Skeletal Muscle Myofibrils Research Articles

A-Band assembly in avian skeletal muscles observed with super-resolution microscopy.

Myofibrils in vertebrate skeletal muscle are organized in aligned arrays of filaments formed from multiple protein components. Despite considerable information describing individual proteins, how they assemble de novo into mature myofibrils is still a challenge. Studies in our lab of sarcomeric protein localization during myofibril assembly led us to propose a three-step progression: premyofibrils to nascent myofibrils, culminating in mature myofibrils. Premyofibrils, forming at the spreading edges of muscle cells, are composed of minisarcomeres containing small bands of non-muscle myosin II filaments alternating with muscle-specific α-actinin Z-Bodies attached to barbed ends of actin filaments, establishing bipolar F-actin arrangements in sarcomeres. Assembly of nascent myofibrils occurs with addition of muscle-specific myosin II, F-actin, titin, and the alignment of Z-Bodies in adjacent fibrils to form beaded Z-Bands. Muscle-specific myosin II filaments in nascent myofibrils appear in an overlapping arrangement when viewed with wide-field and confocal microscopes. In mature myofibrils, non-muscle myosin II is absent, and M-Band proteins localize to the muscle myosin II filaments, aiding their alignment by cross-linking them into A-Bands. Super-resolution microscopy (SIM and STED) revealed muscle myosin II in mini-A-Bands in nascent myofibrils. In contrast to previous reports that vertebrate muscle myosin thick filaments form at their final 1.6 μm lengths, mini-A-Bands are first detected at a length of about 0.4 μm, and gradually increase four-fold in length to 1.6 μm in mature myofibrils. These new discoveries in avian skeletal muscle cells share a common characteristic with invertebrate muscles where some A-Bands can grow to lengths reaching 25 μm.

Immunoglobulin domain unfolding and refolding in passively stretched myofibrils.

In isolated myofibrils, passive force is attributed solely to the protein titin. Mechanically, titin is a non-linear biological spring, with two primary mechanical regions: the PEVK region, and the immunoglobulin (IG) domain region. The PEVK region is approximately elastic, and the IG domain region contains domains that unfold and refold depending on force. Under passive tension, skeletal muscle myofibrils exhibit stress-relaxation, particularly at longer lengths (>3.6 µm/sarcomere) where IG domains begin to unfold. During muscle shortening, IG domains refold, and the PEVK region shortens. We investigated the rate of IG domain unfolding and refolding in myofibrils. Paired tests on myofibrils isolated from the psoas muscle of rabbits (n=7). Passive force was measured using force cantilevers for two test protocols: a 2 s-4 s-2 s stretch, hold, release and a 2 s-0 s-2 s stretch, no-hold, release, both with a target length of 4.5μm/sarcomere. Stretches were performed using a piezoelectric motor. Both test protocols were compared to simulated values using the worm-like chain model. Differences between the two test protocols were apparent specifically in the rate of IG domain refolding. Passive force was present experimentally during the entirety of the two second shortening, with the myofibrils remaining taught over the entire duration of both tests. This behavior was not apparent in simulated tests, where myofibrils were predicted to become slack (no passive force) after 0.79 s and 0.9 s during the 2-s shortening period for the 4-s hold protocol and the no-hold protocol, respectively. This suggests IG domain refolding in myofibrils happens considerably quicker in situ than anticipated from isolated titin testing. Our data show that IG domain refolding at this higher rate likely contributes to substantial positive mechanical work during myofibril shortening for both protocols.

Regulation of troponin and myosin conformation in thin- and thick-filament domains in the sarcomere of skeletal muscle myofibrils.

