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.

Adipokines as Predictive Biomarkers for Training Adaptation in Subjects with Multimorbidity-A Hypothesis-Generating Study.

BACKGROUND: Physical exercise exerts a positive effect on many chronic conditions, specifically lifestyle-related diseases such as overweight and obesity, type 2 diabetes mellitus (T2DM), cardiovascular conditions and osteoarthritis (OA). As a result of common risk factors, most of these patients present with multiple conditions. Exercise- and disease-related biomarkers, such as adipokines, are emerging tools in training supervision and regulation; however, their significance in subjects with multimorbidities is unknown. SUBJECTS AND METHODS: To address this issue, adipokines leptin, adiponectin and resistin were assessed in a cohort of subjects with multimorbidities (n = 39) presenting with at least two of the abovementioned conditions or relevant risk factors before and after a six-month exercise and lifestyle intervention program ('MultiPill-Exercise'), and correlated with training adaptation, namely changes in relative maximum oxygen uptake (V·O2max). RESULTS: There was a significant negative correlation between baseline leptin concentrations and training effect for relative V·O2max (after three months: rho = -0.54, p = 0.020 *; after six months: rho = -0.45, p = 0.013 *), with baseline leptin explaining 35% of the variance in delta relative V·O2max after three months and 23% after six months. CONCLUSIONS: Leptin might be a suitable surrogate biomarker in the context of exercise-based lifestyle intervention programs in subjects with multimorbidity.

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.

Proof of Concept of a 6-Month Person-Oriented Exercise Intervention 'MultiPill-Exercise' among Patients at Risk of or with Multiple Chronic Diseases: Results of a One-Group Pilot Trial.

Physical exercise has been shown to be effective in the treatment of non-communicable chronic diseases. However, patients with multiple chronic diseases (multimorbidity) have received little attention in health policy. This pilot trial served as a proof of concept of a 6-months person-oriented exercise intervention for people at risk of or with diagnosed cardiovascular diseases, diabetes mellitus type 2, overweight and/or hip/knee osteoarthritis, regarding effects on health outcomes as well as adherence and safety. The intervention ('MultiPill-Exercise') was designed to promote physical exercise participation, considering an individual perspective by addressing personal and environmental factors. Outcomes were assessed at baseline (t0) and after three- (t3) and six-months (t6). The primary outcome was self-reported physical exercise participation in minutes/week comparing t3 and t6 vs. t0. Secondary outcomes included cardio-respiratory fitness (maximum oxygen uptake VO2peak during incremental cycling ergometry), isometric peak torque of knee extensors and flexors, health-related quality of life (Veterans Rand 12 with its subscales of perceived general health (GH), mental health (MCS), and physical health (PCS)) and blood levels. Adherence to exercise (% of attended sessions during the first 12-weeks of the intervention) and adverse events were monitored as well. Data were analyzed using a non-parametric procedure for longitudinal data, estimating rank means (MRank) and relative treatment effects (RTE) as well as linear-mixed effect models for parametric data. The primary endpoint of physical exercise participation was significantly higher at t3 and t6 compared to baseline (t3 vs. t0: MRank = 77.1, p < 0.001, RTE: 0.66; t6 vs. t0: MRank = 70.6, p < 0.001, RTE = 0.60). Improvements at both follow-up time points compared to t0 were also found for relative VO2peak (t3 vs. t0 = 2.6 mL/kg/min, p < 0.001; t6 vs. t0 = 2.0 mL/kg/min, p = 0.001), strength of knee extensors (t3 vs. t0 = 11.7 Nm, p = 0.007; t6 vs. t0= 18.1 Nm, p < 0.001) and GH (t3 vs. t0 = 16.2, p = 0.003; t6 vs. t0 = 13.4, p = 0.008). No changes were found for MCS, PCS and for blood levels. Overall exercise adherence was 77%. No serious adverse events were recorded. Results of this pilot trial represent a first proof of concept for the intervention 'MultiPill-Exercise' that will now be implemented and evaluated in a real-world health care setting.

