Biology subjects

Ochala, J.

Publications and source records attributed to Ochala, J..

14 recordsLinked to original sources

bioRxiv2026

Integrated Single-Fiber Multi-Omics Links an Inflammatory-Associated Myofiber State to Altered Myosin Dynamics in Patients with ICU-acquired weakness

Skeletal muscle dysfunction is a pervasive complication of critical illness that worsens survival and recovery, yet remains poorly explained by current clinical or molecular markers. To directly connect disease-associated molecular states to the contractile machinery, this study combined sequential functional, transcriptomic, and proteomic profiling of the same single human skeletal myofibers from critically ill patients in the intensive care unit with acquired weakness (ICU-AW) and controls. Despite marked donor-level heterogeneity, integrated analysis revealed a subtle yet conserved myofiber state enriched in ICU-AW, characterized by inflammatory and chemotactic gene programs, intracellular structural remodeling, and bioenergetic adaptation. Nineteen features were significantly altered at both RNA and protein levels from the same myofiber, linking an inflammatory transcriptional landscape to a proteomic shift toward mitochondrial and translational machinery and away from membrane-associated signaling. Functionally, fibers in this state displayed selectively disrupted myosin dynamics, evidenced by prolonged ATP turnover time of myosin heads in their super-relaxed conformation, implicating altered myosin energetics as a contributor to muscle dysfunction. These findings define a discrete, disease-associated myofiber state and establish an integrative single-fiber framework for connecting multi-omic heterogeneity to molecular motor function in complex human disease. Graphical AbstractSingle-fiber multi-omic and functional analysis reveals a stress-adapted myofiber state in ICU-AW. Specifically, for the present study, myofibers from ICU-AW donors and control donors were isolated and functionally profiled for myosin dynamics before being split for simultaneous transcriptomic and proteomic analysis. Integrated analysis then identified a reproducible fiber phenotype enriched in ICU-AW, characterized by inflammatory transcriptional signatures coordinated with mitochondrial proteomic remodeling and altered myosin super-relaxed state energetics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/706099v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@95715forg.highwire.dtl.DTLVardef@1462ff8org.highwire.dtl.DTLVardef@f74996org.highwire.dtl.DTLVardef@10055ec_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Multi-omics Insight into Cardiac Myofibril Remodeling in Post-Prandial Burmese Pythons

Burmese pythons exhibit rapid cardiac remodeling in response to a dramatic increase in metabolic rate during digestion. Here, we performed single-myofibril mechanics measurements and myosin heavy chain metabolic assays to evaluate the impact of feeding on the cardiomyocyte sarcomere - the fundamental molecular unit of muscle contraction - using two experimental paradigms: normal feeding (one meal per month) and frequent feeding (eight meals per month). Myofibril tension and rate of relaxation increased during digestion in both paradigms, while frequent feeding was further associated with slower myofibril activation kinetics and faster myosin heavy chain ATP turnover. To identify molecular changes at the sarcomere and gain potential mechanistic insight, we performed multi-omics analyses. RNA sequencing identified increased expression of some sarcomere genes during digestion; however, proteomics analysis suggested a delay in sarcomere protein synthesis at the peak of remodeling, as expression of many sarcomere proteins decreased. Analysis of post-translational modifications (ubiquitinomics, phospho-proteomics, acetylomics) identified hundreds of significantly regulated sites on sarcomere proteins during digestion, including many on the tension-regulating titin and myosin heavy chain proteins. Our results detail the molecular underpinnings of cardiac remodeling in digesting Burmese pythons and suggest that natures solution for rapidly increasing cardiac contractility is a post-translational sarcomere tuning program.

molecular biology↗

bioRxiv2024

Site-specific quantification of the in vivo UFMylome reveals myosin modification in ALS

