. This response seems to be partly mediated by increased endogenous production of IGF-1 and HGF. Contrary to c-Kit, Isl1 was not up-regulated by pregnancy or myocardial infarction while ischemia-reperfusion injury induced not a global but a focal up-regulation in the outflow tract and also in the peri-ischemic region, correlating with the up-regulation of endogenous IGF-1. The addition of IGF-1 and HGF did boost the endogenous expression of IGF and HGF correlating to focal up-regulation of Isl1. c-Kit expression was not further influenced by the exogenous growth factors. This indicates that there is a spatial mismatch between on one hand c-Kit and Nkx2.5 expression and on the other hand Isl1 expression. In conclusion, ischemia-reperfusion injury was the strongest stimulus with both global and focal cardiomyocyte progenitor cell marker up-regulations, correlating to the endogenous up-regulation of the growth factors IGF-1 and HGF. Also pregnancy induced a general up-regulation of c-Kit and early Nkx2.5+ cardiomyocytes throughout the heart. Utilization of these pathways could provide new strategies for the treatment of cardiac disease.

Estrogen receptors do not influence angiogenesis after myocardial infarction.

BACKGROUND: There is controversy on whether estrogen receptors are present and functioning in the myocardium. Aims. To explore if after myocardial infarction (MI) estrogen receptors α (ERα) and ß (ERß) are upregulated in myocardial tissue and to explore if the presence/ absence of ERα or ERß influences angiogenesis after MI. METHODS: MI was induced by ligation of the left anterior descending artery in knockout (KO) mice, ERαKO and ERßKO, respectively, and non-KO littermate-controls, C57Bl/6 mice. The hearts were harvested after 12 days. A part of the periinfarct tissue was collected for ERα and ERß mRNA expression determination by real-time polymerase chain reaction. Using immunohistochemistry, ERα and ERß protein expression and capillary and arteriolar densities were blindly determined in the periinfarct area. RESULTS: In myocardium disrupted mRNA was upregulated in both ERαKO and ERßKO, (p < 0.005) and did not change after MI. There was no change in mRNA expression of ERα or ERß in wild type mice after MI. Expression of ERß in ERαKO and of ERα in ERßKO did not change. Following MI ERα or ERß could not be demonstrated by immunohistochemistry in either wild type or ERαKO or ERßKO. The capillary and arteriolar densities after MI did not differ between the groups in the periinfarct area. CONCLUSIONS: Although disrupted ER mRNA is upregulated in myocardium of ER knockout mice, no change in these or native receptors occurs following MI. At least in this model ER therefore seems not to have a role in myocardial arteriogenesis and angiogenesis after MI.

Depressed expression of angiogenic growth factors in the subacute phase of myocardial ischemia: a mechanism behind the remodeling plateau?

BACKGROUND AND AIMS: To investigate whether, in the subacute phase of acute myocardial infarction, in the peri-infarcted area the expressions of the vascular endothelial growth factor (VEGF-A) and angiopoietin (Ang) ligand receptors are depressed, and whether overexpression of these angiogens counteracts a downregulation of myocardial function. METHODS: Acute myocardial infarction was induced by left anterior descending artery ligation and overexpression through injection of human VEGF-A165 and Ang-1 plasmids. The capillary and arteriolar densities, Akt-1 phosphorylation and citrate synthase activity were measured concurrent with the expression of VEGF-A, VEGFR1 and R2, Ang-1, Ang-2 and Tie-2. RESULTS: One day after AMI, VEGR-2 was unchanged but all other measured factors in the two families were upregulated. After day 2, the Ang-2 expression increased but other measured factors decreased. After gene transfer, the vascular supply, Akt phosphorylation and citrate synthase activity were higher in the peri-infarcted area, where also the endogenous angiogenic growth factor expressions were increased. CONCLUSION: A rapid decrease in angiogenic stimulating factors occurs in the subacute phase of AMI and is related to a progressive decrease in myocardial contraction. A negative consequence of such a circuit is a successive reduction in the vascular supply and contractility in areas with reduced perfusion. These negative adaptations can be counteracted by angiogen overexpression.

Activation of the erythropoietin receptor in human skeletal muscle.

OBJECTIVE: Erythropoietin receptor (EPOR) expression in non-hematological tissues has been shown to be activated by locally produced and/or systemically delivered EPO. Improved oxygen homeostasis, a well-established consequence of EPOR activation, is very important for human skeletal muscle performance. In the present study we investigate whether human skeletal muscle fibers and satellite cells express EPOR and if it is activated by exercise. DESIGN AND METHODS: Ten healthy males performed 65 min of cycle exercise. Biopsies were obtained from the vastus lateralis muscle and femoral arterio-venous differences in EPO concentrations were estimated. RESULTS: The EPOR protein was localized in areas corresponding to the sarcolemma and capillaries. Laser dissection identified EPOR mRNA expression in muscle fibers. Also, EPOR mRNA and protein were both detected in human skeletal muscle satellite cells. In the initial part of the exercise bout there was a release of EPO from the exercising leg to the circulation, possibly corresponding to an increased bioavailability of EPO. After exercise, EPOR mRNA and EPOR-associated JAK2 phosphorylation were increased. CONCLUSIONS: Interaction with JAK2 is required for EPOR signaling and the increase found in phosphorylation is therefore closely linked to the activation of EPOR. The receptor activation by acute exercise suggests that signaling through EPOR is involved in exercise-induced skeletal muscle adaptation, thus extending the biological role of EPO into the skeletal muscle.