Implementation and evaluation of an individualized physical exercise promotion program in people with manifested risk factors for multimorbidity (MultiPill-Exercise): a study protocol for a pragmatic randomized controlled trial.

BACKGROUND: Multimorbidity is a major problem in Europe, increasing the need for prevention and rehabilitation programs. In Germany no guidelines have been developed that focus on patients with multiple chronic non-communicable diseases (NCDs). Benefits of physical activity (PA) and exercise in NCDs have been proven, but most interventions focus on single conditions. The evaluation of the effectiveness, efficiency and safety of PA programs in patients suffering from multiple NCDs and the feasibility of the implementation within the health care service remain open research questions. METHODS: The multi-site randomized controlled pragmatic trial includes 320 sedentary subjects with at least two of the following NCDs, either manifested or in a pre-stage with evident risk factors: Cardio-vascular disease, Diabetes mellitus type 2, knee/ hip osteoarthritis and obesity. Participants will be recruited from general practitioners and medical specialists and randomized to standard care of a statutory health insurance or MultiPill-Exercise. Standard care includes a choice of one or a maximum of two 8- to 12-week health programs, including nutrition, exercise, relaxation or special disease management programs. MultiPill-Exercise is based on the bio-psycho-social health model, considering a person-oriented perspective in light of given individual characteristics and context factors. The 24-weeks intervention focuses on aerobic and strengthening exercises in line with the WHO PA recommendations. Psychological and pedagogical elements along with behavior change techniques are implemented to ease the initiation and maintenance of exercise participation and lifestyle change, including nutrition. Primary outcome will be short- and long-term PA measured with the European Health Interview Survey-Physical Activity Questionnaire (EHIS-PAQ). Secondly, the effectiveness of the program on generic, disease specific, economic, and exercise behavioral parameters, as well as program adherence and safety will be evaluated. DISCUSSION: Results of this trial evaluate the PA intervention program in people with multiple NCDs in a real-life scenario. It will serve as a proof of concept with the opportunity of translation into routine practice. This approach, as a multi-site RCT with its rigorous methods and standardized operating procedures for the conduction of the intervention, will allow valid conclusions for the implementation of PA interventions in people with multimorbidity. TRIAL REGISTRATION: The trial was registered at www.drks.de (ID: DRKS00025033 ) on 30th September 2021.

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.

Responders and non-responders to aerobic exercise training: beyond the evaluation of V˙O2max.

The evaluation of the maximal oxygen uptake ( V˙O2max ) following exercise training is the classical assessment of training effectiveness. Research has lacked in investigating whether individuals that do not respond to the training intervention ( V˙O2max ), also do not improve in other health-related parameters. We aimed to investigate the cardiovascular and metabolic adaptations (i.e., performance, body composition, blood pressure, vascular function, fasting blood markers, and resting cardiac function and morphology) to exercise training among participants who showed different levels of V˙O2max responsiveness. Healthy sedentary participants engaged in a 6-week exercise training program, three times a week. Our results showed that responders had a greater increase in peak power output, second lactate threshold, and microvascular responsiveness, whereas non-responders had a greater increase in cycling efficiency. No statistical differences were observed in body composition, blood pressure, fasting blood parameters, and resting cardiac adaptations. In conclusion, our study showed, for the first time, that in addition to the differences in the V˙O2max , a greater increase in microvascular responsiveness in responders compared to non-responders was observed. Additionally, responders and non-responders did not show differences in the adaptations on metabolic parameters. There is an increasing need for personalized training prescription, depending on the target clinical outcome.

Individual cardiovascular responsiveness to work-matched exercise within the moderate- and severe-intensity domains.