UFMylation is a ubiquitin-like protein post-translational modification of Ubiquitin Fold Modifier 1 (UFM1) applied to substrate proteins. The UFMylation system is important for normal development and plays a critical role in a variety of cellular processes including regulating telomere length, stress responses and protein quality control. Here, we describe the development of an antibody-based enrichment approach to immunoprecipitate in vivo remnant UFMylated peptides and identification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). We used this approach to identify >200 UFMylation sites from various mouse tissues revealing extensive modification in skeletal muscle. Furthermore, we show that UFMylation is increased in skeletal muscle biopsies from people living with Amyotrophic Lateral Sclerosis (plwALS). Quantification of UFMylation sites in these participant biopsies with multiplexed isotopic labeling and LC-MS/MS reveal prominent increases in myosin UFMylation. Finally, in silico modelling suggest UFMylation of myosin directly adjacent to the ATP-binding site may regulate stability and/or function. Our data suggest that although UFMylation is not as widespread as ubiquitylation, its in vivo status is more complex than previously thought.

biochemistry↗

bioRxiv2024

Integrated single cell functional-proteomic profiling of human skeletal muscle reveals a shift in cellular specificity in nemaline myopathy

Skeletal muscle is a complex syncytial arrangement of an array of cell types and, in the case of muscle specific cells (myofibers), sub-types. There exists extensive heterogeneity in skeletal muscle functional behaviour and molecular landscape, at the cell composition, myofiber sub-type and intra-myofiber sub-type level. This heterogeneity highlights limitations in currently applied methodological approaches, which has stagnated our understanding of fundamental skeletal muscle biology in both healthy and myopathic contexts. Here, we developed a novel approach that combines a fluorescence based assay for the biophysical examination of the sarcomeric protein, myosin, coupled with same-myofiber high sensitivity proteome profiling, termed Single Myofiber Protein Function-Omics (SMPFO). Successfully applying this approach to healthy human skeletal muscle tissue, we identify the integrate relationship between myofiber functionality and the underlying proteomic landscape that guides divergent, but physiologically important, behaviour in myofiber sub-types. By applying SMPFO to two forms of human nemaline myopathy (ACTA1 and TNNT1 mutations), we reveal significant reduction in the divergence of myofiber sub-types, across both biophysical and proteomic behaviour. Collectively, we develop SMPFO as a novel approach to study skeletal muscle with greater specificity, accuracy and resolution then currently applied methods, facilitating that advancement in understanding of SkM tissue in both healthy and diseased states.

physiology↗

bioRxiv2024

Dysregulated Skeletal Muscle Myosin Super-relaxation in Type II, but Not Type I, Diabetes Mellitus

Disrupted energy balance is critical for the onset and development of Type II diabetes. The exact underlying metabolic mechanisms remain incomplete but skeletal muscle is thought to play an important pathogenic role. As the super-relaxed state of its most abundant protein, myosin, regulates cellular energetics, here, we aimed to investigate whether it is altered in patients with type II diabetes. For that, we used vastus lateralis biopsy specimens (obtained from patients with type II diabetes and matched controls) and run a combination of structural and functional assays consisting of loaded Mant-ATP chase experiments, X-ray diffraction and LC-MS/MS proteomics in isolated muscle fibres. Our studies revealed a greater muscle myosin super-relaxation and decreased cellular ATP demand in patients than controls. Subsequent proteomic analyses indicated that these (mal)adaptations likely originated from remodeled sarcomeric proteins and greater myosin glycation levels in patients than controls. Overall, our findings emphasize a complex molecular dysregulation of myosin super-relaxed state and energy consumption in type II diabetes. Ultimately, pharmacological targeting of myosin could benefit skeletal muscle and whole-body metabolic health through the enhancement of ATP consumption. Significance StatementMyosin super-relaxation, essential for the regulation of skeletal muscle metabolic rate, is disrupted in type II diabetes due to protein hyper-glycation. As a consequence, myosin ATP demand is significantly lowered. Overall, our findings provide a strong rationale for the use of activators of myosin ATPase to enhance basal energy expenditure in type II diabetes.

cell biology↗

bioRxiv2024

Myosin ATPase inhibition fails to rescue the metabolically dysregulated proteome of nebulin-deficient muscle

