MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia

The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle

OBJECTIVE: Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle. METHODS: We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression. RESULTS:We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes. CONCLUSIONS: We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca2+-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression

An integrated proteomic and genomic approach to study FAP patients without APC and MutHY mutations

Familial Adenomatous Polyposis (FAP) is one of the most important clinical forms of inherited susceptibility to colorectal cancer, that is characterized by the development of hundreds to thousands of adenomas in the colon and rectum during the second decade of life. FAP is due to a germline mutation in the APC gene or to biallelic variations of MutYH gene. Almost all patients will develop cancer if the disease is not identified and surgically treated at an early stage. The aim of this study was to characterize, by peptidomic and genetic approaches, 4 pa-tients that, although at the colonoscopy showed many polyps, they did not present any mutations of APC and MutYH genes (defined here unresolved FAP). Regarding the peptidomic study, MALDI-TOF analysis was performed on mutated and unresolved FAP patients. These data were compared with the one from adenoma patients, CRC patients and healthy control subjects. The peptide fingerprint of mutated FAP patients was obtained after performing statistical analysis. A subset of 45 ionic species was found differently expressed in the four groups considered, 12 of them peculiar of FAP patients. Four ionic species were found significantly different in the switch between adenoma and malignant carcinoma. In this study, the potentially prognostic peptides identified derive mainly from circulating proteins and some of them are involved in the inflammatory response. In particular, proteins such as Complement C3 and C4 are known to be cleaved by exoproteases that seem pathology-related. In the case of unresolved FAP patients, in order to better define a specific pattern, the data from MALDI-TOF were combined with whole exome sequencing. The peptidomics data clearly mark a substantial difference between mutated and unresolved FAP patients. Indeed, unresolved FAP patients have characteristics similar to the control subjects, adenoma patients, CRC patients but not to mutated FAP patients. To understand the possible molecular pathway involved in the unresolved FAP cases, the whole exome sequencing (WES) was performed. From WES data analysis, 285 genes present in all the four unresolved FAP patients were filtered and selected. Among them, the O-linked glycans pathway of the mucins was the most represented. In conclusion, in this study it was defined for the first time a specific panel of peptides for mutated FAP patients, that could be useful to monitor and predict the pathological evolution of adenocarcinoma malignancy. Furthermore, it was possible to characterize a preliminary genetic variations pattern for unresolved FAP patients, in which mucin genes might represent the key of the molecular pathway involved. However, further study are necessary to relate the identified mucin gene variations to their possible causative role in the polyposis. Future analysis of this pattern will be helpful, indeed, to better understand the interatome (the biological network that in-cludes the whole set of direct and indirect molecular interactions in a cell) of these un-resolved FAP patients.La poliposi adenomatosa familiare (FAP) è una delle più importanti forme cliniche di cancro colo-rettale ereditario ed è caratterizzata dallo sviluppo di centinaia/migliaia di polipi adenomatosi nel colon e nel retto durante la seconda decade di vita. La FAP è causata da una mutazione germinale del gene APC o da varianti bialleliche del gene MutYH. Quasi tutti i pazienti FAP sviluppano il cancro se la patologia non viene precocemente identificata e trattata chirurgicamente. Lo scopo di questo lavoro è stato caratterizzare 4 pazienti in cui, nonostante l’esame colonscopico presentasse una poliposi conclamata, non risultavano mutazioni nei gene APC e MutYH (in questa tesi definiti pazienti FAP irrisolti) utilizzando un approccio integrato di peptidomica e genomica. Riguardo la peptidomica, il MALDI-TOF è stato utilizzato per studiare il profilo peptidico plasmatico di pazienti FAP mutati ed irrisolti comparando i dati ottenuti con quelli derivanti dallo studio di pazienti con adenoma, cancro colo-rettale e soggetti sani di controllo. Dopo analisi statistica è stato ottenuto il fingerprint peptidico dei pazienti FAP mutati. Sono state ottenute 45 specie ioniche differentemente espresse nei quattro gruppi considerati, 12 delle quali peculiari per i pazienti FAP. L’intensità di segnale di quattro di queste specie ioniche è stata trovata statisticamente alterata nello switch tra adenoma e carcinoma maligno. I peptidi potenzialmente prognostici identificati in questo studio derivano principalmente da proteine circolanti, alcune delle quali implicate nella risposta infiammatoria. In particolare è noto dalla letteratura che proteine del sistema del complemento come C3 e C4 vengono tagliate da esoproteasi che sembrano essere patologia correlate. Riguardo ai pazienti FAP irrisolti, per definirne un pattern specifico, i dati derivanti dall’analisi con il MALDI-TOF sono stati combinati con quelli ottenuti dal sequenzia-mento dell’esoma. I dati di peptidomica hanno chiaramente evidenziato le differenze tra pazienti FAP mutati e FAP irrisolti. Infatti i pazienti FAP irrisolti presentano caratteristiche simili a quelle dei soggetti di controllo, dei pazienti con adenoma e cancro colo rettale ma non a quelle dei pazienti FAP mutati. Allo scopo di capire la via di trasduzione del segnale implicata, è stato quindi eseguito il sequenziamento dell'esoma dei pazienti FAP irrisolti. Da questa analisi sono stati selezionati 285 geni variati in tutti i pazienti e tra questi la via di trasduzione del segnale della O-glicosilazione delle mucine è risultata la più rappresentata. In conclusione, in questo studio è stato definito per la prima volta un set peptidico specifico per i pazienti FAP mutati che potrebbe essere utilizzato per monitorare e predire l’evoluzione patologica della malattia. Inoltre è stato possibile caratterizzare un pattern preliminare per i pazienti FAP irrisolti in cui i geni delle mucine potrebbero rappresentare la chiave della via di trasduzione del segnale implicata. Ulteriori studi saranno necessari per correlare i geni delle mucine con la poliposi e costruire l'interatoma (network biologico definito come l’insieme di tutte le interazioni molecolari dirette e indirette che ci sono all'interno di una cellula e di un organismo) di questi pazienti FAP irrisolti

