A Laing distal myopathy-associated proline substitution in the ß-myosin rod perturbs myosin cross-bridging activity.

Proline substitutions within the coiled-coil rod region of the ß-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.

Remodeling of skeletal muscle myosin metabolic states in hibernating mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Many animals use hibernation as a tactic to survive harsh winters. During this dormant, inactive state, animals reduce or limit body processes, such as heart rate and body temperature, to minimise their energy use. To conserve energy during hibernation, animals can use different approaches. For example, garden dormice undergo periodic states of extremely low core temperatures (down to 4­8^oC); whereas Eurasian brown bears see milder temperature drops (down to 23­25^oC). An important organ that changes during hibernation is skeletal muscle. Skeletal muscle typically uses large amounts of energy, making up around 50% of body mass. To survive, hibernating animals must change how their skeletal muscle uses energy. Traditionally, active myosin ­ a protein found in muscles that helps muscles to contract ­ was thought to be responsible for most of the energy use by skeletal muscle. But, more recently, resting myosin has also been found to use energy when muscles are relaxed. Lewis et al. studied myosin and skeletal muscle energy use changes during hibernation and whether they could impact the metabolism of hibernating animals. Lewis et al. assessed myosin changes in muscle samples from squirrels, dormice and bears during hibernation and during activity. Experiments showed changes in resting myosin in squirrels and dormice (whose temperature drops to 4­8^oC during hibernation) but not in bears. Further analysis revealed that cooling samples from non-hibernating muscle to 4­8^oC increased energy use in resting myosin, thereby generating heat. However, no increase in energy use was found after cooling hibernating muscle samples to 4­8^oC. This suggest that resting myosin generates heat at cool temperatures ­ a mechanism that is switched off in hibernating animals to allow them to cool their body temperature. These findings reveal key insights into how animals conserve energy during hibernation. In addition, the results show that myosin regulates energy use in skeletal muscles, which indicates myosin may be a potential drug target in metabolic diseases, such as obesity.

Aberrant myonuclear domains and impaired myofiber contractility despite marked hypertrophy in MYMK-related, Carey-Fineman-Ziter Syndrome.

Carey Fineman Ziter Syndrome (CFZS) is a rare autosomal recessive disease caused by mutations in the MYMK locus which encodes the protein, myomaker. Myomaker is essential for fusion and concurrent myonuclei donation of muscle progenitors during growth and development. Strikingly, in humans, MYMK mutations appear to prompt myofiber hypertrophy but paradoxically, induce generalised muscle weakness. As the underlying cellular mechanisms remain unexplored, the present study aimed to gain insights by combining myofiber deep-phenotyping and proteomic profiling. Hence, we isolated individual muscle fibers from CFZS patients and performed mechanical, 3D morphological and proteomic analyses. Myofibers from CFZS patients were ~ 4x larger than controls and possessed ~ 2x more myonuclei than those from healthy subjects, leading to disproportionally larger myonuclear domain volumes. These greater myonuclear domain sizes were accompanied by smaller intrinsic cellular force generating-capacities in myofibers from CFZS patients than in control muscle cells. Our complementary proteomic analyses indicated remodelling in 233 proteins particularly those associated with cellular respiration. Overall, our findings suggest that myomaker is somewhat functional in CFZS patients, but the associated nuclear accretion may ultimately lead to non-functional hypertrophy and altered energy-related mechanisms in CFZS patients. All of these are likely contributors of the muscle weakness experienced by CFZS patients.

Muscle fibre size and myonuclear positioning in trained and aged humans.