Nemaline myopathy (NM) is a genetic muscle disease, primarily caused by mutations in the NEB gene (NEB-NM) and with muscle myosin dysfunction as a major molecular pathogenic mechanism. Recently, we have observed that the myosin biochemical super-relaxed state was significantly impaired in NEB-NM, inducing an aberrant increase in ATP consumption and remodelling of the energy proteome in diseased muscle fibres. As the small-molecule Mavacamten is known to promote the myosin super-relaxed state and reduce the ATP demand, here, we tested its potency in the context of NEB-NM. We first conducted in vitro experiments in isolated single myofibres from patients and found that Mavacamten successfully reversed the myosin ATP over-consumption. Following this, we assessed its short-term in vivo effects by using the conditional nebulin knock-out (cNeb KO) mouse model and by subsequently performing global proteomics profiling in dissected soleus myofibres. After a four-week treatment period, we observed a remodelling of a large number of proteins in both cNeb KO mice and their wild-type siblings. Nevertheless, these changes were not related to the energy proteome, indicating that short-term Mavacamten treatment is not sufficient to properly counterbalance the metabolically dysregulated proteome of cNeb KO mice. Taken together, our findings emphasize Mavacamten potency in vitro but challenge its short-term efficacy in vivo. Key points summaryO_LINo cure exists for nemaline myopathy, a type of genetic skeletal muscle disease mainly derived from mutations in genes encoding myofilament proteins. C_LIO_LIApplying Mavacamten, a small molecule directly targeting the myofilament, to isolated membrane-permeabilized muscle fibres from human patients restored myosin energetic disturbances. C_LIO_LITreating a mouse model of nemaline myopathy in vivo with Mavacamten for four weeks, remodeled the skeletal muscle fibre proteome without any noticeable effects on energetic proteins. C_LIO_LIShort-term Mavacamten treatment may not be sufficient to reverse the muscle phenotype in nemaline myopathy. C_LI

physiology↗

bioRxiv2023

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{degrees}C). Upon repeating loaded Mant-ATP chase experiments at 8{degrees}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.

physiology↗

bioRxiv2023

Beyond Myosin Heavy Chains: Ribosomal Specialization Drives Human Skeletal Muscle Fiber Heterogeneity

Skeletal muscle is an inherently heterogenous tissue comprised primarily of myofibers, which are historically classified into three distinct fiber types in humans: one "slow" (type 1) and two "fast" (type 2A and type 2X), delineated by the expression of myosin heavy chain isoforms (MYHs). However, heterogeneity between and within traditional fiber types remains underexplored. Indeed, whether MYHs are the main classifiers of skeletal muscle fibers has not been examined in an unbiased manner. Through the development and application of novel transcriptomic and proteomic workflows, applied to 1050 and 1038 single muscle fibers from human vastus lateralis, respectively, we show that MYHs are not the only principal drivers of skeletal muscle fiber heterogeneity. Instead, metabolic, ribosomal, and cell junction proteins are a source of multi-dimensional variation between skeletal muscle fibers. Furthermore, whilst slow and fast fiber clusters can be identified, described by their contractile and metabolic profiles, our data suggests that type 2X fibers are not phenotypically distinct to other fast fibers at an omics level. Moreover, MYH-based classifications do not adequately describe the phenotype of skeletal muscle fibers in one of the most common genetic muscle diseases, nemaline myopathy, with fibers shifting towards a non-oxidative phenotype independently of MYH-based fiber type. We also characterize novel transcriptomic and proteomic features of slow and fast skeletal muscle fibers, including identifying several muscle fiber type-specific polypeptides, termed microproteins, encoded by transcripts annotated as non-coding RNA. Overall, our data indicates that skeletal muscle fiber heterogeneity is multi-dimensional with sources of variation beyond myosin heavy chain isoforms.