An integrated proteomic and genomic approach to study FAP patients without APC and MutHY mutations

Familial Adenomatous Polyposis (FAP) is one of the most important clinical forms of inherited susceptibility to colorectal cancer, that is characterized by the development of hundreds to thousands of adenomas in the colon and rectum during the second decade of life. FAP is due to a germline mutation in the APC gene or to biallelic variations of MutYH gene. Almost all patients will develop cancer if the disease is not identified and surgically treated at an early stage. The aim of this study was to characterize, by peptidomic and genetic approaches, 4 pa-tients that, although at the colonoscopy showed many polyps, they did not present any mutations of APC and MutYH genes (defined here unresolved FAP). Regarding the peptidomic study, MALDI-TOF analysis was performed on mutated and unresolved FAP patients. These data were compared with the one from adenoma patients, CRC patients and healthy control subjects. The peptide fingerprint of mutated FAP patients was obtained after performing statistical analysis. A subset of 45 ionic species was found differently expressed in the four groups considered, 12 of them peculiar of FAP patients. Four ionic species were found significantly different in the switch between adenoma and malignant carcinoma. In this study, the potentially prognostic peptides identified derive mainly from circulating proteins and some of them are involved in the inflammatory response. In particular, proteins such as Complement C3 and C4 are known to be cleaved by exoproteases that seem pathology-related. In the case of unresolved FAP patients, in order to better define a specific pattern, the data from MALDI-TOF were combined with whole exome sequencing. The peptidomics data clearly mark a substantial difference between mutated and unresolved FAP patients. Indeed, unresolved FAP patients have characteristics similar to the control subjects, adenoma patients, CRC patients but not to mutated FAP patients. To understand the possible molecular pathway involved in the unresolved FAP cases, the whole exome sequencing (WES) was performed. From WES data analysis, 285 genes present in all the four unresolved FAP patients were filtered and selected. Among them, the O-linked glycans pathway of the mucins was the most represented. In conclusion, in this study it was defined for the first time a specific panel of peptides for mutated FAP patients, that could be useful to monitor and predict the pathological evolution of adenocarcinoma malignancy. Furthermore, it was possible to characterize a preliminary genetic variations pattern for unresolved FAP patients, in which mucin genes might represent the key of the molecular pathway involved. However, further study are necessary to relate the identified mucin gene variations to their possible causative role in the polyposis. Future analysis of this pattern will be helpful, indeed, to better understand the interatome (the biological network that in-cludes the whole set of direct and indirect molecular interactions in a cell) of these un-resolved FAP patients.La poliposi adenomatosa familiare (FAP) è una delle più importanti forme cliniche di cancro colo-rettale ereditario ed è caratterizzata dallo sviluppo di centinaia/migliaia di polipi adenomatosi nel colon e nel retto durante la seconda decade di vita. La FAP è causata da una mutazione germinale del gene APC o da varianti bialleliche del gene MutYH. Quasi tutti i pazienti FAP sviluppano il cancro se la patologia non viene precocemente identificata e trattata chirurgicamente. Lo scopo di questo lavoro è stato caratterizzare 4 pazienti in cui, nonostante l’esame colonscopico presentasse una poliposi conclamata, non risultavano mutazioni nei gene APC e MutYH (in questa tesi definiti pazienti FAP irrisolti) utilizzando un approccio integrato di peptidomica e genomica. Riguardo la peptidomica, il MALDI-TOF è stato utilizzato per studiare il profilo peptidico plasmatico di pazienti FAP mutati ed irrisolti comparando i dati ottenuti con quelli derivanti dallo studio di pazienti con adenoma, cancro colo-rettale e soggetti