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Muscular atrophy is a mortality risk factor that happens with disuse, chronic disease, and aging. Recovery from atrophy requires changes in several cell types including muscle fibers, and satellite and immune cells. Here we show that Zfp697/ZNF697 is a damage-induced regulator of muscle regeneration, during which its expression is transiently elevated. Conversely, sustained Zfp697 expression in mouse muscle leads to a gene expression signature of chemokine secretion, immune cell recruitment, and extracellular matrix remodeling. Myofiber-specific Zfp697 ablation hinders the inflammatory and regenerative response to muscle injury, compromising functional recovery. We uncover Zfp697 as an essential interferon gamma mediator in muscle cells, interacting primarily with ncRNAs such as the pro-regenerative miR-206. In sum, we identify Zfp697 as an integrator of cell-cell communication necessary for tissue regeneration. ONE SENTENCE SUMMARY: Zfp697 is necessary for interferon gamma signaling and muscle regeneration.

This study examined acute molecular responses to concurrent exercise involving different muscles. Eight men participated in a randomized crossover-trial with two sessions, one where they performed interval cycling followed by upper body resistance exercise (ER-Arm), and one with upper body resistance exercise only (R-Arm). Biopsies were taken from the triceps prior to and immediately, 90- and 180-min following exercise. Immediately after resistance exercise, the elevation in S6K1 activity was smaller and the 4E-BP1:eIF4E interaction greater in ER-Arm, but this acute attenuation disappeared during recovery. The protein synthetic rate in triceps was greater following exercise than at rest, with no difference between trials. The level of PGC-1α1 mRNA increased to greater extent in ER-Arm than R-Arm after 90 min of recovery, as was PGC-1α4 mRNA after both 90 and 180 min. Levels of MuRF-1 mRNA was unchanged in R-Arm, but elevated during recovery in ER-Arm, whereas MAFbx mRNA levels increased slightly in both trials. RNA sequencing in a subgroup of subjects revealed 862 differently expressed genes with ER-Arm versus R-Arm during recovery. These findings suggest that leg cycling prior to arm resistance exercise causes systemic changes that potentiate induction of specific genes in the triceps, without compromising the anabolic response.

Additional file 1: Figure S1. Characterization of differentiated SH-SY5Y cells. Figure S2. Cytotoxicity of compounds evaluated in the HTS. Figure S3. Effects of luteolin in respiratory capacity and ΔΨm. Figure S4. Effects of luteolin in mitochondrial biogenesis and cristae organization. Figure S5. Effects of luteolin on calcium levels and characterization of isolated mitochondrial fractions.

Increasing exercise capacity promotes healthy aging and is strongly associated with lower mortality rates. In this study, we analyzed skeletal muscle transcriptomics coupled to exercise performance in humans and rats to dissect the inherent and response components of aerobic exercise capacity. Using rat models selected for intrinsic and acquired aerobic capacity, we determined that the high aerobic capacity muscle transcriptome is associated with pathways for tissue oxygenation and vascularization. Conversely, the low capacity muscle transcriptome indicated immune response and metabolic dysfunction. Low response to training was associated with an inflammatory signature and revealed a potential link to circadian rhythm. Next, we applied bioinformatics tools to predict potential secreted factors (myokines). The predicted secretome profile for exercise capacity highlighted circulatory factors involved in lipid metabolism and the exercise response secretome was associated with extracellular matrix remodelling. Lastly, we utilized human muscle mitochondrial respiration and transcriptomics data to explore molecular mediators of exercise capacity and response across species. Human transcriptome comparison highlighted epigenetic mechanisms linked to exercise capacity and the damage repair for response. Overall, our findings from this cross-species transcriptome analysis of exercise capacity and response establish a foundation for future studies on the mechanisms that link exercise and health.

