Smyd1: Implications for novel approaches in rhabdomyosarcoma therapy.

Rhabdomyosarcoma (RMS), a tumor that consists of poorly differentiated skeletal muscle cells, is the most common soft-tissue sarcoma in children. Despite considerable progress within the last decades, therapeutic options are still limited, warranting the need for novel approaches. Recent data suggest deregulation of the Smyd1 protein, a sumoylation target as well as H3K4me2/3 methyltransferase and transcriptional regulator in myogenesis, and its binding partner skNAC, in RMS cells. Here, we show that despite the fact that most RMS cells express at least low levels of Smyd1 and skNAC, failure to upregulate expression of these genes in reaction to differentiation-promoting signals can always be observed. While overexpression of the Smyd1 gene enhances many aspects of RMS cell differentiation and inhibits proliferation rate and metastatic potential of these cells, functional integrity of the putative Smyd1 sumoylation motif and its SET domain, the latter being crucial for HMT activity, appear to be prerequisites for most of these effects. Based on these findings, we explored the potential for novel RMS therapeutic strategies, employing small-molecule compounds to enhance Smyd1 activity. In particular, we tested manipulation of (a) Smyd1 sumoylation, (b) stability of H3K4me2/3 marks, and (c) calpain activity, with calpains being important targets of Smyd1 in myogenesis. We found that specifically the last strategy might represent a promising approach, given that suitable small-molecule compounds will be available for clinical use in the future.

The Acute, Short-, and Long-Term Effects of Endurance Exercise on Skeletal Muscle Transcriptome Profiles.

A better understanding of the cellular and molecular mechanisms that are involved in skeletal muscle adaptation to exercise is fundamentally important to take full advantage of the enormous benefits that exercise training offers in disease prevention and therapy. The aim of this study was to elucidate the transcriptional signatures that distinguish the endurance-trained and untrained muscles in young adult males (24 ± 3.5 years). We characterized baseline differences as well as acute exercise-induced transcriptome responses in vastus lateralis biopsy specimens of endurance-trained athletes (ET; n = 8; VO2max, 67.2 ± 8.9 mL/min/kg) and sedentary healthy volunteers (SED; n = 8; VO2max, 40.3 ± 7.6 mL/min/kg) using microarray technology. A second cohort of SED volunteers (SED-T; n = 10) followed an 8-week endurance training program to assess expression changes of selected marker genes in the course of skeletal muscle adaptation. We deciphered differential baseline signatures that reflected major differences in the oxidative and metabolic capacity of the endurance-trained and untrained muscles. SED-T individuals in the training group displayed an up-regulation of nodal regulators of oxidative adaptation after 3 weeks of training and a significant shift toward the ET signature after 8 weeks. Transcriptome changes provoked by 1 h of intense cycling exercise only poorly overlapped with the genes that constituted the differential baseline signature of ETs and SEDs. Overall, acute exercise-induced transcriptional responses were connected to pathways of contractile, oxidative, and inflammatory stress and revealed a complex and highly regulated framework of interwoven signaling cascades to cope with exercise-provoked homeostatic challenges. While temporal transcriptional programs that were activated in SEDs and ETs were quite similar, the quantitative divergence in the acute response transcriptomes implicated divergent kinetics of gene induction and repression following an acute bout of exercise. Together, our results provide an extensive examination of the transcriptional framework that underlies skeletal muscle plasticity.

The ROS scavenger PDTC affects adaptation to treadmill running in mice: distinct effects on murine body mass, resting heart rate and skeletal muscle fiber type composition.