PURPOSE: We investigated the cardiovascular individual response to 6 weeks (3×/week) of work-matched within the severe-intensity domain (high-intensity interval training, HIIT) or moderate-intensity domain (moderate-intensity continuous training, MICT). In addition, we analyzed the cardiovascular factors at baseline underlying the response variability. METHODS: 42 healthy sedentary participants were randomly assigned to HIIT or MICT. We applied the region of practical equivalence-method for identifying the levels of responders to the maximal oxygen uptake (V̇O2max) response. For investigating the influence of cardiovascular markers, we trained a Bayesian machine learning model on cardiovascular markers. RESULTS: Despite that HIIT and MICT induced significant increases in V̇O2max, HIIT had greater improvements than MICT (p < 0.001). Greater variability was observed in MICT, with approximately 50% classified as "non-responder" and "undecided". 20 "responders", one "undecided" and no "non-responders" were observed in HIIT. The variability in the ∆V̇O2max was associated with initial cardiorespiratory fitness, arterial stiffness, and left-ventricular (LV) mass and LV end-diastolic diameter in HIIT; whereas, microvascular responsiveness and right-ventricular (RV) excursion velocity showed a significant association in MICT. CONCLUSION: Our findings highlight the critical influence of exercise-intensity domains and biological variability on the individual V̇O2max response. The incidence of "non-responders" in MICT was one third of the group; whereas, no "non-responders" were observed in HIIT. The incidence of "responders" was 11 out of 21 participants in MICT, and 20 out of 21 participants in HIIT. The response in HIIT showed associations with baseline fitness, arterial stiffness, and LV-morphology; whereas, it was associated with RV systolic function in MICT.

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.

Stability of Smyd1 in endothelial cells is controlled by PML-dependent SUMOylation upon cytokine stimulation.

Smyd1 is an epigenetic modulator of gene expression that has been well-characterized in muscle cells. It was recently reported that Smyd1 levels are modulated by inflammatory processes. Since inflammation affects the vascular endothelium, this study aimed to characterize Smyd1 expression in endothelial cells. We detected Smyd1 in human endothelial cells (HUVEC and EA.hy926 cells), where the protein was largely localized in PML nuclear bodies (PML-NBs). By transfection of EA.hy926 cells with expression vectors encoding Smyd1, PML, SUMO1, active or mutant forms of the SUMO protease SuPr1 and/or the SUMO-conjugation enzyme UBC9, as well as Smyd1- or PML-specific siRNAs, in the presence or absence of the translation blocker cycloheximide or the proteasome-inhibitor MG132, and supported by computational modeling, we show that Smyd1 is SUMOylated in a PML-dependent manner and thereby addressed for degradation in proteasomes. Furthermore, transfection with Smyd1-encoding vectors led to PML up-regulation at the mRNA level, while PML transfection lowered Smyd1 protein stability. Incubation of EA.hy926 cells with the pro-inflammatory cytokine TNF-α resulted in a constant increase in Smyd1 mRNA and protein over 24 h, while incubation with IFN-γ induced a transient increase in Smyd1 expression, which peaked at 6 h and decreased to control values within 24 h. The IFN-γ-induced increase in Smyd1 was accompanied by more Smyd1 SUMOylation and more/larger PML-NBs. In conclusion, our data indicate that in endothelial cells, Smyd1 levels are regulated through a negative feedback mechanism based on SUMOylation and PML availability. This molecular control loop is stimulated by various cytokines.

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.

The iReAct study - A biopsychosocial analysis of the individual response to physical activity.

BACKGROUND: Physical activity is a substantial promoter for health and well-being. Yet, while an increasing number of studies shows that the responsiveness to physical activity is highly individual, most studies focus this issue from only one perspective and neglect other contributing aspects. In reference to a biopsychosocial framework, the goal of our study is to examine how physically inactive individuals respond to two distinct standardized endurance trainings on various levels. Based on an assessment of activity- and health-related biographical experiences across the life course, our mixed-method study analyzes the responsiveness to physical activity in the form of a transdisciplinary approach, considering physiological, epigenetic, motivational, affective, and body image-related aspects. METHODS: Participants are randomly assigned to two different training programs (High Intensity Interval Training vs. Moderate Intensity Continuous Training) for six weeks. After this first training period, participants switch training modes according to a two-period sequential-training-intervention (STI) design and train for another six weeks. In order to analyse baseline characteristics as well as acute and adaptive biopsychosocial responses, three extensive mixed-methods diagnostic blocks take place at the beginning (t0) of the study and after the first (t1) and the second (t2) training period resulting in a net follow-up time of 15 weeks. The study is divided into five modules in order to cover a wide array of perspectives. DISCUSSION: The study's transdisciplinary mixed-method design allows to interlace a multitude of subjective and objective data and therefore to draw an integrated picture of the biopsychosocial efficacy of two distinct physical activity programs. The results of our study can be expected to contribute to the development and design of individualised training programs for the promotion of physical activity. TRIAL REGISTRATION: The study was retrospectively registered in the German Clinical Trials Register on 12 June 2019 (DRKS00017446).