Significance Skeletal muscle cells are unique among human cells because they contain hundreds of nuclei distributed over large distances within a shared cytoplasm. This unusual organization raises questions about how gene expression is coordinated between nuclei and how parts of the muscle cell can become functionally distinct. Since transcription factors and other nuclear proteins control gene expression, we investigated how far nuclear proteins can travel between nuclei within a single muscle cell. We found that we could predict how far a nuclear protein would travel based on its size, nuclear import speed, and ability to diffuse across the nuclear pore. These findings can help explain how muscle cells are organized and provide design parameters for engineering gene therapies in skeletal muscle., Skeletal muscle cells contain hundreds of myonuclei within a shared cytoplasm, presenting unique challenges for regulating gene expression. Certain transcriptional programs (e.g., postsynaptic machinery) are segregated to specialized domains, while others (e.g., contractile proteins) do not show spatial confinement. Furthermore, local stimuli, such as denervation, can induce transcriptional responses that are propagated along the muscle cells. Regulated transport of nuclear proteins (e.g., transcription factors) between myonuclei represents a potential mechanism for coordinating gene expression. However, the principles underlying the transport of nuclear proteins within multinucleated cells remain poorly defined. Here we used a mosaic transfection model to create myotubes that contained exactly one myonucleus expressing a fluorescent nuclear reporter and monitored its distribution among all myonuclei. We found that the transport properties of these model nuclear proteins in myotubes depended on molecular weight and nuclear import rate, as well as on myotube width. Interestingly, muscle hypertrophy increased the transport of high molecular weight nuclear proteins, while atrophy restricted the transport of smaller nuclear proteins. We have developed a mathematical model of nuclear protein transport within a myotube that recapitulates the results of our in vitro experiments. To test the relevance to nuclear proteins expressed in skeletal muscle, we studied the transport of two transcription factors—aryl hydrocarbon receptor nuclear translocator and sine oculis homeobox 1—and found that their distributions were similar to the reporter proteins with corresponding molecular weights. Together, these results define a set of variables that can be used to predict the spatial distributions of nuclear proteins within a myotube.

Objective We examined whether skeletal muscle overexpression of PGC-1α1 or PGC-1α4 affected myokine secretion and neuromuscular junction (NMJ) formation. Methods A microfluidic device was used to model endocrine signaling and NMJ formation between primary mouse myoblast-derived myotubes and embryonic stem cell-derived motor neurons. Differences in hydrostatic pressure allowed for fluidic isolation of either cell type or unidirectional signaling in the fluid phase. Myotubes were transduced to overexpress PGC-1α1 or PGC-1α4, and myokine secretion was quantified using a proximity extension assay. Morphological and functional changes in NMJs were measured by fluorescent microscopy and by monitoring muscle contraction upon motor neuron stimulation. Results Skeletal muscle transduction with PGC-1α1, but not PGC-1α4, increased NMJ formation and size. PGC-1α1 increased muscle secretion of neurturin, which was sufficient and necessary for the effects of muscle PGC-1α1 on NMJ formation. Conclusions Our findings indicate that neurturin is a mediator of PGC-1α1-dependent retrograde signaling from muscle to motor neurons., Highlights • A microfluidic device is used to model neuromuscular junction formation. • Muscle expression of PGC-1α1 increases neuromuscular junction formation and size. • Skeletal muscle transduction with PGC-1α4 does not elicit the same responses. • PGC-1α1 promotes neuromuscular junctions by increasing secretion of neurturin.

Transcriptional co-activator PGC-1α1 has been shown to regulate energy metabolism and to mediate metabolic adaptations in pathological and physiological cardiac hypertrophy but other functional implications of PGC-1α1 expression are not known. Transgenic PGC-1α1 overexpression within the physiological range in mouse heart induces purposive changes in contractile properties, electrophysiology and calcium signalling but does not induce substantial metabolic remodelling. The phenotype of the PGC-1α1 transgenic mouse heart recapitulates most of the functional modifications usually associated with the exercise-induced heart phenotype, but does not protect the heart against load-induced pathological hypertrophy. Transcriptional effects of PGC-1α1 show clear dose-dependence with diverse changes in genes in circadian clock, heat shock, excitability, calcium signalling and contraction pathways at low overexpression levels, while metabolic genes are recruited at much higher PGC-1α1 expression levels. These results imply that the physiological role of PGC-1α1 is to promote a beneficial excitation-contraction coupling phenotype in the heart.The transcriptional coactivator PGC-1α1 has been identified as a central factor mediating metabolic adaptations of the heart. However, to what extent physiological changes in PGC-1α1 expression levels actually contribute to the functional adaptation of the heart is still mostly unresolved. The aim of this study was to characterize the transcriptional and functional effects of physiologically relevant, moderate PGC-1α1 expression in the heart. In vivo and ex vivo physiological analysis shows that expression of PGC-1α1 within a physiological range in mouse heart does not induce the expected metabolic alterations, but instead induces a unique excitation-contraction (EC) coupling phenotype recapitulating features typically seen in physiological hypertrophy. Transcriptional screening of PGC-1α1 overexpressing mouse heart and myocyte cultures with higher, acute adenovirus-induced PGC-1α1 expression, highlights PGC-1α1 as a transcriptional coactivator with a number of binding partners in various pathways (such as heat shock factors and the circadian clock) through which it acts as a pleiotropic transcriptional regulator in the heart, to both augment and repress the expression of its target genes in a dose-dependent fashion. At low levels of overexpression PGC-1α1 elicits a diverse transcriptional response altering the expression state of circadian clock, heat shock, excitability, calcium signalling and contraction pathways, while metabolic targets of PGC-1α1 are recruited at higher PGC-1α1 expression levels. Together these findings demonstrate that PGC-1α1 elicits a dual effect on cardiac transcription and phenotype. Further, our results imply that the physiological role of PGC-1α1 is to promote a beneficial EC coupling phenotype in the heart.