Changes in myonuclear architecture and positioning are associated with exercise adaptations and ageing. However, data on the positioning and number of myonuclei following exercise are inconsistent. Additionally, whether myonuclear domains (MNDs; i.e., the theoretical volume of cytoplasm within which a myonucleus is responsible for transcribing DNA) and myonuclear positioning are altered with age remains unclear. The aim of this investigation was to investigate relationships between age and activity status and myonuclear domains and positioning. Vastus lateralis muscle biopsies from younger endurance-trained (YT) and older endurance-trained (OT) individuals were compared with age-matched untrained counterparts (YU and OU; OU samples were acquired during surgical operation). Serial, optical z-slices were acquired throughout isolated muscle fibres and analysed to give three-dimensional coordinates for myonuclei and muscle fibre dimensions. The mean cross-sectional area (CSA) of muscle fibres from OU individuals was 33%-53% smaller compared with the other groups. The number of nuclei relative to fibre CSA was 90% greater in OU compared with YU muscle fibres. Additionally, scaling of MND volume with fibre size was altered in older untrained individuals. The myonuclear arrangement, in contrast, was similar across groups. Fibre CSA and most myonuclear parameters were significantly associated with age in untrained individuals, but not in trained individuals. These data indicate that regular endurance exercise throughout the lifespan might better preserve the size of muscle fibres in older age and maintain the relationship between fibre size and MND volumes. Inactivity, however, might result in reduced muscle fibre size and altered myonuclear parameters.

Remodelling of Skeletal Muscle Myosin Metabolic States in Hibernating Mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20°C). Upon repeating loaded Mant-ATP chase experiments at 8°C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Ryanodine receptor type 1 content decrease-induced endoplasmic reticulum stress is a hallmark of myopathies.

BACKGROUND: Decreased ryanodine receptor type 1 (RyR1) protein levels are a well-described feature of recessive RYR1-related myopathies. The aim of the present study was twofold: (1) to determine whether RyR1 content is also decreased in other myopathies and (2) to investigate the mechanisms by which decreased RyR1 protein triggers muscular disorders. METHODS: We used publicly available datasets, muscles from human inflammatory and mitochondrial myopathies, an inducible muscle-specific RYR1 recessive mouse model and RyR1 knockdown in C2C12 muscle cells to measure RyR1 content and endoplasmic reticulum (ER) stress markers. Proteomics, lipidomics, molecular biology and transmission electron microscopy approaches were used to decipher the alterations associated with the reduction of RyR1 protein levels. RESULTS: RYR1 transcripts were reduced in muscle samples of patients suffering from necrotizing myopathy (P = 0.026), inclusion body myopathy (P = 0.003), polymyositis (P < 0.001) and juvenile dermatomyositis (P < 0.001) and in muscle samples of myotonic dystrophy type 2 (P < 0.001), presymptomatic (P < 0.001) and symptomatic (P < 0.001) Duchenne muscular dystrophy, Becker muscular dystrophy (P = 0.004) and limb-girdle muscular dystrophy type 2A (P = 0.004). RyR1 protein content was also significantly decreased in inflammatory myopathy (-75%, P < 0.001) and mitochondrial myopathy (-71%, P < 0.001) muscles. Proteomics data showed that depletion of RyR1 protein in C2C12 myoblasts leads to myotubes recapitulating the common molecular alterations observed in myopathies. Mechanistically, RyR1 protein depletion reduces ER-mitochondria contact length (-26%, P < 0.001), Ca2+ transfer to mitochondria (-48%, P = 0.002) and the mitophagy gene Parkinson protein 2 transcripts (P = 0.037) and induces mitochondrial accumulation (+99%, P = 0.005) and dysfunction (P < 0.001). This was associated to the accumulation of deleterious sphingolipid species. Our data showed increased levels of the ER stress marker chaperone-binding protein/glucose regulated protein 78, GRP78-Bip, in RyR1 knockdown myotubes (+45%, P = 0.046), in mouse RyR1 recessive muscles (+58%, P = 0.001) and in human inflammatory (+96%, P = 0.006) and mitochondrial (+64%, P = 0.049) myopathy muscles. This was accompanied by increased protein levels of the pro-apoptotic protein CCAAT-enhancer-binding protein homologous protein, CHOP-DDIT3, in RyR1 knockdown myotubes (+27%, P < 0.001), mouse RyR1 recessive muscles (+63%, P = 0.009), human inflammatory (+50%, P = 0.038) and mitochondrial (+51%, P = 0.035) myopathy muscles. In publicly available datasets, the decrease in RYR1 content in myopathies was also associated to increased ER stress markers and RYR1 transcript levels are inversely correlated with ER stress markers in the control population. CONCLUSIONS: Decreased RyR1 is commonly observed in myopathies and associated to ER stress in vitro, in mouse muscle and in human myopathy muscles, suggesting a potent role of RyR1 depletion-induced ER stress in the pathogenesis of myopathies.