Physical Exercise and Epigenetic Modifications in Skeletal Muscle.

Physical activity and sports play major roles in the overall health status of humans. It is well known that regular exercise helps to lower the risk for a broad variety of health problems, such as cardiovascular disease, type 2 diabetes, and cancer. Being physically active induces a wide variety of molecular adaptations, for example fiber type switches or other metabolic alterations, in skeletal muscle tissue. These adaptations are based on exercise-induced changes to the skeletal muscle transcriptome. Understanding their nature is crucial to improve the development of exercise-based therapeutic strategies. Recent research indicates that specifically epigenetic mechanisms, i.e., pathways that induce changes in gene expression patterns without altering the DNA base sequence, might play a major role in controlling skeletal muscle transcriptional patterns. Epigenetic mechanisms include DNA and histone modifications, as well as expression of specific microRNAs. They can be modulated by environmental factors or external stimuli, such as exercise, and eventually induce specific and fine-tuned changes to the transcriptional response. In this review, we highlight current knowledge on epigenetic changes induced in exercising skeletal muscle, their target genes, and resulting phenotypic changes. In addition, we raise the question of whether epigenetic modifications might serve as markers for the design and management of optimized and individualized training protocols, as prognostic tools to predict training adaptation, or even as targets for the design of "exercise mimics".

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.

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.

siRNA-mediated inhibition of skNAC and Smyd1 expression disrupts myofibril organization: Immunofluorescence and electron microscopy study in C2C12 cells.

skNAC (skeletal and heart muscle-specific variant of nascent polypeptide-associated complex) and Smyd1 (SET and MYND domain-containing 1) form a protein dimer which is specific for striated muscle cells. Its function is largely unknown. On the one hand, skNAC-Smyd1 appears to control transcriptional processes in the nucleus, on the other hand, specifically at later stages of myogenic differentiation, both proteins translocate to the sarcoplasm and at least Smyd1 specifically associates with sarcomeric structures and might control myofibrillogenesis and/or sarcomere architecture. Here, using immunofluorescence and electron microscopy, we analyzed sarcomere formation and myofibril organization after siRNA-mediated knockdown of skNAC or Smyd1 expression in murine C2C12 skeletal muscle cells. We found that inhibition of skNAC or Smyd1 expression indeed prevents myofibrillogenesis and sarcomere formation, leading to a disorganized array of myofilaments predominantly within the region immediately beneath the plasma membrane.

Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells.

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

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.

skNAC and Smyd1 in transcriptional control.

Skeletal and heart muscle-specific variant of the alpha subunit of nascent polypeptide associated complex (skNAC) is exclusively found in striated muscle cells. Its function, however, is largely unknown. Previous reports could demonstrate that skNAC binds to Smyd1 (SET and MYND domain containing protein 1). The facts that (a) SET domains have histone methyltransferase activity, and (b) MYND domains are known recruiters of histone deacetylases (HDACs), implicate the skNAC-Smyd1 complex in transcriptional control. To study potential target genes, we carried out cDNA microarray analysis on differentiating C2C12 myoblasts in which expression of the skNAC gene had been knocked down. We found and confirmed a series of targets, specifically genes encoding regulators of inflammation, cellular metabolism, and cell migration. Mechanistically, as shown by Western blot, ELISA, and ChIP analysis at selected promoter regions, transcriptional control by skNAC-Smyd1 appears to be exerted at least in part by affecting a series of histone modifications, specifically H3K4 di- and trimethylation and potentially also histone acetylation. Taken together, our data suggest that the skNAC-Smyd1 complex is involved in transcriptional regulation both via the control of histone methylation and histone (de)acetylation.