Regular exercise induces a broad spectrum of adaptation reactions in a variety of tissues and organs. However, the respective mechanisms are incompletely understood. In the context of their analysis, animal model systems, specifically rodent treadmill running protocols, play an important role. However, few researchers have studied different aspects of adaptation, such as cardiorespiratory and skeletal muscle training effects, within one set of experiments. Here, we analyzed physiological adaptation to 10 weeks of regular, moderate-intensity, uphill treadmill running in mice, a widely used model for endurance exercise training. To study the effects of reactive oxygen species (ROS), which have been suggested to be major regulators of training adaptation, a subgroup of mice was treated with the ROS scavenger PDTC (pyrrolidine dithiocarbamate). We found that mass gain in mice that exercised under PDTC treatment lagged behind that of all other experimental groups. In addition, both exercise and PDTC significantly and additively decreased resting heart rate. Furthermore, there was a trend towards an enhanced proportion of type 2A skeletal muscle fibers and differential expression of metabolism-associated genes, indicating metabolic and functional adaptation of skeletal muscle fibers. By contrast, there were no effects on grip strength and relative mass of individual muscles, suggesting that our protocol of uphill running did not increase skeletal muscle hypertrophy and strength. Taken together, our data suggest that a standard protocol of moderate-intensity uphill running induces adaptation reactions at multiple levels, part of which might be modulated by ROS, but does not enhance skeletal muscle hypertrophy and force.

TRAF6: A player in CVB3-induced myocarditis?

Coxsackievirus B3 (CVB3) is an important inducer of myocarditis, which, in susceptible individuals, can chronify and eventually lead to the development of dilated cardiomyopathy and heart failure. The respective mechanisms are not completely understood. Here, we analyzed expression of the TRAF6 gene, encoding TNF receptor-associated factor 6 (TRAF6), a signal transduction scaffold protein that acts downstream of cytokine receptors, in heart tissue of susceptible and non-susceptible mouse strains. We found that after infection, TRAF6 expression was upregulated in both non-susceptible C57BL/6 wildtype and susceptible A.BY/SnJ and C57BL/6-TLR3 (-/-) mice, however, to different degrees. In infected HeLa cells, we also found moderately elevated TRAF6 levels after infection, in addition, activity of the transcription factor nuclear factor kappa B (NFκB), which can be activated downstream of TRAF6, was strongly enhanced in infected cells. To functionally analyze the role of TRAF6 with regard to infection progression, TRAF6 expression was knocked down in cultured HeLa cells using specific siRNAs. We found that reduction of TRAF6 expression had no effect on NFκB activation in response to infection. Taken together, our data suggest that CVB3 infection enhances TRAF6 levels, however, this induction might not be necessary for infection-induced NFκB activation.

Skeletal Muscle MicroRNA Patterns in Response to a Single Bout of Exercise in Females: Biomarkers for Subsequent Training Adaptation?

microRNAs (miRs) have been proposed as a promising new class of biomarkers in the context of training adaptation. Using microarray analysis, we studied skeletal muscle miR patterns in sedentary young healthy females (n = 6) before and after a single submaximal bout of endurance exercise ('reference training'). Subsequently, participants were subjected to a structured training program, consisting of six weeks of moderate-intensity continuous endurance training (MICT) and six weeks of high-intensity interval training (HIIT) in randomized order. In vastus lateralis muscle, we found significant downregulation of myomiRs, specifically miR-1, 133a-3p, and -5p, -133b, and -499a-5p. Similarly, exercise-associated miRs-23a-3p, -378a-5p, -128-3p, -21-5p, -107, -27a-3p, -126-3p, and -152-3p were significantly downregulated, whereas miR-23a-5p was upregulated. Furthermore, in an untargeted approach for differential expression in response to acute exercise, we identified n = 35 miRs that were downregulated and n = 20 miRs that were upregulated by factor 4.5 or more. Remarkably, KEGG pathway analysis indicated central involvement of this set of miRs in fatty acid metabolism. To reproduce these data in a larger cohort of all-female subjects (n = 29), qPCR analysis was carried out on n = 15 miRs selected from the microarray, which confirmed their differential expression. Furthermore, the acute response, i.e., the difference between miR concentrations before and after the reference training, was correlated with changes in maximum oxygen uptake (V̇O2max) in response to the training program. Here, we found that miRs-199a-3p and -19b-3p might be suitable acute-response candidates that correlate with individual degrees of training adaptation in females.

miRNAs as markers for the development of individualized training regimens: A pilot study.

