Spectroscopic Studies of the Super-Relaxed State of Skeletal Muscle

In the super-relaxed state of myosin, ATPase activity is strongly inhibited by binding of the myosin heads to the core of the thick filament in a structure known as the interacting-heads motif. In the disordered relaxed state myosin heads are not bound to the core of the thick filament and have an ATPase rate that is 10 fold greater. In the interacting-heads motif the two regulatory light chains appear to bind to each other. We have made single cysteine mutants of the regulatory light chain, placed both paramagnetic and fluorescent probes on them, and exchanged them into skinned skeletal muscle fibers. Many of the labeled light chains tended to disrupt the stability of the super-relaxed state, and showed spectral changes in the transition from the disordered relaxed state to the super-relaxed state. These data support the putative interface between the two regulatory light chains identified by cryo electron microscopy and show that both the divalent cation bound to the regulatory light chain and the N-terminus of the regulatory light chain play a role in the stability of the super-relaxed state. One probe showed a shift to shorter wavelengths in the super-relaxed state such that a ratio of intensities at 440nm to that at 520nm provided a measure of the population of the super-relaxed state amenable for high throughput screens for finding potential pharmaceuticals. The results provide a proof of concept that small molecules that bind to this region can destabilize the super-relaxed state and provide a method to search for small molecules that do so leading to a potentially effective treatment for Type 2 diabetes and obesity

Drug Repurposing for Duchenne Muscular Dystrophy: The Monoamine Oxidase B Inhibitor Safinamide Ameliorates the Pathological Phenotype in mdx Mice and in Myogenic Cultures From DMD Patients

Oxidative stress and mitochondrial dysfunction play a crucial role in the pathophysiology of muscular dystrophies. We previously reported that the mitochondrial enzyme monoamine oxidase (MAO) is a relevant source of reactive oxygen species (ROS) not only in murine models of muscular dystrophy, in which it directly contributes to contractile impairment, but also in muscle cells from Collagen VI-deficient patients. Here we now assessed the efficacy of a novel MAO-B inhibitor, safinamide, using in vivo and in vitro models of Duchenne muscular dystrophy (DMD). Specifically, we found that administration of safinamide in 3-month old mdx mice reduced myofiber damage and oxidative stress, and improved muscle functionality. In vitro studies with myogenic cultures from mdx mice and DMD patients showed that even cultured dystrophic myoblasts were more susceptible to oxidative stress than matching cells from healthy donors. Indeed, upon exposure to the MAO substrate tyramine or to hydrogen peroxide, DMD muscle cells displayed a rise in ROS levels and a consequent mitochondrial depolarization. Remarkably, both phenotypes normalized when cultures were treated with safinamide. Given that safinamide is already in clinical use for neurological disorders, our findings could pave the way towards a promising translation into clinical trials for DMD patients as a classic case of drug repurposing

Changes in the fraction of strongly attached cross bridges in mouse atrophic and hypertrophic muscles as revealed by continuous wave electron paramagnetic resonance.

Electron paramagnetic resonance (EPR), coupled with site-directed spin labeling, has been proven to be a particularly suitable technique to extract information on the fraction of myosin heads strongly bound to actin upon muscle contraction. The approach can be used to investigate possible structural changes occurring in myosin of fiber s altered by diseases and aging. In this work, we labeled myosin at position Cys707, located in the SH1-SH2 helix in the myosin head cleft, with iodoacetamide spin label, a spin label that is sensitive to the reorientational motion of this protein during the ATPase cycle and characterized the biochemical states of the labeled myosin head by means of continuous wave EPR. After checking the sensitivity and the power of the technique on different muscles and species, we investigated whether changes in the fraction of strongly bound myosin heads might explain the contractile alterations observed in atrophic and hypertrophic murine muscles. In both conditions, the difference in contractile force could not be justified simply by the difference in muscle mass. Our results showed that in atrophic muscles the decrease in force generation was attributable to a lower fraction of strongly bound cross bridges during maximal activation. In contrast in hypertrophic muscles, the increase in force generation was likely due to several factors, as pointed out by the comparison of the EPR experiments with the tension measurements on single skinned fibers

Drug Repurposing for Duchenne Muscular Dystrophy: The Monoamine Oxidase B Inhibitor Safinamide Ameliorates the Pathological Phenotype in mdx Mice and in Myogenic Cultures From DMD Patients