sani di controllo. Dopo analisi statistica è stato ottenuto il fingerprint peptidico dei pazienti FAP mutati. Sono state ottenute 45 specie ioniche differentemente espresse nei quattro gruppi considerati, 12 delle quali peculiari per i pazienti FAP. L’intensità di segnale di quattro di queste specie ioniche è stata trovata statisticamente alterata nello switch tra adenoma e carcinoma maligno. I peptidi potenzialmente prognostici identificati in questo studio derivano principalmente da proteine circolanti, alcune delle quali implicate nella risposta infiammatoria. In particolare è noto dalla letteratura che proteine del sistema del complemento come C3 e C4 vengono tagliate da esoproteasi che sembrano essere patologia correlate. Riguardo ai pazienti FAP irrisolti, per definirne un pattern specifico, i dati derivanti dall’analisi con il MALDI-TOF sono stati combinati con quelli ottenuti dal sequenzia-mento dell’esoma. I dati di peptidomica hanno chiaramente evidenziato le differenze tra pazienti FAP mutati e FAP irrisolti. Infatti i pazienti FAP irrisolti presentano caratteristiche simili a quelle dei soggetti di controllo, dei pazienti con adenoma e cancro colo rettale ma non a quelle dei pazienti FAP mutati. Allo scopo di capire la via di trasduzione del segnale implicata, è stato quindi eseguito il sequenziamento dell'esoma dei pazienti FAP irrisolti. Da questa analisi sono stati selezionati 285 geni variati in tutti i pazienti e tra questi la via di trasduzione del segnale della O-glicosilazione delle mucine è risultata la più rappresentata. In conclusione, in questo studio è stato definito per la prima volta un set peptidico specifico per i pazienti FAP mutati che potrebbe essere utilizzato per monitorare e predire l’evoluzione patologica della malattia. Inoltre è stato possibile caratterizzare un pattern preliminare per i pazienti FAP irrisolti in cui i geni delle mucine potrebbero rappresentare la chiave della via di trasduzione del segnale implicata. Ulteriori studi saranno necessari per correlare i geni delle mucine con la poliposi e costruire l'interatoma (network biologico definito come l’insieme di tutte le interazioni molecolari dirette e indirette che ci sono all'interno di una cellula e di un organismo) di questi pazienti FAP irrisolti

Peptide Patterns as Discriminating Biomarkers in Plasma of Patients With Familial Adenomatous Polyposis

BACKGROUND: Familial adenomatous polyposis (FAP) is one of the most important clinical forms of inherited susceptibility to colorectal cancer. So far, no accepted prognostic markers are present to monitor patients with FAP. Consequently, the major problem in managing patients with FAP is the difficulty to predict when the switch between adenoma and malignant carcinoma occurs, leading to the necessity of preventive surgery. Proteomics is one of the most suitable approaches to identify biomarkers, and it is widely used in cancer research. In this investigation, we studied the circulating plasma peptides in samples collected from patients with FAP and compared the obtained results with adenoma, colorectal cancer, and control samples to discover peptides able to distinguish different phenotypes. MATERIALS AND METHODS: The peptide fingerprint was obtained by matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry. After statistical analysis, a subset of 45 ionic species was found differently expressed in the 4 groups considered, 12 of them peculiar to patients with FAP. Moreover, 4 ionic species were found significantly changed in the switch between adenoma and malignant carcinoma. RESULTS: Potentially prognostic peptides identified by this study derive mainly from circulating proteins, some of which are involved in the inflammatory response, such as complement C3 and C4 subjected to an exoprotease activity that seemed pathology related. CONCLUSIONS: In this study, we defined for the first time a specific panel of peptides for monitoring patients with FAP that could be profitably used to monitor and predict the pathologic evolution in adenocarcinoma malignancy

Inducible activation of Akt increases skeletal muscle mass and force without satellite cell activation.