The contractility of striated muscle is controlled by a dual-filament regulatory mechanism which couples calcium-dependent structural changes in thin filaments with those in thick filaments triggering the release of folded-helical myosin motors from the filament surface. The folded-helical conformation of motors in relaxed muscle may be stabilized in the region of the thick filament containing myosin-binding protein-C (C-zone). Moreover, in active muscle actin-bound myosin motors might locally alter the activation level of the thin filament. To test these hypotheses we developed a novel fluorescence-based approach to measure troponin and myosin orientation in subdomains of the sarcomere in isolated myofibrils. Bifunctional rhodamine probes labelling troponin-C (TnC) and the myosin regulatory light chain (RLC) were exchanged into myofibrils from rabbit psoas muscle, and the spatial distribution of probe orientations in the sarcomere was measured by fluorescence polarization microscopy. In the absence of ATP, the RLCs of actin-attached myosin motors were perpendicular to the filament axis and the orientation of the TnC probes in the region of overlap with thick filaments was markedly different from that in the rest of the sarcomere, indicating that myosin binding to actin alters the conformation of troponin in the thin filament. In relaxing conditions all TnC probes had the same conformation which was insensitive to temperature. In contrast, the RLC probes became more parallel to the filament axis as temperature was increased above 12°C, and at 30°C in the presence of 4% Dextran their orientation was more parallel in the C-zone, indicating enrichment of folded myosin motors in this filament domain. These results show directly that troponin and myosin have distinct conformations in different sarcomere subdomains in situ (supported by the Wellcome Trust and the Royal Society, UK).

Reprint of: The Content of Troponin, Tropomyosin, Actin, and Myosin in Rabbit Skeletal Muscle Myofibrils

A stacking sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to resolve and quantify all the major myofibrillar protein components (actin, myosin, tropomyosin, and troponin C, T, and I). Quantification was achieved by densitometry of the fast green-stained gels calibrated with the use of purified proteins. The approximate molar ratios of these proteins in rabbit muscle are: actin : myosin: tropomyosin: troponin T: troponin I: troponin C = 7:1:1:1:1:1. On the basis of these results and available structural information one obtains an estimate of 254 myosin molecules per thick filament.

On 'The content of troponin, tropomyosin, actin, and myosin in rabbit skeletal muscle myofibrils' by James D. Potter

After the discovery of troponin by Ebashi almost sixty years ago the field of striated muscle regulation has made significant progress. In the 1970's the nascent troponin field gained momentum, including contributions by James D. Potter who established the stoichiometry of contractile proteins in the myofibril (Arch Biochem Biophys. 1974 Jun; 162(2):436-41. https://doi.org/10.1016/0003-9861(7490202-1)). This opened the door to refinement of competing models that described possible thick filament configurations. This study suggested the presence of one myosin per cross bridge and provided accurate calculations of the molar ratios of each protein - myosin: actin: tropomyosin: troponin T: troponin I: troponin C.

Copper ions impair zebrafish skeletal myofibrillogenesis via epigenetic regulation.

Unbalanced copper (Cu2+ ) homeostasis is associated with the developmental defects of vertebrate myogenesis, but the underlying molecular mechanisms remain elusive. In this study, it was found that Cu2+ stressed zebrafish embryos and larvae showed reduced locomotor speed as well as loose and decreased myofibrils in skeletal muscle, coupled with the downregulated expression of muscle fiber markers mylpfa and smyhc1l and the irregular arrangement of myofibril and sarcomere. Meanwhile, the Cu2+ stressed zebrafish embryos and larvae also showed significant reduction in the expression of H3K4 methyltransferase smyd1b transcripts and H3K4me3 protein as well as in the binding enrichment of H3K4me3 on gene mylpfa promoter in skeletal muscle cells, suggesting that smyd1b-H3K4me3 axis mediates the Cu2+ -induced myofibrils specification defects. Additionally, whole genome DNA methylation sequencing unveiled that the gene smyd5 exhibited significant promoter hyper-methylation and increased expression in Cu2+ stressed embryos, and the ectopic expression of smyd5 in zebrafish embryos also induced the myofibrils specification defects as those observed in Cu2+ stressed embryos. Moreover, Cu2+ was shown to suppress myofibrils specification and smyd1b promoter transcriptional activity directly independent of the integral function of copper transporter cox17 and atp7b. All these data may shed light on the linkage of unbalanced copper homeostasis with specific gene promoter methylation and epigenetic histone protein modification as well as the resultant signaling transduction and the myofibrillogenesis defects.