physiology↗

bioRxiv2023

Muscle fibre size and myonuclear positioning in trained and aged humans

Myonuclear domain (MND) is the theoretical volume of cytoplasm within which a myonucleus is responsible for transcribing DNA. Changes in myonuclear number, organisation, and myonuclear domain size are associated with exercise adaptations and ageing. However, data on satellite cell activation, changes in MND volumes and myonuclear arrangement following exercise are inconsistent. Additionally, whether MNDs and myonuclear arrangement are altered with age remains unclear. The aim of the present investigation was therefore to investigate relationships between age and activity status and myonuclear numbers and organisation. Muscle fibres from younger trained (YT) and older trained (OT) individuals were compared with age-matched untrained counterparts (YU and OU). Serial, optical z-slices were acquired throughout isolated muscle fibres and analysed to give 3D coordinates for myonuclei and muscle fibre dimensions, respectively. As expected, mean cross-sectional area (CSA) (m2) of muscle fibres from OU was 29-42% smaller compared to the other groups. Number of nuclei relative to fibre CSA was 87% greater in OU compared to YU muscle fibres (P < 0.05). Additionally, scaling of myonuclear domain volume with fibre size was altered in older untrained individuals. Myonuclear arrangement, on the other hand, was similar across groups. These data indicate that regular endurance exercise throughout the lifespan may preserve the size of single muscle fibres in older age and maintain the relationship between fibre size and MND volumes. Inactivity, however, may result in reduced muscle fibre size and disrupted relationship between fibre size and MND volumes. Plain Language SummaryIn this study, we examined the relationship between physical activity and the characteristics of muscle fibres in individuals of different age groups. We focused on a concept called the myonuclear domain (MND), which refers to the volume surrounding muscle nuclei or myonuclei that house the genome. We wanted to understand how changes in myonuclear number, organisation, and MND size were influenced by exercise and aging. To do this, we compared muscle fibres from younger trained individuals, older trained individuals, and age-matched untrained individuals. The results showed that the average size of muscle fibres in the untrained older individuals was smaller compared to the other groups. Moreover, the number of nuclei relative to fibre size was significantly higher in the untrained older individuals. However, myonuclear arrangement was similar across all groups. These findings suggest that regular endurance exercise throughout life may help maintain muscle fibre size, myonuclear numbers, MND volumes, and myonuclear organisation in older individuals. Conversely, inactivity can lead to reduced muscle fibre size and disrupted relationship between fibre size and MND volumes. These results have important implications for understanding the effects of exercise and aging on muscle health.

physiology↗

bioRxiv2023

Human light meromyosin mutations linked to skeletal myopathies disrupt the coiled coil structure and myosin head sequestration

Myosin heavy chains encoded by MYH7 and MYH2 are among the most abundant proteins in human skeletal muscle. After decades of intense research using a wide range of biophysical and biological approaches, their functions have begun to be elucidated. Despite this, it remains unclear how mutations in these genes and resultant proteins disrupt myosin structure and function, inducing pathological states and skeletal myopathies termed myosinopathies. Here, we have analysed the effects of several common MYH7 and MYH2 mutations located in light meromyosin (LMM) using a broad range of approaches. We determined the secondary structure and filament forming capabilities of expressed and purified LMM constructs in vitro, performed in-silico modelling of LMM constructs, and evaluated the incorporation of eGFP-myosin heavy chain constructs into sarcomeres in cultured myotubes. Using muscle biopsies from patients, we applied Mant-ATP chase protocols to estimate the proportion of myosin heads that were super-relaxed, X-ray diffraction measurements to estimate myosin head order and myofibre mechanics to investigate contractile function. We found that human MYH7 and MYH2 LMM mutations commonly disrupt myosin coiled-coil structure and packing of filaments in vitro; decrease the myosin super-relaxed state in vivo and increase the basal myosin ATP consumption; but are not associated with myofibre contractile deficits. Altogether, these findings indicate that the structural remodelling resulting from 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 in the design of future therapies for myosinopathies.

physiology↗

bioRxiv2023

Predominant myosin super-relaxed 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 non-failing controls (NF). 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 super-relaxed (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 non-sequestered but ATP-conserving SRX state, that can be counterbalanced by EMD-57033 demonstrating the potential for a myosin-centered pharmacological treatment of DCM. New & noteworthyThe key finding of the present study is that, in left ventricles of dogs with a naturally occurring dilated cardiomyopathy, relaxed myosin molecules favor a non-sequestered super-relaxed state potentially impairing sarcomeric contractility. This alteration is rescuable by applying a small molecule activating myosin known as EMD-57033.