Small, non-coding RNAs (microRNAs) have been shown to regulate gene expression in response to exercise in various tissues and organs, thus possibly coordinating their adaptive response. Thus, it is likely that differential microRNA expression might be one of the factors that are responsible for different training responses of different individuals. Consequently, determining microRNA patterns might be a promising approach toward the development of individualized training strategies. However, little is known on (1) microRNA patterns and their regulation by different exercise regimens and (2) possible correlations between these patterns and individual training adaptation. Here, we present microarray data on skeletal muscle microRNA patterns in six young, female subjects before and after six weeks of either moderate-intensity continuous or high-intensity interval training on a bicycle ergometer. Our data show that n = 36 different microRNA species were regulated more than twofold in this cohort (n = 28 upregulated and n = 8 downregulated). In addition, we correlated baseline microRNA patterns with individual changes in VO2 max and identified some specific microRNAs that might be promising candidates for further testing and evaluation in the future, which might eventually lead to the establishment of microRNA marker panels that will allow individual recommendations for specific exercise regimens.

Short-term treadmill running as a model for studying cell-free DNA kinetics in vivo.

BACKGROUND: Increased plasma concentrations of cell-free DNA (cf-DNA) are considered a hallmark of various clinical conditions. Despite intensive research in this field, limited data are available concerning the time course of release and clearance of cf-DNA in vivo. METHODS: We extracted cf-DNA from plasma samples taken before and immediately after a 10-km cross-country run, and from samples taken before, immediately after, and 30 min after exhaustive short-term treadmill exercise. The contribution of nuclear (nDNA) and mitochondrial DNA (mtDNA) was measured by quantitative real-time PCR. The incremental treadmill exercise setup was exploited to delineate the precise sequencing and timing of cf-nDNA, lactate, and high-mobility group box 1 protein (HMGB1) release during the exercise and recovery phases. RESULTS: Postexercise plasma cf-nDNA concentrations in cross-country and treadmill runners were significantly increased, by 7.6-fold and 9.9-fold, respectively (P < 0.001). cf-nDNA concentrations were not correlated with age, sex, or body mass index. Plasma concentrations of cf-nDNA and HMGB1 in postexercise samples of treadmill runners were significantly correlated (r = 0.84; P = 0.004). cf-mtDNA concentrations were not affected by treadmill exercise. Time-course analyses demonstrated that cf-nDNA is released within minutes after the onset of exercise and is rapidly cleared from the circulation after the cessation of exercise. Nearly congruent kinetics for cf-nDNA, lactate, and HMGB1 were observed during the exercise phase. CONCLUSIONS: A single bout of exhaustive short-term treadmill exercise constitutes a versatile model system suitable for addressing basic questions about cf-DNA biology.

Skeletal muscle effects of two different 10-week exercise regimens, voluntary wheel running, and forced treadmill running, in mice: A pilot study.

Physical activity and exercise induce a complex pattern of adaptation reactions in a broad variety of tissues and organs, particularly the cardiovascular and the musculoskeletal systems. The underlying mechanisms, however, specifically the molecular changes that occur in response to training, are still incompletely understood. Animal models help to systematically elucidate the mechanisms of exercise adaptation. With regard to endurance-based running exercise in mice, two basic regimens have been established: forced treadmill running (FTR), usually consisting of several sessions per week, and voluntary wheel running (VWR). However, the effects of these two programs on skeletal muscle molecular adaptation patterns have never been directly compared. To address this issue, in a pilot study, we analyzed the effects of two ten-week training regimens in juvenile, male, C57BL/6 mice: moderate-intensity forced treadmill running three-times-a-week, employing a protocol that has been widely used in similar studies before, and voluntary wheel running. Our data suggest that there are similarities, but also characteristic differences in the molecular responses of different skeletal muscle species to the two training regimens. In particular, we found that VWR induces a significant fiber type shift toward more type IIX fibers in the slow, oxidative soleus muscle (p = .0053), but not in the other three muscles analyzed. In addition, while training-induced expression patterns of the two metabolic markers Ppargc1a, encoding Pgc-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and Nr4a3 (nuclear receptor subfamily 4 group A member 3) were roughly similar, downregulation of the Mstn (myostatin) gene and the "atrogene" Fbox32 could only be observed in response to VWR in specific muscles, such as in the gastrocnemius (p = .0015 for Mstn) and in the tibialis anterior (p = .0053 for Fbox32) muscles, suggesting that molecular adaptation reactions to the two training regimens show distinct characteristics.