Effect of Resistance Exercise and Vitamin C Intake on Expression of Telomerase Reverse Transcriptase and Telomere Repeat Binding Factor-2 Genes and the Diameter and Number of Myofibrils in Old Rats

Objective: Cell aging is one of the most important and fundamental step in cellular behavior and reduces muscle mass and myofibrils. This study aims to investigate the effect of resistance exercise along with vitamin C consumption on the expression of Telomerase Reverse Transcriptase (TERT) and Telomere Repeat Binding Factor-2 (TRF2) genes and the diameter and number of skeletal muscle myofibrils in old male Wistar rats. Methods: This is an experimental study using control groups. Twenty-five male Wistar rats (280-320 g and aged 24 weeks) were randomly divided into five groups of young control, old+resistance exercise, old+vitamin intake, old+resistance exercise+vitamin intake, and old control. In the supplementation groups, rats received liposomal vitamin C daily by gavage per body weight. Weight attached to the tail gradually increased during 8 weeks of exercise (weeks 1 and 2 by 50%; weeks 3 and 4 by 50%; weeks 5 and 6 by 75%; weeks 7 and 8 by 100% of total body weight). Exercises were performed at 3-5 sets of 8-12 repetitions, 3 or 4 days a week. The Quantitative polymerase chain reaction method was used to evaluate the expression of TERT and TRF2 genes in muscle tissue. One-way ANOVA was used to examine the difference between the groups and Tukey’s post hoc test was used to determine between which groups the difference was significant. Results: Aging significantly reduced the expression of TERT, TRF2 genes and the diameter and number of myofibrils in skeletal muscle of rats (P=0.001). Resistance exercise along with vitamin C intake had no significant effect on TERT and TRF2 expression (P≥0.05), but caused a significant increase in the diameter and number of myofibrils (P= 0.001). Conclusion: Eight weeks of resistance exercise along with vitamin C supplementation can significantly increase the number and diameter of skeletal muscle myofibrils in old rats.

SPEG binds with desmin and its deficiency causes defects in triad and focal adhesion proteins.

Striated preferentially expressed gene (SPEG), a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle-specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here, we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amount of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1 and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and β1 integrin, both of which are critical components of the focal adhesion complex. Further, β1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.

Extraction of Thick Filaments in Individual Sarcomeres Affects Force Production by Single Myofibrils

It has been accepted that the force produced by a skeletal muscle myofibril depends on its cross-sectional area but not on the number of active sarcomeres because they are arranged in series. However, a previous study performed by our group showed that blocking actomyosin interactions within an activated myofibril and depleting the thick filaments in one sarcomere unexpectedly reduced force production. In this study, we examined in detail how consecutive depletion of thick filaments in individual sarcomeres within a myofibril affects force production. Myofibrils isolated from rabbit psoas were activated and relaxed using a perfusion system. An extra microperfusion needle filled with a high-ionic strength solution was used to erase thick filaments in individual sarcomeres in real time before myofibril activation. The isometric forces were measured upon activation. The force produced by myofibrils with intact sarcomeres was significantly higher than the force produced by myofibrils with one or more sarcomeres lacking thick filaments (p < 0.0001) irrespective of the number of contractions imposed on the myofibrils and their initial sarcomere length. Our results suggest that the myofibril force is affected by intersarcomere dynamics and the number of active sarcomeres in series.

Force generated by myosin cross-bridges is reduced in myofibrils exposed to ROS/RNS.

Skeletal muscle weakness is associated with oxidative stress and oxidative posttranslational modifications on contractile proteins. There is indirect evidence that reactive oxygen/nitrogen species (ROS/RNS) affect skeletal muscle myofibrillar function, although the details of the acute effects of ROS/RNS on myosin-actin interactions are not known. In this study, we examined the effects of peroxynitrite (ONOO-) on the contractile properties of individual skeletal muscle myofibrils by monitoring myofibril-induced displacements of an atomic force cantilever upon activation and relaxation. The isometric force decreased by ~50% in myofibrils treated with the ONOO- donor (SIN-1) or directly with ONOO-, which was independent of the cross-bridge abundancy condition (i.e., rigor or relaxing condition) during SIN-1 or ONOO- treatment. The force decrease was attributed to an increase in the cross-bridge detachment rate (gapp) in combination with a conservation of the force redevelopment rate (kTr) and hence, an increase in the population of cross-bridges transitioning from force-generating to non-force-generating cross-bridges during steady-state. Taken together, the results of this study provide important information on how ROS/RNS affect myofibrillar force production which may be of importance for conditions where increased oxidative stress is part of the pathophysiology.