Background: Hypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear. Methods: The ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student’s t-test and ANOVA. Results: In the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells. Conclusions: Our data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis. publishedVersion

Tese de doutoramento, Ciências Biológicas, Faculdade de Ciências e Tenologia, Universidade do Algarve, 2014 Sedentary lifestyles, combined with an environment of caloric abundance, have led to a remarkable increase in the incidence of obesity and diabetes. The resistance to insulin action observed in type 2 diabetes is a complex and multifactorial metabolic disorder. Several factors have been implicated in the development of insulin resistance, including systemic low-grade inflammation and ectopic lipid accumulation. The transcriptional regulators FXR and PGC-1α have been shown to promote beneficial effects on energy metabolism, which can be explored to improve metabolic status in settings of insulin resistance. Here, we show that FXR regulates hepatic metabolism in an isoform-dependent manner. Expression of each human FXR variant in primary hepatocytes promoted robust and discrete changes in target gene expression. Importantly, expression of FXRα2 and FXRα4 decreased hepatocyte lipid accumulation and improved insulin sensitivity. Furthermore, FXRα2 expression increased fatty acid oxidation and β-hydroxybutyrate production. In line with these observations, we observed that FXR splicing was dynamically regulated in mouse liver in response to fasting and physical exercise, with an increase in the relative expression of FXRα2. In addition, FXR expression is elevated in white adipose tissue of skeletal muscle-specific PGC-1α1 transgenic mice (MCK-PGC-1α1) and regulates adipocyte expression of major lipolytic mediators. Finally, we show that PGC-1α1 promotes an anti-inflammatory biological response in skeletal muscle, repressing the classical NFκB pathway. MCK-PGC-1α1 mice have decreased IKKβ, p50 and p65 expression, which is accompanied by a strong increase in IκBα protein levels. Importantly, PGC-1α1 enhances insulin-stimulated glycogen synthesis in cultured myotubes upon pro-inflammatory stimulation. Taken together, our results suggest that FXRα2 and PGC-1α1 improve metabolic conditions implicated in the development of insulin resistance and therefore are attractive therapeutic targets for the treatment of obesityassociated metabolic disorders. O sedentarismo e o elevado consumo calórico têm conduzido a um aumento alarmante na incidência de obesidade e diabetes. A resistência à acção da insulina, que ocorre em diabetes tipo 2, é uma anomalia metabólica complexa e multifactorial. Vários factores estão associados ao desenvolvimento de resitência à insulina, incluindo inflamação sistémica e acumulação ectópica de lípidos. Os reguladores transcricionais FXR e PGC-1α promovem efeitos benéficos ao nível do metabolismo energético, que podem ser explorados para melhorar a condição metabólica em estados de resitência à insulina. Nestes estudos, demonstramos que a regulação exercida pelo FXR ao nível do metabolismo lipídico em hepatócitos depende da isoforma expressa. A expressão de cada isoforma do FXR em hepatócitos primários promove alterações distintas na expressão de genes alvo. A expressão de FXRα2 ou FXRα4 diminui a acumulação lipídica em hepatócitos, o que resulta num aumento da sensibilidade à insulina. Ademais, a expressão de FXRα2 promove a oxidação de ácidos gordos e produção de β-hidroxibutirato. Em linha com estas observações, o splicing do FXR é alterado em situações de jejum e exercício físico, com um aumento da expressão relativa de FXRα2. Também observámos que a expressão do FXR se encontra elevada no tecido adiposo de ratinhos com expressão transgénica de PGC-1α1 especificamente no músculo esquelético (MCK-PGC-1α1), participando na regulação de importantes fatores lipolíticos. Finalmente, PGC-1α1 promove uma resposta anti-inflamatória em músculo esquelético, caracterizada por um decréscimo na expressão de IKKβ, p50 e p65 e por um aumento dos níveis proteicos de IκBα, o que sugere um decréscimo significativo na atividade do sistema NFκB. Estas alterações resultam num aumento de sensibilidade à insulina em miotubos submetidos a estímulos pro-inflamatórios. Estes resultados sugerem que FXRα2 e PGC-1α1 promovem melhorias em condições metabólicas associadas com o desenvolvimento de resistência à insulina, sendo portanto alvos terapêuticos interessantes para o tratamento de doenças metabólicas associadas à obesidade. Fundação Para a Ciência e Tecnologia (FCT): SFRH/BD/44825/2008