AMP deaminase deficiency is associated with lower sprint cycling performance in healthy subjects.

AMP deaminase (AMPD) deficiency is an inherited disorder of skeletal muscle found in approximately 2% of the Caucasian population. Although most AMPD-deficient individuals are asymptomatic, a small subset has exercise-related cramping and pain without any other identifiable neuromuscular complications. This heterogeneity has raised doubts about the physiological significance of AMPD in skeletal muscle, despite evidence for disrupted adenine nucleotide catabolism during exercise in deficient individuals. Previous studies have evaluated the effect of AMPD deficiency on exercise performance with mixed results. This study was designed to circumvent the perceived limitations in previous reports by measuring exercise performance during a 30-s Wingate test in 139 healthy, physically active subjects of both sexes, with different AMPD1 genotypes, including 12 AMPD-deficient subjects. Three of the deficient subjects were compound heterozygotes characterized by the common c.34C>T mutation in one allele and a newly discovered AMPD1 mutation, c.404delT, in the other. While there was no significant difference in peak power across AMPD1 genotypes, statistical analysis revealed a faster power decrease in the AMPD-deficient group and a difference in mean power across the genotypes (P = 0.0035). This divergence was most striking at 15 s of the 30-s cycling. Assessed by the fatigue index, the decrease in power output at 15 s of exercise was significantly greater in the deficient group compared with the other genotypes (P = 0.0006). The approximate 10% lower mean power in healthy AMPD-deficient subjects during a 30-s Wingate cycling test reveals a functional role for the AMPD1 enzyme in sprint exercise.

Fatty acid binding protein 4 in human skeletal muscle.

The mechanisms that regulate intramyocellular triglycerol (IMTG) storage and mobilization are largely unknown. However, during the last decades several intracellular fatty acid binding proteins (FABPs) have been identified. FABP3 is the dominating FABP in skeletal muscle. Expression of additional FABPs is suggested from findings in FABP3-null mutated mice. In the present study, our aims were to investigate if FABP4 is expressed within skeletal muscle fibers and if FABP3 and FABP4 are more abundant in skeletal muscle fibers in endurance-trained than in control subjects. We show that FABP4 protein is expressed within the skeletal muscle fibers and that FABP4 mRNA and protein are more abundant in the endurance trained subjects. Still, FABP4 is markedly less expressed than FABP3, which is the generally accepted dominating FABP in skeletal muscle tissue.

Modulation of extracellular matrix genes reflects the magnitude of physiological adaptation to aerobic exercise training in humans.

BACKGROUND: Regular exercise reduces cardiovascular and metabolic disease partly through improved aerobic fitness. The determinants of exercise-induced gains in aerobic fitness in humans are not known. We have demonstrated that over 500 genes are activated in response to endurance-exercise training, including modulation of muscle extracellular matrix (ECM) genes. Real-time quantitative PCR, which is essential for the characterization of lower abundance genes, was used to examine 15 ECM genes potentially relevant for endurance-exercise adaptation. Twenty-four sedentary male subjects undertook six weeks of high-intensity aerobic cycle training with muscle biopsies being obtained both before and 24 h after training. Subjects were ranked based on improvement in aerobic fitness, and two cohorts were formed (n = 8 per group): the high-responder group (HRG; peak rate of oxygen consumption increased by +0.71 +/- 0.1 L min(-1); p < 0.0001) while the low-responder group (LRG; peak rate of oxygen consumption did not change, +0.17 +/- 0.1 L min(-1), ns). ECM genes profiled included the angiopoietin 1 and related genes (angiopoietin 2, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1) and 2 (TIE2), vascular endothelial growth factor (VEGF) and related receptors (VEGF receptor 1, VEGF receptor 2 and neuropilin-1), thrombospondin-4, alpha2-macroglobulin and transforming growth factor beta2. RESULTS: neuropilin-1 (800%; p < 0.001) and VEGF receptor 2 (300%; p < 0.01) transcript abundance increased only in the HRG, whereas levels of VEGF receptor 1 mRNA actually declined in the LRG (p < 0.05). TIE1 and TIE2 mRNA levels were unaltered in the LRG, whereas transcription levels of both genes were increased by 2.5-fold in the HRG (p < 0.01). Levels of thrombospondin-4 (900%; p < 0.001) and alpha2-macroglobulin (300%, p < 0.05) mRNA increased substantially in the HRG. In contrast, the amount of transforming growth factor beta2 transcript increased only in the HRG (330%; p < 0.01), whereas it remained unchanged in the LRG (-80%). CONCLUSION: We demonstrate for the first time that aerobic training activates angiopoietin 1 and TIE2 genes in human muscle, but only when aerobic capacity adapts to exercise-training. The fourfold-greater increase in aerobic fitness and markedly differing gene expression profile in the HRG indicates that these ECM genes may be critical for physiological adaptation to exercise in humans. In addition, we show that, without careful demonstration of physiological adaptation, conclusions derived from gene expression profiling of human skeletal muscle following exercise may be of limited value. We propose that future studies should (a) investigate the mechanisms that underlie the apparent link between physiological adaptation and gene expression and (b) use the genes profiled in this paper as candidates for population genetic studies.