The Role of Nesprin in the Facial Branchiomotor Neuron (FBMN) Migration of Danio rerio (zebrafish) development

Nuclear envelope spectrin‐repeat proteins (Nesprins) are part of the LINC (LInker of Nucleoskeleton and Cytoskeleton) complex, which plays a vital role in the binding of F‐actin cytoskeleton to the nuclear envelope. Nesprins are crucial in maintaining the integrity of the nuclear membrane. Nuclear membrane movement is a fundamental aspect in cell migration and muscle development. Previous work has demonstrated that Nesprins may be associated with myosin‐dependent tension, which has implications in muscular dystrophy due to the role that nesprins play in muscle cells. We hypothesize that expressing dominant‐negative nesprin in Danio rerio (zebrafish) embryos would result in defects in the migration of facial branchiomotor neurons and also cause disruptions in nuclear positioning in multinucleated muscle cells. To test this hypothesis, we use transient transgenic expression of dominant Nesprin in cranial motor neurons using the islet1 promoter. We quantify the extent of migration in neurons with inactive Nesprin. In addition, we express dominant‐negative Nesprin in muscle cells using a heat shock promoter and explore the positioning of nuclei in developing skeletal muscle myofibrils. Our preliminary data shows that transgenic zebrafish embryos with expression of dominant‐negative nesprin have disruptions and defects in tangential FBMN migration. However, with further experimentation and data collection in progress, we hope to analyze our findings for statistical significance. Our goal is to gain insight into the function of Nesprins in embryo development to better devise therapeutic strategies for patients with mutations in Nesprins.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of elamipretide on skeletal muscle in dogs with experimentally induced heart failure.

AimsElamipretide (ELAM), an aromatic–cationic tetrapeptide, interacts with cardiolipin and normalizes dysfunctional mitochondria of cardiomyocytes. This study examined the effects of ELAM on skeletal muscle mitochondria function in dogs with chronic heart failure (HF).Methods and resultsStudies were performed in skeletal muscle biopsy specimens obtained from normal dogs (n = 7) and dogs with chronic intracoronary microembolization‐induced HF (n = 14) treated with subcutaneous ELAM 0.5 mg/kg (HF + ELAM, n = 7) or vehicle (normal saline control, HF‐CON, n = 7). After 3 months of therapy, triceps skeletal muscle samples were obtained from all dogs, and the proportion of type 1 and type 2 fibres was assessed. Mitochondria isolated from myofibrils of the vastus lateralis skeletal muscle exposed in vitro to ELAM for 1 h were used to assess mitochondrial function. The proportion of skeletal muscle type 1 fibres was lower in HF‐CON dogs compared with normal dogs (23 ± 4 vs. 32 ± 5%, P < 0.05). Treatment with ELAM restored a near‐normal fibre‐type composition (31 ± 7%, P < 0.05 vs. HF‐CON). Skeletal muscle mitochondria showed significantly lower levels of adenosine diphosphate‐dependent mitochondrial respiration (100 ± 9 vs. 164 ± 15 natom O/min/mg protein, P < 0.05), mitochondrial membrane potential (0.17 ± 0.03 vs. 0.53 ± 0.03 red/green fluorescence ratio, P < 0.05), mitochondrial permeability transition pore (38 ± 3 vs. 62 ± 2 relative light units, P < 0.05), maximum rate of adenosine triphosphate synthesis (3284 ± 418 vs. 8835 ± 423 RLU/μg protein, P < 0.05), and cytochrome c oxidase activity (1390 ± 108 vs. 2459 ± 210 natom O/min/mg protein, P < 0.05) compared with normal dogs. Exposure of skeletal muscle myofibrillar mitochondria from HF dogs to ELAM showed a dose‐dependent improvement/normalization of all measures of mitochondrial function. In mitochondria from skeletal muscle of HF dogs exposed to 0.10 μM ELAM, adenosine diphosphate‐dependent mitochondrial respiration increased to 183 ± 18 natom O/min/mg protein, membrane potential increased to 0.30 ± 0.03 red/green fluorescence ratio, mitochondrial permeability transition pore increased to 54 ± 4 RLU, maximum rate of adenosine triphosphate synthesis increased to 4423 ± 414, and cytochrome c oxidase activity increased to 2033 ± 191 natom O/min/mg protein. Exposure of skeletal muscle myofibrillar mitochondria from normal dogs to ELAM had no effect on mitochondrial function parameters.ConclusionsThe results indicate that ELAM, previously shown to positively influence mitochondrial function of the failing heart, can also positively impact mitochondrial function of skeletal muscle and potentially help restore skeletal muscle function and improve exercise tolerance.