Oxidative stress and mitochondrial dysfunction play a crucial role in the pathophysiology of muscular dystrophies. We previously reported that the mitochondrial enzyme monoamine oxidase (MAO) is a relevant source of reactive oxygen species (ROS) not only in murine models of muscular dystrophy, in which it directly contributes to contractile impairment, but also in muscle cells from collagen VI-deficient patients. Here, we now assessed the efficacy of a novel MAO-B inhibitor, safinamide, using in vivo and in vitro models of Duchenne muscular dystrophy (DMD). Specifically, we found that administration of safinamide in 3-month-old mdx mice reduced myofiber damage and oxidative stress and improved muscle functionality. In vitro studies with myogenic cultures from mdx mice and DMD patients showed that even cultured dystrophic myoblasts were more susceptible to oxidative stress than matching cells from healthy donors. Indeed, upon exposure to the MAO substrate tyramine or to hydrogen peroxide, DMD muscle cells displayed a rise in ROS levels and a consequent mitochondrial depolarization. Remarkably, both phenotypes normalized when cultures were treated with safinamide. Given that safinamide is already in clinical use for neurological disorders, our findings could pave the way toward a promising translation into clinical trials for DMD patients as a classic case of drug repurposing

Genetic association of conformation scores with growth traits in Angus breed cattle

O objetivo deste trabalho foi determinar a associação genética entre escores visuais de conformação e as características de ganho de peso médio diário e de velocidade de crescimento em bovinos da raça Angus à desmama e ao sobreano. Os componentes de covariância foram estimados por modelo animal de análise tetracaracterística, com uso do método de inferência bayesiana, tendo-se assumido o modelo linear para: ganho de peso médio diário do nascimento à desmama (GMD) e da desmama ao sobreano (GMS); e velocidade de ganho de peso do nascimento à desmama (VD) e da desmama ao sobreano (VS). Um modelo não linear (de limiar) foi utilizado para os escores de conformação à desmama (CD) e ao sobreano (CS). As médias a posteriori, para a herdabilidade direta, foram: 0,12±0,023 (CD), 0,15±0,020 (GMD), 0,15±0,024 (VD), 0,17±0,020 (CS), 0,17±0,023(GMS), e 0,17±0,023 (VS). A correlação genética variou de ‑0,09±0,11 a 0,60±0,06, entre os escores CD e CS e as características de ganho médio diário de peso e velocidade de ganho de peso. A correlação entre CD e CS foi 0,52±0,08. A seleção direta para escores visuais de conformação, ganho médio diário e velocidade de ganho responde de forma lenta à seleção, tanto à desmama como ao sobreano.The objective of this work was determine the genetic association between visual scores of conformation and the traits of daily average weight gain and weight gain rate in Angus breed cattle at weaning and yearling. The components of covariance were estimated by a multitrait animal model using the Bayesian inference method, assuming a linear model for: daily average weight gain from birth to weaning (BWG) and from weaning to yearling (WYG); and weight gain rate from birth to weaning (BWR) and from weaning to yearling (WYR). A nonlinear model (threshold) was used for conformation scores at weaning (WC) and at yearling (YC). The a posteriori means for heritability were: 0.12±0.023 (WC), 0.15±0.020 (BWG), 0.15±0.024 (BWR), 0.17±0.020 (YC), 0.17±0.023 (WYG) and 0.17±0,023 (WYR). The genetic correlations ranged from ‑0.09±0.11 to 0.60±0.06, between WC, YC, and the daily average weight gain and weight gain rate. The correlation between WC and YC was 0.52±0.089. The direct selection for visual scores of conformation, average daily gain and weight gain rate respond slowly to selection, both at weaning and yearling

Genetic association of conformation scores with growth traits in Angus breed cattle