Effects of the anti-oxidant PDTC in combination with a single bout of treadmill running on murine skeletal muscle.

Objectives: Skeletal muscle adaptation to physical activity is dependent on various factors. Important signaling mediators are reactive oxygen species (ROS). However, recent research suggests that ROS have both beneficial and deleterious effects on exercise adaptation, dependent on training intensity and training status, so that the question of whether anti-oxidants should be taken in connection with exercise cannot easily be answered. Thus, it is important to gain more insight into the complex roles of ROS in regulating training adaptation. Methods: The effects of ROS inhibition on skeletal muscle training adaptation were analyzed by applying the anti-oxidant PDTC, which is also an inhibitor of the ROS-activated transcription factor nuclear factor kappa B (NFκB), to juvenile mice in connection with a single bout of treadmill running. Results: We found that PDTC inhibits exercise-mediated induction of specific stress- and inflammation-associated genes. Other genes, specifically those encoding metabolic and mitochondrial factors, were affected to a lesser extent and there appeared to be little effect on the microRNA (miR) profile. Discussion: Our data suggest that anti-oxidants regulate distinct sets of adaptation-relevant genes, which might have important implications for the design of exercise-based preventive and therapeutic approaches.

Establishing a novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure for the direct detection of gene doping.

So far, the abuse of gene transfer technology in sport, so-called gene doping, is undetectable. However, recent studies in somatic gene therapy indicate that long-term presence of transgenic DNA (tDNA) following various gene transfer protocols can be found in DNA isolated from whole blood using conventional PCR protocols. Application of these protocols for the direct detection of gene doping would require almost complete knowledge about the sequence of the genetic information that has been transferred. Here, we develop and describe the novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure that overcomes this difficulty. Apart from the interesting perspectives that this spiPCR procedure offers in the fight against gene doping, this technology could also be of interest in biodistribution and biosafety studies for gene therapeutic applications.

Effects of 10 weeks of regular running exercise with and without parallel PDTC treatment on expression of genes encoding sarcomere-associated proteins in murine skeletal muscle.

Physical exercise can induce various adaptation reactions in skeletal muscle tissue, such as sarcomere remodeling. The latter involves degradation of damaged sarcomere components, as well as de novo protein synthesis and sarcomere assembly. These processes are controlled by specific protease systems in parallel with molecular chaperones that assist in folding of newly synthesized polypeptide chains and their incorporation into sarcomeres. Since acute exercise induces oxidative stress and inflammation, leading to activation of the transcription factor NFκB (nuclear factor kappa B), we speculated that this transcription factor might also play a role in the regulation of long-term adaptation to regular exercise. Thus, we studied skeletal muscle adaptation to running exercise in a murine model system, with and without parallel treatment with the NFκB-inhibitory, anti-oxidant and anti-inflammatory drug pyrrolidine dithiocarbamate (PDTC). In control mice, 10 weeks of uphill (15° incline) treadmill running for 60 min thrice a week at a final speed of 14 m/min had differential, but only minor effects on many genes encoding molecular chaperones for sarcomere proteins, and/or factors involved in the degradation of the latter. Furthermore, there were marked differences between individual muscles. PDTC treatment modulated gene expression patterns as well, both in sedentary and exercising mice; however, most of these effects were also modest and there was little effect of PDTC treatment on exercise-induced changes in gene expression. Taken together, our data suggest that moderate-intensity treadmill running, with or without parallel PDTC treatment, had little effect on the expression of genes encoding sarcomere components and sarcomere-associated factors in murine skeletal muscle tissue.

Functional analysis of ZFP36 proteins in keratinocytes.