CHD4 and the NuRD complex directly control cardiac sarcomere formation

Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal α-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development.

Sarcomere Stiffness during Stretching and Shortening of Rigor Skeletal Myofibrils

In this study, we measured the stiffness of skeletal muscle myofibrils in rigor. Using a custom-built atomic force microscope, myofibrils were first placed in a rigor state then stretched and shortened at different displacements (0.1–0.3 μm per sarcomere) and nominal speeds (0.4 and 0.8 μm/s). During stretching, the myofibril stiffness was independent of both displacement and speed (average of 987 nN/μm). During shortening, the myofibril stiffness was independent of displacement, but dependent on speed (1234 nN/μm at 0.4 μm/s; 1106 nN/μm at 0.8 μm/s). Furthermore, the myofibril stiffness during shortening was greater than that during stretching and the difference depended on speed (31% at 0.4 μm/s; 8% at 0.8 μm/s). The results suggest that the myofibrils exhibit nonlinear viscoelastic properties that may be derived from myofibril filaments, similar to what has been observed in muscle fibers.

A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8

Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot–Marie–Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces impairments in autophagy that may aggravate the phenotype.

Microfluidic perfusion shows intersarcomere dynamics within single skeletal muscle myofibrils

The sarcomere is the smallest functional unit of myofibrils in striated muscles. Sarcomeres are connected in series through a network of elastic and structural proteins. During myofibril activation, sarcomeres develop forces that are regulated through complex dynamics among their structures. The mechanisms that regulate intersarcomere dynamics are unclear, which limits our understanding of fundamental muscle features. Such dynamics are associated with the loss in forces caused by mechanical instability encountered in muscle diseases and cardiomyopathy and may underlie potential target treatments for such conditions. In this study, we developed a microfluidic perfusion system to control one sarcomere within a myofibril, while measuring the individual behavior of all sarcomeres. We found that the force from one sarcomere leads to adjustments of adjacent sarcomeres in a mechanism that is dependent on the sarcomere length and the myofibril stiffness. We concluded that the cooperative work of the contractile and the elastic elements within a myofibril rules the intersarcomere dynamics, with important consequences for muscle contraction.

Activated Skeletal Muscle Myofibrils have Different Peak Stresses at Similar Sarcomere Lengths when Lengthened Beyond Myofilament Overlap

We suggested previously that the free-spring region of titin in the I-band can be length-modulated; giving rise to dramatically elevated forces in actively compared to passively stretched myofibrils at sarcomere length (SL) beyond actin-myosin filament overlap (>4µm).[1]If peak stresses were to vary at very long SL (> 4µm) where titin is the sole contributor (in myofibrils) to force, then that would suggest that the free-spring length of titin can be length-modulated somehow. The result being that a shorter free-spring titin would generate higher stress at matched SL compared to a myofibril where titin length was not shortened. We stretched calcium activated (pCa+2 3.5 in all experiments) rabbit psoas myofibrils beyond myofilament overlap (>4µm) while measuring forces and mean SL. Samples were either fully activated (control) or were activated with 2mM BDM. All samples were lengthened (0.1µm/sarcomere/second) following activation. Initial activation stress at SL=2.2µm for BDM was 35.5 ±10.5nN/um2 compared to control 89.3nN/um2. The BDM activated samples had less peak stress (278±32nN/um2) compared to the control (407nN/um2) at similar mean SL 4.7±0.05µm. Previous data [1] for myofibrils lengthened passively showed less peak stress compared to all experiments here; for mean SL 4.6µm, the mean stress was 135±47nN/um2.Based on these results, we suggest that titin is a molecular spring whose stiffness may be regulated by changes in effective length and that regulation is not an “all-or-none” model. We suggest that titin is a molecular spring whose stiffness is regulated by changes in effective length controlled by force-dependent actin-titin interactions.