O objetivo deste trabalho foi determinar a associação genética entre escores visuais de conformação e as características de ganho de peso médio diário e de velocidade de crescimento em bovinos da raça Angus à desmama e ao sobreano. Os componentes de covariância foram estimados por modelo animal de análise tetracaracterística, com uso do método de inferência bayesiana, tendo-se assumido o modelo linear para: ganho de peso médio diário do nascimento à desmama (GMD) e da desmama ao sobreano (GMS); e velocidade de ganho de peso do nascimento à desmama (VD) e da desmama ao sobreano (VS). Um modelo não linear (de limiar) foi utilizado para os escores de conformação à desmama (CD) e ao sobreano (CS). As médias a posteriori, para a herdabilidade direta, foram: 0,12±0,023 (CD), 0,15±0,020 (GMD), 0,15±0,024 (VD), 0,17±0,020 (CS), 0,17±0,023(GMS), e 0,17±0,023 (VS). A correlação genética variou de -0,09±0,11 a 0,60±0,06, entre os escores CD e CS e as características de ganho médio diário de peso e velocidade de ganho de peso. A correlação entre CD e CS foi 0,52±0,089. A seleção direta para escores visuais de conformação, ganho médio diário e velocidade de ganho responde de forma lenta à seleção, tanto à desmama como ao sobreano.The objective of this work was determine the genetic association between visual scores of conformation and the traits of daily average weight gain and weight gain rate in Angus breed cattle at weaning and yearling. The components of covariance were estimated by a multitrait animal model using the Bayesian inference method, assuming a linear model for: daily average weight gain from birth to weaning (BWG) and from weaning to yearling (WYG); and weight gain rate from birth to weaning (BWR) and from weaning to yearling (WYR). A nonlinear model (threshold) was used for conformation scores at weaning (WC) and at yearling (YC). The a posteriori means for heritability were: 0.12±0.023 (WC), 0.15±0.020 (BWG), 0.15±0.024 (BWR), 0.17±0.020 (YC), 0.17±0.023 (WYG) and 0.17±0,023 (WYR). The genetic correlations ranged from -0.09±0.11 to 0.60±0.06, between WC, YC, and the daily average weight gain and weight gain rate. The correlation between WC and YC was 0.52±0.089. The direct selection for visual scores of conformation, average daily gain and weight gain rate respond slowly to selection, both at weaning and yearling

Spectroscopic studies of the super relaxed state of skeletal muscle: a possible target for obesity and type 2 diabetes treatments