Human skeletal myopathy myosin mutations disrupt myosin head sequestration.

Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyze the effects of common MYH7 and MYH2 mutations in the light meromyosin (LMM) region of myosin. Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in silico modeling showed that myosin coiled coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients and fluorescent ATP analog chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with x-ray diffraction measurements to estimate myosin head order, we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofiber mechanics experiments to investigate contractile function showed that myofiber contractility was not affected. These findings indicate that the structural remodeling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.

Abnormal myosin post-translational modifications and ATP turnover time associated with human congenital myopathy-related RYR1 mutations.

AIM: Conditions related to mutations in the gene encoding the skeletal muscle ryanodine receptor 1 (RYR1) are genetic muscle disorders and include congenital myopathies with permanent weakness, as well as episodic phenotypes such as rhabdomyolysis/myalgia. Although RYR1 dysfunction is the primary mechanism in RYR1-related disorders, other downstream pathogenic events are less well understood and may include a secondary remodeling of major contractile proteins. Hence, in the present study, we aimed to investigate whether congenital myopathy-related RYR1 mutations alter the regulation of the most abundant contractile protein, myosin. METHODS: We used skeletal muscle tissues from five patients with RYR1-related congenital myopathy and compared those with five controls and five patients with RYR1-related rhabdomyolysis/myalgia. We then defined post-translational modifications on myosin heavy chains (MyHCs) using LC/MS. In parallel, we determined myosin relaxed states using Mant-ATP chase experiments and performed molecular dynamics (MD) simulations. RESULTS: LC/MS revealed two additional phosphorylations (Thr1309-P and Ser1362-P) and one acetylation (Lys1410-Ac) on the ß/slow MyHC of patients with congenital myopathy. This method also identified six acetylations that were lacking on MyHC type IIa of these patients (Lys35-Ac, Lys663-Ac, Lys763-Ac, Lys1171-Ac, Lys1360-Ac, and Lys1733-Ac). MD simulations suggest that modifying myosin Ser1362 impacts the protein structure and dynamics. Finally, Mant-ATP chase experiments showed a faster ATP turnover time of myosin heads in the disordered-relaxed conformation. CONCLUSIONS: Altogether, our results suggest that RYR1 mutations have secondary negative consequences on myosin structure and function, likely contributing to the congenital myopathic phenotype.

Predominant myosin superrelaxed state in canine myocardium with naturally occurring dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a naturally occurring heart failure condition in humans and dogs, notably characterized by a reduced contractility and ejection fraction. As the identification of its underlying cellular and molecular mechanisms remain incomplete, the aim of the present study was to assess whether the molecular motor myosin and its known relaxed conformational states are altered in DCM. For that, we dissected and skinned thin cardiac strips from left ventricle obtained from six DCM Doberman Pinschers and six nonfailing (NF) controls. We then used a combination of Mant-ATP chase experiments and X-ray diffraction to assess both energetic and structural changes of myosin. Using the Mant-ATP chase protocol, we observed that in DCM dogs, the amount of myosin molecules in the ATP-conserving conformational state, also known as superrelaxed (SRX), is significantly increased when compared with NF dogs. This alteration can be rescued by applying EMD-57033, a small molecule activating myosin. Conversely, with X-ray diffraction, we found that in DCM dogs, there is a higher proportion of myosin heads in the vicinity of actin when compared with NF dogs (1,0 to 1,1 intensity ratio). Hence, we observed an uncoupling between energetic (Mant-ATP chase) and structural (X-ray diffraction) data. Taken together, these results may indicate that in the heart of Doberman Pinschers with DCM, myosin molecules are potentially stuck in a nonsequestered but ATP-conserving SRX state, that can be counterbalanced by EMD-57033 demonstrating the potential for a myosin-centered pharmacological treatment of DCM.NEW & NOTEWORTHY The key finding of the present study is that, in left ventricles of dogs with a naturally occurring dilated cardiomyopathy, relaxed myosin molecules favor a nonsequestered superrelaxed state potentially impairing sarcomeric contractility. This alteration is rescuable by applying a small molecule activating myosin known as EMD-57033.

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle.

OBJECTIVE: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. METHODS: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. RESULTS: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. CONCLUSIONS: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

Physical activity impacts resting skeletal muscle myosin conformation and lowers its ATP consumption.