Software-based measurement of thin filament lengths: an open-source GUI for Distributed Deconvolution analysis of fluorescence images.

The periodically arranged thin filaments within the striated myofibrils of skeletal and cardiac muscle have precisely regulated lengths, which can change in response to developmental adaptations, pathophysiological states, and genetic perturbations. We have developed a user-friendly, open-source ImageJ plugin that provides a graphical user interface (GUI) for super-resolution measurement of thin filament lengths by applying Distributed Deconvolution (DDecon) analysis to periodic line scans collected from fluorescence images. In the workflow presented here, we demonstrate thin filament length measurement using a phalloidin-stained cryosection of mouse skeletal muscle. The DDecon plugin is also capable of measuring distances of any periodically localized fluorescent signal from the Z- or M-line, as well as distances between successive Z- or M-lines, providing a broadly applicable tool for quantitative analysis of muscle cytoarchitecture. These functionalities can also be used to analyse periodic fluorescence signals in nonmuscle cells.

The role of sarcomere length non-uniformities in residual force enhancement of skeletal muscle myofibrils.

The sarcomere length non-uniformity theory (SLNT) is a widely accepted explanation for residual force enhancement (RFE). RFE is the increase in steady-state isometric force following active muscle stretching. The SLNT predicts that active stretching of a muscle causes sarcomere lengths (SL) to become non-uniform, with some sarcomeres stretched beyond actin–myosin filament overlap (popping), causing RFE. Despite being widely known, this theory has never been directly tested. We performed experiments on isolated rabbit muscle myofibrils (n = 12) comparing SL non-uniformities for purely isometric reference contractions (I-state) and contractions following active stretch producing RFE (FE-state). Myofibrils were activated isometrically along the descending limb of the force–length relationship (mean ± 1 standard deviation (SD) = 2.8 ± 0.3 µm sarcomere−1). Once the I-state was reached, myofibrils were shortened to an SL on the plateau of the force–length relationship (2.4 µm sarcomere−1), and then were actively stretched to the reference length (2.9 ± 0.3 µm sarcomere−1). We observed RFE in all myofibrils (39 ± 15%), and saw varying amounts of non-uniformity (1 SD = 0.9 ± 0.5 µm) that was not significantly correlated with the amount of RFE, but through pairwise comparisons was found to be significantly greater than the non-uniformity measured for the I-state (0.7 ± 0.4 µm). Three myofibrils exhibited no increase in non-uniformity. Active stretching was accompanied by sarcomere popping in four myofibrils, and seven had popped sarcomeres in the I-state. These results suggest that, while non-uniformities are present with RFE, they are also present in the I-state. Furthermore, non-uniformity is not associated with the magnitude of RFE, and myofibrils that had no increase in non-uniformity with stretch still showed normal RFE. Therefore, it appears that SL non-uniformity is a normal associate of muscle contraction, but does not contribute to RFE following active stretching of isolated skeletal muscle myofibrils.

Mouse myofibers lacking the SMYD1 methyltransferase are susceptible to atrophy, internalization of nuclei and myofibrillar disarray.

ABSTRACTThe Smyd1 gene encodes a lysine methyltransferase specifically expressed in striated muscle. Because Smyd1-null mouse embryos die from heart malformation prior to formation of skeletal muscle, we developed a Smyd1 conditional-knockout allele to determine the consequence of SMYD1 loss in mammalian skeletal muscle. Ablation of SMYD1 specifically in skeletal myocytes after myofiber differentiation using Myf6cre produced a non-degenerative myopathy. Mutant mice exhibited weakness, myofiber hypotrophy, prevalence of oxidative myofibers, reduction in triad numbers, regional myofibrillar disorganization/breakdown and a high percentage of myofibers with centralized nuclei. Notably, we found broad upregulation of muscle development genes in the absence of regenerating or degenerating myofibers. These data suggest that the afflicted fibers are in a continual state of repair in an attempt to restore damaged myofibrils. Disease severity was greater for males than females. Despite equivalent expression in all fiber types, loss of SMYD1 primarily affected fast-twitch muscle, illustrating fiber-type-specific functions for SMYD1. This work illustrates a crucial role for SMYD1 in skeletal muscle physiology and myofibril integrity.