Myosin and muscles have been studied since the beginning of the last century. The high amount of myosin and its optimized purification protocol started the huge field of the molecular motors around 50 years ago. The importance of myosin is not only due to its physiological role but also for its large contribution to the general biochemistry knowledge. Since myosin is a molecular motor, it represented an important step in the connection between chemistry and physics as fields of studies applied to biology. Myosin, as the fulcrum of biochemical conversion of energy into physical work, is the perfect example of the intimate connection between biochemistry and biophysics. Several techniques have been used to study molecular muscle functions, and so, myosin function. Some of them are common but very powerful, as fluorescence. Some others are less used in biology, such as electronic paramagnetic resonance (EPR). Back in the 1980s, the first papers about EPR measurements on muscle fibers were published. Muscle fibers, and myosin filaments, are not an ordinary biological sample because of their physiological microscopic and macroscopic order. In such samples, EPR, being sensitive to orientation, becomes really informative. EPR was first used on muscle samples to describe the freedom of the myosin head in all the different physiological states it has to go through to complete a cycle. Using these and other techniques, scientists have been working on myosin, characterizing most aspects of the muscle motor protein. There are several models which try to describe the myosin cycle. They may be really detailed and complex. Generally, they all agree on the steps that the myosin has to go through to perform a power stroke. Recently, a new state of myosin has been identified. This state has been called the “Super Relaxed” (SRX) of myosin because when myosin enters this state the ATPase activity is very low. In this thesis, we aim to characterize myosin behavior especially by studying the stability and the structural details of the super relaxed state. First we wanted to explore the super relaxed state by modifying one of its components. We expressed mutants of a subunit associated with myosin and that has an important role in the stability of the super relaxed state. The protein has been exchanged into muscle fibers. The idea is to find a way to sense whether the myosin is in the super relaxed state or not. By collecting data on the effects of these mutants labeled with several probes, we were able to identify the mutant/probe couple able to sense the super relaxed state. We used that fluorimetric assay in a high throughput screening, we were looking for a molecule that was able to destabilize the state. Our belief is that the muscle tissue is a good target for increasing basal metabolism in humans, lowering the stability of the slow ATPase state to improve life quality of people affected by obesity and diabetes. A molecule has been identified and it has been shown to be a good leading compound for further pharmacological studies.Sin dall’inizio dello scorso secolo la miosina ed il tessuto muscolare sono stati oggetto di ricerca scientifica. Circa 50 anni fa, l’abbondanza della miosina ed il perfezionamento dei protocolli di purificazione hanno contribuito ad iniziare lo studio del grande campo dei motori molecolari. L’importanza della miosina non è solo dovuta al suo ruolo fisiologico, ma anche per il suo grande contributo alla crescita delle conoscenze biochimiche. La miosina è un motore molecolare ed ha rappresentato e rappresenta tuttora un importante anello di congiunzione tra chimica e fisica in quanto campi di studio applicabili alla biologia. La miosina è il fulcro della conversione di energia chimica in lavoro nel tessuto muscolare ed è l’esempio perfetto della intima connessione fra biochimica e biofisica. Per lo studio delle funzioni molecolari del muscolo e della miosina si sono utilizzate numerose tecniche. Alcune di queste sono ormai piuttosto comuni, e anche molto potenti, quali la fluorescenza. Alcune altre sono meno conosciute in ambito biologico, come l’electron paramagnetic resonance (EPR) o risonanza paramagnetica elettronica, detta anche risonanza di spin elettronico. I primi lavori che vedono protagonista l’EPR su campioni di muscolo risalgono agli anni 80. Le fibre muscolari ed i miofilamenti sono campioni biologici particolari perchè sono fisiologicamente caratterizzati da un elevatissimo ordine microscopico e macroscopico. In tali campioni, l’EPR, essendo sensibile all'orientamento spaziale della molecola paramagnetica che porta l'elettrone spaiato, diventa realmente informativo. L’EPR è stato usato su campioni muscolari per descrivere la libertà di movimento della miosina durante i differenti stati del ciclo di interazione con l'actina. Usando questa ed altre tecniche, gli scienziati hanno studiato la miosina, ottenendo una profonda caratterizzazione di questo motore molecolare. Ci sono diversi modelli che descrivono tutti gli stati o passi che la miosina deve attraversare per completare un ciclo di interazione con l'actina. Recentemente, è stato identificato un nuovo stato della miosina. E’ stato denominato stato “Super Relaxed State” o SRX per via del fatto che è caratterizzato da una attività ATPasica molto bassa. Lo scopo di questa tesi è di caratterizzare il comportamento della miosina specialmente per quanto riguarda la stabilità e i dettagli strutturali dello stato Super Relaxed. Il lavoro sperimentale è iniziato con lo studio sullo stato SRX attraverso la modifica di uno dei suoi componenti. Abbiamo espresso mutanti di una proteina associata alla miosina, la catena leggera regolatoria o fosforilabile, che svolge un ruolo fisiologicamente importante per la stabilità dello stato. La proteina mutata è stata sostituita alla forma naturale in fibre muscolari. Lo scopo è di trovare un insieme di proteina mutante marcata con una sonda in grado di identificare quando lo stato SRX è presente. Abbiamo sviluppato quindi un saggio fluorimetrico e abbiamo effettuato un "high throughput screening" al fine di trovare una molecola in grado di destabilizzare lo stato rilassato. Il tessuto muscolare può essere un ottimo bersaglio per aumentare il metabolismo basale, destabilizzando lo stato SRX a basso consumo di ATP e quindi aumentando moderatamente la spesa energetica. Come possibile applicazione, questo potrebbe avere un impatto positivo sulla salute delle persone affette da obesità e diabete di tipo 2. Abbiamo identificato una molecola candidato e abbiamo dimostrato che ha le caratteristiche per essere un buon punto di partenza per sviluppare ulteriori studi farmacologici

Spectroscopic studies of the super relaxed state of skeletal muscle: a possible target for obesity and type 2 diabetes treatments