A better understanding of the signaling pathways that control muscle growth is required to identify appropriate countermeasures to prevent or reverse the loss of muscle mass and force induced by aging, disuse, or neuromuscular diseases. However, two major issues in this field have not yet been fully addressed. The first concerns the pathways involved in leading to physiological changes in muscle size. Muscle hypertrophy based on perturbations of specific signaling pathways is either characterized by impaired force generation, e.g., myostatin knockout, or incompletely studied from the physiological point of view, e.g., IGF-1 overexpression. A second issue is whether satellite cell proliferation and incorporation into growing muscle fibers is required for a functional hypertrophy. To address these issues, we used an inducible transgenic model of muscle hypertrophy by short-term Akt activation in adult skeletal muscle. In this model, Akt activation for 3 wk was followed by marked hypertrophy ( approximately 50% of muscle mass) and by increased force generation, as determined in vivo by ankle plantar flexor stimulation, ex vivo in intact isolated diaphragm strips, and in single-skinned muscle fibers. No changes in fiber-type distribution and resistance to fatigue were detectable. Bromodeoxyuridine incorporation experiments showed that Akt-dependent muscle hypertrophy was accompanied by proliferation of interstitial cells but not by satellite cell activation and new myonuclei incorporation, pointing to an increase in myonuclear domain size. We can conclude that during a fast hypertrophic growth myonuclear domain can increase without compromising muscle performance

No evidence for inositol 1,4,5-trisphosphate-dependent Ca2+ release in isolated fibers of adult mouse skeletal muscle

The presence and role of functional inositol 1,4,5-trisphosphate (IP3) receptors (IP(3)Rs) in adult skeletal muscle are controversial. The current consensus is that, in adult striated muscle, the relative amount of IP(3)Rs is too low and the kinetics of Ca2+ release from IP3R is too slow compared with ryanodine receptors to contribute to the Ca2+ transient during excitation-contraction coupling. However, it has been suggested that IP3-dependent Ca2+ release may be involved in signaling cascades leading to regulation of muscle gene expression. We have reinvestigated IP3-dependent Ca2+ release in isolated flexor digitorum brevis (FDB) muscle fibers from adult mice. Although Ca2+ transients were readily induced in cultured C2C12 muscle cells by (a) UTP stimulation, (b) direct injection of IP3, or (c) photolysis of membrane-permeant caged IP3, no statistically significant change in calcium signal was detected in adult FDB fibers. We conclude that the IP3-IP3R system does not appear to affect global calcium levels in adult mouse skeletal muscle

Eccentric contractions lead to myofibrillar dysfunction in muscular dystrophy.

It is commonly accepted that skeletal muscles from dystrophin-deficient mdx mice are more susceptible than those from wild-type mice to damage from eccentric contractions. However, the downstream mechanisms involved in this enhanced force drop remain controversial. We studied the reduction of contractile force induced by eccentric contractions elicited in vivo in the gastrocnemius muscle of wild-type mice and three distinct models of muscle dystrophy: mdx, alpha-sarcoglycan (Sgca)-null, and collagen 6A1 (Col6a1)-null mice. In mdx and Sgca-null mice, force decreased 35% compared with 14% in wild-type mice. Drop of force in Col6a1-null mice was comparable to that in wild-type mice. To identify the determinants of the force drop, we measured force generation in permeabilized fibers dissected from gastrocnemius muscle that had been exposed in vivo to eccentric contractions and from the contralateral unstimulated muscle. A force loss in skinned fibers after in vivo eccentric contractions was detectable in fibers from mdx and Sgca-null, but not wild-type and Col6a1-null, mice. The enhanced force reduction in mdx and Sgca-null mice was observed only when eccentric contractions were elicited in vivo, since eccentric contractions elicited in vitro had identical effects in wild-type and dystrophic skinned fibers. These results suggest that 1) the enhanced force loss is due to a myofibrillar impairment that is present in all fibers, and not to individual fiber degeneration, and 2) the mechanism causing the enhanced force reduction is active in vivo and is lost after fiber permeabilization