pathology↗

bioRxiv2022

Preponderant Myosin Super-Relaxed State In Skeletal Muscle From Endurance Athletes

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 super-relaxed (SRX) and disordered-relaxed (DRX) states and are finely balanced to optimize skeletal muscle metabo-lism. Indeed, SRX myosins are thought to have a 10-fold reduction in ATP turnover compared to 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 mus-cle fibres from various athletic and sedentary populations and ran a loaded Mant-ATP chase proto-col. We observed that, in endurance-trained athletes, the amounts of myosin molecules in the SRX state was significantly greater than in age-matched sedentary individuals or than in strength athletes. To further assess whether this change would have an impact on the potency of a SRX-inducing pharmacological compound, Mavacamten, we performed similar analyses as above with and without the drug in muscle fibres from endurance athletes. Surprisingly, we found that 0.3 M of Mava-camten had only marginal effects. Altogether, our results indicate that chronic endurance training-status influences resting skeletal myosin conformations, and Mavacamten potency. Our findings also emphasize that environmental stimuli such as exercise can re-wire the molecular metabolism of human skeletal muscle through myosin. SummaryLewis et al., investigate how training-status influences myosin conformations involved in the resting metabolism of skeletal muscle. They find that, in endurance-trained athletes, skeletal myosin preferentially adopts an energy-saving conformation known as super-relaxed state, lowering the metabolic rate and affecting the potency of a super-relaxed state-inducing drug, Mavacamten.

physiology↗

bioRxiv2022

Exercise induces myonuclear remodelling in humans independently of age

Age-related decline in skeletal muscle structure and function can be mitigated by regular exercise. However, the precise mechanisms that govern this are not fully understood. The nucleus plays an active role in translating forces into biochemical signals (mechanotransduction), with nuclear lamina protein Lamin A regulating nuclear shape, nuclear mechanics, and ultimately gene expression. Defective Lamin A expression causes muscle pathologies and premature ageing syndromes, but the roles of nuclear structure and function in physiological ageing and in exercise adaptations remain obscure. Here, we isolated single muscle fibres and carried out detailed morphological and functional analyses on myonuclei from young and older exercise-trained individuals. Strikingly, myonuclei from trained individuals were more spherical, less deformable, and contained a thicker nuclear lamina than untrained individuals. Complementary to this, exercise resulted in increased levels of Lamin A and increased myonuclear stiffness in mice. We conclude that exercise is associated with myonuclear remodelling, independently of age, which may contribute to the preservative effects of exercise on muscle function throughout the lifespan. Key pointsO_LIThe nucleus plays an active role in translating forces into biochemical signals C_LIO_LIMyonuclear aberrations in a group of muscular dystrophies called laminopathies suggest that the shape and mechanical properties of myonuclei are important for maintaining muscle function. C_LIO_LIHere, we present striking differences in myonuclear shape and mechanics associated with exercise, in both young and old humans. C_LIO_LIMyonuclei from trained individuals were more spherical, less deformable, and contained a thicker nuclear lamina than untrained individuals. C_LIO_LIWe conclude that exercise is associated with age-independent myonuclear remodelling, which may help to maintain muscle function throughout the lifespan. C_LI

cell biology↗

bioRxiv2019

Impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy

Nemaline myopathy (NM) is a genetically heterogeneous skeletal muscle disorder caused by mutations predominately affecting contractile filaments, in particular thin filament structure and/or regulation. The underlying cellular pathophysiology of this disease remains largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mice and patients with NM, including disrupted nuclear envelope, altered chromatin arrangement, and disorganisation of the cortical cytoskeleton. We demonstrate that such nuclear defects are caused by impairment of muscle fibre contractility, and that cytoskeletal organisation determines nuclear morphology. Our results overlap with findings in diseases caused by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to underlie some of the hallmarks of NM, which include broad transcriptional alterations, arrested muscle fibre growth, contractile filament disarray and altered mechanical properties.

cell biology↗