It has recently been established that myosin, the molecular motor protein, is able to exist in two conformations in relaxed skeletal muscle. These conformations are known as the super-relaxed (SRX) and disordered-relaxed (DRX) states and are finely balanced to optimize ATP consumption and skeletal muscle metabolism. Indeed, SRX myosins are thought to have a 5- to 10-fold reduction in ATP turnover compared with DRX myosins. Here, we investigated whether chronic physical activity in humans would be associated with changes in the proportions of SRX and DRX skeletal myosins. For that, we isolated muscle fibers from young men of various physical activity levels (sedentary, moderately physically active, endurance-trained, and strength-trained athletes) and ran a loaded Mant-ATP chase protocol. We observed that in moderately physically active individuals, the amount of myosin molecules in the SRX state in type II muscle fibers was significantly greater than in age-matched sedentary individuals. In parallel, we did not find any difference in the proportions of SRX and DRX myosins in myofibers between highly endurance- and strength-trained athletes. We did however observe changes in their ATP turnover time. Altogether, these results indicate that physical activity level and training type can influence the resting skeletal muscle myosin dynamics. Our findings also emphasize that environmental stimuli such as exercise have the potential to rewire the molecular metabolism of human skeletal muscle through myosin.

Binding pocket dynamics along the recovery stroke of human ß-cardiac myosin.

The druggability of small-molecule binding sites can be significantly affected by protein motions and conformational changes. Ligand binding, protein dynamics and protein function have been shown to be closely interconnected in myosins. The breakthrough discovery of omecamtiv mecarbil (OM) has led to an increased interest in small molecules that can target myosin and modulate its function for therapeutic purposes (myosin modulators). In this work, we use a combination of computational methods, including steered molecular dynamics, umbrella sampling and binding pocket tracking tools, to follow the evolution of the OM binding site during the recovery stroke transition of human ß-cardiac myosin. We found that steering two internal coordinates of the motor domain can recapture the main features of the transition and in particular the rearrangements of the binding site, which shows significant changes in size, shape and composition. Possible intermediate conformations were also identified, in remarkable agreement with experimental findings. The differences in the binding site properties observed along the transition can be exploited for the future development of conformation-selective myosin modulators.

Revisiting specific force loss in human permeabilized single skeletal muscle fibers obtained from older individuals.

Specific force (SF) has been shown to be reduced in some but not all studies of human aging using chemically skinned single muscle fibers. This may be due, in part, not only to the health status/physical activity levels of different older cohorts, but also from methodological differences in studying skinned fibers. The aim of the present study was to compare SF in fibers from older hip fracture patients (HFP), healthy master cyclists (MC), and healthy nontrained young adults (YA) using two different activating solutions. Quadriceps muscle samples and 316 fibers were obtained from HFPs (74.6 ± 4 years, n = 5), MCs (74.8 ± 1, n = 5), and YA (25.5 ± 2, n = 6). Fibers were activated (pCa 4.5, 15°C) in solutions containing either 60 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid pH buffer (TES) or 20 mM imidazole. SF was determined by normalizing force to fiber cross-sectional area (CSA) assuming either an elliptical or circular shape and to fiber myosin heavy chain content. Activation in TES resulted in significantly higher MHC-I SF in all groups and YA MHC-IIA fibers, irrespective of normalization method. Although there were no differences in SF between the participant groups, the ratio of SF between the TES and imidazole solutions was lower in HFPs compared with YAs (MHC-I P < 0.05; MHC-IIA P = 0.055). Activating solution composition, as opposed to donor characteristics, had a more notable effect on single fiber SF. However, this two-solution approach revealed an age-related difference in sensitivity in HFPs, which was not shown in MCs. This suggests further novel approaches may be required to probe age/activity-related differences in muscle contractile quality.NEW & NOTEWORTHY Whether specific force (SF) decreases with advancing age in human single skeletal muscle fibers is uncertain. Equivocal published findings may be due to the different physical activity levels of the elderly cohorts studied and/or different chemical solutions used to measure force. We compared single fiber SF between young adults, elderly cyclists, and hip fracture patients (HFP) using two solutions. The solution used significantly affected force and revealed a difference in sensitivity of HFP muscle fibers.