The ZFP36 family of zinc finger proteins, including ZFP36, ZFP36L1, and ZFP36L2, regulates the production of growth factors and cytokines via destabilization of the respective mRNAs. We could recently demonstrate that in cultured keratinocytes, expression of the ZFP36, ZFP36L1, and ZFP36L2 genes is induced by growth factors and cytokines and that ZFP36L1 is a potent regulator of keratinocyte VEGF production. We now further analyzed the localization and function of ZFP36 proteins in the skin, specifically in epidermal keratinocytes. We found that in human epidermis, the ZFP36 protein could be detected in basal and suprabasal keratinocytes, whereas ZFP36L1 and ZFP36L2 were expressed mainly in the basal layer, indicating different and non-redundant functions of the three proteins in the epidermis. Consistently, upon inhibition of ZFP36 or ZFP36L1 expression using specific siRNAs, there was no major effect on expression of the respective other gene. In addition, we demonstrate that both ZFP36 and ZFP36L1 influence keratinocyte cell cycle, differentiation, and apoptosis in a distinct manner. Finally, we show that similarly as ZFP36L1, ZFP36 is a potent regulator of keratinocyte VEGF production. Thus, it is likely that both proteins regulate angiogenesis via paracrine mechanisms. Taken together, our results suggest that ZFP36 proteins might control reepithelialization and angiogenesis in the skin in a multimodal manner.

Neutrophil extracellular traps: a walk on the wild side of exercise immunology.

Intense exercise evokes a rapid and transient increase in circulating cell-free DNA (cf-DNA), a phenomenon that is commonly observed in a variety of acute and chronic inflammatory conditions. While the potential value of cf-DNA for the prediction of disease outcome and therapeutic response is well documented, the release mechanisms and biological relevance of cf-DNA have long remained enigmatic. The discovery of neutrophil extracellular traps (NETs) provided a novel mechanistic explanation for increased cf-DNA levels. Now there is increasing evidence that NETs may contribute to cf-DNA in diverse infectious, non-infectious and autoinflammatory conditions, as well as in response to acute exercise. NETs have now been firmly established as a fundamental immune mechanism used by neutrophils to respond to infection and tissue injury. On the other side, aberrant formation of NETs appears to be a driving force in the pathogenesis of autoimmunity and cardiovascular disease. Thus, the emergence of NETs in the 'exercising vasculature' raises important questions considering beneficial effects, as well as occasional adverse effects, of exercise on immune homeostasis. This review gives an overview of the current state of research into the mechanisms of how NETs are released, contribute to host defence and participate in inflammatory disorders. We discuss the impact of exercise-induced NETs, considering a potentially beneficial role in the prevention of lifestyle-related diseases, as well as putative detrimental effects that may arise in elite sports. Finally, we propose that exercise-induced cf-DNA responses could be exploited for diagnostic/prognostic purposes to identify individuals who are at increased risk of cardiovascular events or autoimmunity.

Neutrophils release extracellular DNA traps in response to exercise.

Intense exercise evokes a rapid and transient increase in circulating cell-free DNA (cf-DNA), a phenomenon that is commonly observed in a variety of acute and chronic inflammatory conditions. In this study, we aimed to shed new light on the release and clearance mechanisms of cf-DNA in response to exercise. We hypothesized that activated neutrophils may primarily contribute to exercise-evoked cf-DNA levels by releasing neutrophil extracellular traps (NETs). Analysis of plasma and/or serum samples from male athletes at rest and in response to exhaustive treadmill exercise revealed an immediate and transient increase in cf-DNA that was concomitantly counterbalanced by an increase in serum DNase activity. Consistently, rapid release and clearance kinetics for cf-DNA could also be observed in response to intensive cycling exercise, with no significant differences between endurance-trained (V̇o2max >57 ml·min(-1)·kg(-1)) and healthy (V̇o2max <49 ml·min(-1)·kg(-1)) sedentary individuals. In postexercise blood smear samples, we detected seemingly intact neutrophils displaying morphological signs of NET release, as indicated by abnormal swollen nuclei and emanating DNA fibers. In support, we observed a striking correlation of postexercise cf-DNA concentrations with plasma levels of the granule-derived enzyme myeloperoxidase. Our study indicates that intense exercise induces liberation of NETs, which is sufficiently counterbalanced in healthy individuals by a concomitant rise in serum DNase activity. As aberrant release of NETs has been linked to diverse disease states, monitoring of cf-DNA/DNase levels or activities in response to standardized exercise testing could provide a valuable tool to identify people who are at increased risk for cardiac ischemia, thrombosis, autoimmunity, or chronic fatigue.