Human muscle gene expression responses to endurance training provide a novel perspective on Duchenne muscular dystrophy.

Global gene expression profiling is used to generate novel insight into a variety of disease states. Such studies yield a bewildering number of data points, making it a challenge to validate which genes specifically contribute to a disease phenotype. Aerobic exercise training represents a plausible model for identification of molecular mechanisms that cause metabolic-related changes in human skeletal muscle. We carried out the first transcriptome-wide characterization of human skeletal muscle responses to 6 wk of supervised aerobic exercise training in 8 sedentary volunteers. Biopsy samples before and after training allowed us to identify approximately 470 differentially regulated genes using the Affymetrix U95 platform (80 individual hybridization steps). Gene ontology analysis indicated that extracellular matrix and calcium binding gene families were most up-regulated after training. An electronic reanalysis of a Duchenne muscular dystrophy (DMD) transcript expression dataset allowed us to identify approximately 90 genes modulated in a nearly identical fashion to that observed in the endurance exercise dataset. Trophoblast noncoding RNA, an interfering RNA species, was the singular exception-being up-regulated by exercise and down-regulated in DMD. The common overlap between gene expression datasets may be explained by enhanced alpha7beta1 integrin signaling, and specific genes in this signaling pathway were up-regulated in both datasets. In contrast to these common features, OXPHOS gene expression is subdued in DMD yet elevated by exercise, indicating that more than one major mechanism must exist in human skeletal muscle to sense activity and therefore regulate gene expression. Exercise training modulated diabetes-related genes, suggesting our dataset may contain additional and novel gene expression changes relevant for the anti-diabetic properties of exercise. In conclusion, gene expression profiling after endurance exercise training identified a range of processes responsible for the physiological remodeling of human skeletal muscle tissue, many of which were similarly regulated in DMD. Furthermore, our analysis demonstrates that numerous genes previously suggested as being important for the DMD disease phenotype may principally reflect compensatory integrin signaling.

Approaches to anorexia in rodents: focus on the anx/anx mouse.

Eating disorders constitute major medical health problems in the western world. Even though little is known about the mechanisms behind abnormal eating behavior, it has become clear that the central nervous system (CNS), particularly the hypothalamus, plays a significant role. The anorexic anx/anx mouse is a unique model for studying food intake and energy expenditure. The anx mutation is linked to marked alterations in hypothalamic distributions of signal substances known to have potent regulatory roles in the control of food intake. We have identified a mutation in anx/anx mice that is likely to cause the anorectic phenotype. Using RNA profiling, we have found 29 genes with differential expression in the anx/anx mouse brain. The anx gene, its protein product or molecules in the anx pathway may thus be interesting targets for development of new pharmaceuticals for the treatment of eating disorders. Based on the histochemical alterations found in the anx/anx mouse, we hypothesised and showed that many sera from anorectic/bulimic patients contain antibodies that bind specifically to the hypothalamic food intake regulatory system in rat. This finding represents a novel research avenue that may lead to a better understanding of eating disorders. It also suggests that targeted immunological approaches may be used in therapy.

Cyclin D2 expression in familial and sporadic breast cancer.

In a differential display experiment comparing normal breast epithelial cells with breast tumors and breast cancer cell lines we isolated a gene, cyclin D2, showing a low expression in tumor cells compared to normal cells. This result was confirmed using Northern blotting and RT-PCR. In total, low expression was seen in 48/109 (44%) tumors. Low cyclin D2 expression was seen in 10/39 (25%) sporadic breast cancer, and in 38/70 (54%) of familial breast cancer. In order to find the explanation for the down-regulation of cyclin D2 we screened for inactivating mutations in tumors with low expression, and studied methylation of the promoter region in breast cancer cell lines. We also used growth factors in an attempt to stimulate cell lines to express cyclin D2, and transfected the cell lines with a vector expressing cyclin D2. Treatment with 5-azacytidin for four days resulted in transcription of cyclin D2. Our result suggests that methylation of the cyclin D2 promoter or another regulating gene is associated with loss of cyclin D2 expression in breast cancer.