Slow myosin heavy chain 1 is required for slow myofibril and muscle fibre growth but not for myofibril initiation.

Slow myosin heavy chain 1 (Smyhc1) is the major sarcomeric myosin driving early contraction by slow skeletal muscle fibres in zebrafish. New mutant alleles lacking a functional smyhc1 gene move poorly, but recover motility as the later-formed fast muscle fibres of the segmental myotomes mature, and are adult viable. By motility analysis and inhibiting fast muscle contraction pharmacologically, we show that a slow muscle motility defect persists in mutants until about 1 month of age. Breeding onto a genetic background marking slow muscle fibres with EGFP revealed that mutant slow fibres undergo terminal differentiation, migration and fibre formation indistinguishable from wild type but fail to generate large myofibrils and maintain cellular orientation and attachments. In mutants, initial myofibrillar structures with 1.67 â€‹µm periodic actin bands fail to mature into the 1.96 â€‹µm sarcomeres observed in wild type, despite the presence of alternative myosin heavy chain molecules. The poorly-contractile mutant slow muscle cells generate numerous cytoplasmic organelles, but fail to grow and bundle myofibrils or to increase in cytoplasmic volume despite passive movements imposed by fast muscle. The data show that both slow myofibril maturation and cellular volume increase depend on the function of a specific myosin isoform and suggest that appropriate force production regulates muscle fibre growth.

Myosin post-translational modifications and function in the presence of myopathy-linked truncating MYH2 mutations.

Congenital myopathies are a vast group of genetic muscle diseases. Among the causes are mutations in the MYH2 gene resulting in truncated type IIa myosin heavy chains (MyHCs). The precise cellular and molecular mechanisms by which these mutations induce skeletal muscle symptoms remain obscure. Hence, in the present study, we aimed to explore whether such genetic defects would alter the presence as well as the post-translational modifications of MyHCs and the functionality of myosin molecules. For this, we dissected muscle fibers from four myopathic patients with MYH2 truncating mutations and from five human healthy controls. We then assessed 1) MyHCs presence/post-translational modifications using LC/MS; 2) relaxed myosin conformation and concomitant ATP consumption with a loaded Mant-ATP chase setup; 3) myosin activation with an unloaded in vitro motility assay; and 4) cellular force production with a myofiber mechanical setup. Interestingly, the type IIa MyHC with one additional acetylated lysine (Lys35-Ac) was present in the patients. This was accompanied by 1) a higher ATP demand of myosin heads in the disordered-relaxed conformation; 2) faster actomyosin kinetics; and 3) reduced muscle fiber force. Overall, our findings indicate that MYH2 truncating mutations impact myosin presence/functionality in human adult mature myofibers by disrupting the ATPase activity and actomyosin complex. These are likely important molecular pathological disturbances leading to the myopathic phenotype in patients.

The dawn of the functional genomics era in muscle physiology.

Skeletal muscle is the most abundant component of the mature mammalian phenotype. Designed to generate contractile force and movement, skeletal muscle is crucial for organism health, function and development. One of the great interests for muscle biologists is in understanding how skeletal muscle adapts during periods of stress and stimuli, such as disease, disuse and ageing. To this end, genomic-based experimental and analytical approaches offer one of the most powerful approaches for comprehensively mapping the molecular paradigms that regulate skeletal muscle. With the power, applicability, and robustness of 'omic' technologies continually being developed, we are now in a position to investigate these molecular mechanisms in skeletal muscle to an unprecedented level of accuracy and precision, heralding the dawn of a new era of functional genomics in the field of muscle physiology.

Comparative study of binding pocket structure and dynamics in cardiac and skeletal myosin.