Myosin and muscles have been studied since the beginning of the last century. The high amount of myosin and its optimized purification protocol started the huge field of the molecular motors around 50 years ago. The importance of myosin is not only due to its physiological role but also for its large contribution to the general biochemistry knowledge. Since myosin is a molecular motor, it represented an important step in the connection between chemistry and physics as fields of studies applied to biology. Myosin, as the fulcrum of biochemical conversion of energy into physical work, is the perfect example of the intimate connection between biochemistry and biophysics. Several techniques have been used to study molecular muscle functions, and so, myosin function. Some of them are common but very powerful, as fluorescence. Some others are less used in biology, such as electronic paramagnetic resonance (EPR). Back in the 1980s, the first papers about EPR measurements on muscle fibers were published. Muscle fibers, and myosin filaments, are not an ordinary biological sample because of their physiological microscopic and macroscopic order. In such samples, EPR, being sensitive to orientation, becomes really informative. EPR was first used on muscle samples to describe the freedom of the myosin head in all the different physiological states it has to go through to complete a cycle. Using these and other techniques, scientists have been working on myosin, characterizing most aspects of the muscle motor protein. There are several models which try to describe the myosin cycle. They may be really detailed and complex. Generally, they all agree on the steps that the myosin has to go through to perform a power stroke. Recently, a new state of myosin has been identified. This state has been called the “Super Relaxed” (SRX) of myosin because when myosin enters this state the ATPase activity is very low. In this thesis, we aim to characterize myosin behavior especially by studying the stability and the structural details of the super relaxed state. First we wanted to explore the super relaxed state by modifying one of its components. We expressed mutants of a subunit associated with myosin and that has an important role in the stability of the super relaxed state. The protein has been exchanged into muscle fibers. The idea is to find a way to sense whether the myosin is in the super relaxed state or not. By collecting data on the effects of these mutants labeled with several probes, we were able to identify the mutant/probe couple able to sense the super relaxed state. We used that fluorimetric assay in a high throughput screening, we were looking for a molecule that was able to destabilize the state. Our belief is that the muscle tissue is a good target for increasing basal metabolism in humans, lowering the stability of the slow ATPase state to improve life quality of people affected by obesity and diabetes. A molecule has been identified and it has been shown to be a good leading compound for further pharmacological studies.Sin dall’inizio dello scorso secolo la miosina ed il tessuto muscolare sono stati oggetto di ricerca scientifica. Circa 50 anni fa, l’abbondanza della miosina ed il perfezionamento dei protocolli di purificazione hanno contribuito ad iniziare lo studio del grande campo dei motori molecolari. L’importanza della miosina non è solo dovuta al suo ruolo fisiologico, ma anche per il suo grande contributo alla crescita delle conoscenze biochimiche. La miosina è un motore molecolare ed ha rappresentato e rappresenta tuttora un importante anello di congiunzione tra chimica e fisica in quanto campi di studio applicabili alla biologia. La miosina è il fulcro della conversione di energia chimica in lavoro nel tessuto muscolare ed è l’esempio perfetto della intima connessione fra biochimica e biofisica. Per lo studio delle funzioni molecolari del muscolo e della miosina si sono utilizzate numerose tecniche. Alcune di queste sono ormai piuttosto comuni, e anche molto potenti, quali la fluorescenza. Alcune altre sono meno conosciute in ambito biologico, come l’electron paramagnetic resonance (EPR) o risonanza paramagnetica elettronica, detta anche risonanza di spin elettronico. I primi lavori che vedono protagonista l’EPR su campioni di muscolo risalgono agli anni 80. Le fibre muscolari ed i miofilamenti sono campioni biologici particolari perchè sono fisiologicamente caratterizzati da un elevatissimo ordine microscopico e macroscopico. In tali campioni, l’EPR, essendo sensibile all'orientamento spaziale della molecola paramagnetica che porta l'elettrone spaiato, diventa realmente informativo. L’EPR è stato usato su campioni muscolari per descrivere la libertà di movimento della miosina durante i differenti stati del ciclo di interazione con l'actina. Usando questa ed altre tecniche, gli scienziati hanno studiato la miosina, ottenendo una profonda caratterizzazione di questo motore molecolare. Ci sono diversi modelli che descrivono tutti gli stati o passi che la miosina deve attraversare per completare un ciclo di interazione con l'actina. Recentemente, è stato identificato un nuovo stato della miosina. E’ stato denominato stato “Super Relaxed State” o SRX per via del fatto che è caratterizzato da una attività ATPasica molto bassa. Lo scopo di questa tesi è di caratterizzare il comportamento della miosina specialmente per quanto riguarda la stabilità e i dettagli strutturali dello stato Super Relaxed. Il lavoro sperimentale è iniziato con lo studio sullo stato SRX attraverso la modifica di uno dei suoi componenti. Abbiamo espresso mutanti di una proteina associata alla miosina, la catena leggera regolatoria o fosforilabile, che svolge un ruolo fisiologicamente importante per la stabilità dello stato. La proteina mutata è stata sostituita alla forma naturale in fibre muscolari. Lo scopo è di trovare un insieme di proteina mutante marcata con una sonda in grado di identificare quando lo stato SRX è presente. Abbiamo sviluppato quindi un saggio fluorimetrico e abbiamo effettuato un "high throughput screening" al fine di trovare una molecola in grado di destabilizzare lo stato rilassato. Il tessuto muscolare può essere un ottimo bersaglio per aumentare il metabolismo basale, destabilizzando lo stato SRX a basso consumo di ATP e quindi aumentando moderatamente la spesa energetica. Come possibile applicazione, questo potrebbe avere un impatto positivo sulla salute delle persone affette da obesità e diabete di tipo 2. Abbiamo identificato una molecola candidato e abbiamo dimostrato che ha le caratteristiche per essere un buon punto di partenza per sviluppare ulteriori studi farmacologici