The development of small molecule myosin modulators has seen an increased effort in recent years due to their possible use in the treatment of cardiac and skeletal myopathies. Omecamtiv mecarbil (OM) is the first-in-class cardiac myotrope and the first to enter clinical trials. Its selectivity toward slow/beta-cardiac myosin lies at the heart of its function; however, little is known about the underlying reasons for selectivity to this isoform as opposed to other closely related ones such as fast-type skeletal myosins. In this work, we compared the structure and dynamics of the OM binding site in cardiac and in fasttype IIa skeletal myosin to identify possible reasons for OM selectivity. We found that the different shape, size, and composition of the binding pocket in skeletal myosin directly affects the binding mode and related affinity of OM, which is potentially a result of weaker interactions and less optimal molecular recognition. Moreover, we identified a side pocket adjacent to the OM binding site that shows increased accessibility in skeletal myosin compared with the cardiac isoform. These findings could pave the way to the development of skeletal-selective compounds that can target this region of the protein and potentially be used to treat congenital myopathies where muscle weakness is related to myosin loss of function.

Activation of eIF4E-binding-protein-1 rescues mTORC1-induced sarcopenia by expanding lysosomal degradation capacity.

BACKGROUND: Chronic mTORC1 activation in skeletal muscle is linked with age-associated loss of muscle mass and strength, known as sarcopenia. Genetic activation of mTORC1 by conditionally ablating mTORC1 upstream inhibitor TSC1 in skeletal muscle accelerates sarcopenia development in adult mice. Conversely, genetic suppression of mTORC1 downstream effectors of protein synthesis delays sarcopenia in natural aging mice. mTORC1 promotes protein synthesis by activating ribosomal protein S6 kinases (S6Ks) and inhibiting eIF4E-binding proteins (4EBPs). Whole-body knockout of S6K1 or muscle-specific over-expression of a 4EBP1 mutant transgene (4EBP1mt), which is resistant to mTORC1-mediated inhibition, ameliorates muscle loss with age and preserves muscle function by enhancing mitochondria activities, despite both transgenic mice showing retarded muscle growth at a young age. Why repression of mTORC1-mediated protein synthesis can mitigate progressive muscle atrophy and dysfunction with age remains unclear. METHODS: Mice with myofiber-specific knockout of TSC1 (TSC1mKO), in which mTORC1 is hyperactivated in fully differentiated myofibers, were used as a mouse model of sarcopenia. To elucidate the role of mTORC1-mediated protein synthesis in regulating muscle mass and physiology, we bred the 4EBP1mt transgene or S6k1 floxed mice into the TSC1mKO mouse background to generate 4EBP1mt-TSC1mKO or S6K1-TSC1mKO mice, respectively. Functional and molecular analyses were performed to assess their role in sarcopenia development. RESULTS: Here, we show that 4EBP1mt-TSC1mKO, but not S6K1-TSC1mKO, preserved muscle mass (36.7% increase compared with TSC1mKO, P < 0.001) and strength (36.8% increase compared with TSC1mKO, P < 0.01) at the level of control mice. Mechanistically, 4EBP1 activation suppressed aberrant protein synthesis (two-fold reduction compared with TSC1mKO, P < 0.05) and restored autophagy flux without relieving mTORC1-mediated inhibition of ULK1, an upstream activator of autophagosome initiation. We discovered a previously unidentified phenotype of lysosomal failure in TSC1mKO mouse muscle, in which the lysosomal defect was also conserved in the naturally aged mouse muscle, whereas 4EBP1 activation enhanced lysosomal protease activities to compensate for impaired autophagy induced by mTORC1 hyperactivity. Consequently, 4EBP1 activation relieved oxidative stress to prevent toxic aggregate accumulation (0.5-fold reduction compared with TSC1mKO, P < 0.05) in muscle and restored mitochondrial homeostasis and function. CONCLUSIONS: We identify 4EBP1 as a communication hub coordinating protein synthesis and degradation to protect proteostasis, revealing therapeutic potential for activating lysosomal degradation to mitigate sarcopenia.

Myosin Heavy Chain as a Novel Key Modulator of Striated Muscle Resting State.

After years of intense research using structural, biological, and biochemical experimental procedures, it is clear that myosin molecules are essential for striated muscle contraction. However, this is just the tip of the iceberg of their function. Interestingly, it has been shown recently that these molecules (especially myosin heavy chains) are also crucial for cardiac and skeletal muscle resting state. In the present review, we first overview myosin heavy chain biochemical states and how they influence the consumption of ATP. We then detail how neighboring partner proteins including myosin light chains and myosin binding protein C intervene in such processes, modulating the ATP demand in health and disease. Finally, we present current experimental drugs targeting myosin ATP consumption and how they can treat muscle diseases.