Signaling by reactive oxygen species has emerged as a major physiological process. Due to its high metabolic rate, striated muscle is especially subject to oxidative stress, and there are multiple examples in cardiac and skeletal muscle where oxidative stress modulates contractile function. Here we assessed the potential of cysteine oxidation as a mechanism for modulating contractile function in skeletal and cardiac muscle. Analyzing the cysteine content of the myofilament proteins in striated muscle, we found that cysteine residues are relatively rare, but are very similar between different muscle types and different vertebrate species. To refine this list of cysteines to those that may modulate function, we estimated the accessibility of oxidants to cysteine residues using protein crystal structures, and then sharpened these estimates using fluorescent labeling of cysteines in cardiac and skeletal myofibrils. We demonstrate that cysteine accessibility to oxidants and ATPase rates depend on the contractile state in which preparations are exposed. Oxidant exposure of skeletal and cardiac myofibrils in relaxing solution exposes myosin cysteines not accessible in rigor solution, and these modifications correspond to a decrease in maximum ATPase. Oxidant exposure under rigor conditions produces modifications that increase basal ATPase and calcium sensitivity in ventricular myofibrils, but these effects were muted in fast twitch muscle. These experiments reveal how structural and sequence variations can lead to divergent effects from oxidants in different muscle types.

To understand molecular interactions that organize developing myofibrils, we examined the biosynthesis and interaction of titin and myosin heavy chain in cultures of developing muscle. Use of pulse-labeling, immunoprecipitation, and a reversible cross-linking procedure demonstrates that within minutes of synthesis, titin and myosin heavy chain can be chemically cross-linked into very large, detergent-resistant complexes retaining many features of intact myotubes. These complexes, predominantly of titin and myosin, occur very early in myofibrillogenesis as well as later. These data suggest that synthesis and assembly of titin and myosin are temporally and spatially coordinated in nascent myofibrils and support the hypothesis that titin molecules help to organize sarcomere formation.

How proteins assemble into sarcomeric arrays to form myofibrils is controversial. Immunostaining and transfections of cultures of cardiomyocytes from 10-day avian embryos led us to propose that assembly proceeded in three stages beginning with the formation of premyofibrils followed by nascent myofibrils and culminating in mature myofibrils. However, premyofibril and nascent myofibril arrays have not been detected in early cardiomyocytes examined in situ in the forming avian heart suggesting that the mechanism for myofibrillogenesis differs in cultured and uncultured cells. To address this question of in situ myofibrillogenesis, we applied non-enzymatic procedures and deconvolution imaging techniques to examine early heart forming regions in situ at 2- to 13-somite stages (beating begins at the 9-somite stage), a time span of about 23 h. These approaches enabled us to detect the three myofibril stages in developing hearts supporting a three-step model of myofibrillogenesis in cardiomyocytes, whether they are present in situ, in organ cultures or in tissue culture. We have also discovered that before titin is organized the first muscle myosin filaments are about half the length of the 1.6 mum filaments present in mature A-bands. This supports the proposal that titin may play a role in length determination of myosin filaments.

Cardiomyocytes from human stem cells have applications in regenerative medicine and can provide models for heart disease and toxicity screening. Soluble components of the culture system such as growth factors within serum and insoluble components such as the substrate on which cells adhere to are important variables controlling the biological activity of cells. Using a combinatorial materials approach we develop a synthetic, chemically defined cellular niche for the support of functional cardiomyocytes derived from human embryonic stem cells (hESC-CMs) in a serum-free fully defined culture system. Almost 700 polymers were synthesized and evaluated for their utility as growth substrates. From this group, 20 polymers were identified that supported cardiomyocyte adhesion and spreading. The most promising 3 polymers were scaled up for extended culture of hESC-CMs for 15 days and were characterized using patch clamp electrophysiology and myofibril analysis to find that functional and structural phenotype was maintained on these synthetic substrates without the need for coating with extracellular matrix protein. In addition, we found that hESC-CMs cultured on a co-polymer of isobornyl methacrylate and tert-butylamino-ethyl methacrylate exhibited significantly longer sarcomeres relative to gelatin control. The potential utility of increased structural integrity was demonstrated in an in vitro toxicity assay that found an increase in detection sensitivity of myofibril disruption by the anti-cancer drug doxorubicin at a concentration of 0.05 μM in cardiomyocytes cultured on the co-polymer compared to 0.5 μM on gelatin. The chemical moieties identified in this large-scale screen provide chemically defined conditions for the culture and manipulation of hESC-CMs, as well as a framework for the rational design of superior biomaterials.

Contraction of striated muscles is initiated by an increase in cytosolic Ca2+ concentration, which is regulated by tropomyosin and troponin acting on actin filaments at the sarcomere level. Namely, Ca2+-binding to troponin C shifts the "on-off" equilibrium of the thin filament state toward the "on" state, promoting actomyosin interaction; likewise, an increase in temperature to within the body temperature range shifts the equilibrium to the on state, even in the absence of Ca2+. Here, we investigated the temperature dependence of sarcomere shortening along isolated fast skeletal myofibrils using optical heating microscopy. Rapid heating (25 to 41.5°C) within 2 s induced reversible sarcomere shortening in relaxing solution. Further, we investigated the temperature-dependence of the sliding velocity of reconstituted fast skeletal or cardiac thin filaments on fast skeletal or β-cardiac myosin in an in vitro motility assay within the body temperature range. We found that (a) with fast skeletal thin filaments on fast skeletal myosin, the temperature dependence was comparable to that obtained for sarcomere shortening in fast skeletal myofibrils (Q10 ∼8), (b) both types of thin filaments started to slide at lower temperatures on fast skeletal myosin than on β-cardiac myosin, and (c) cardiac thin filaments slid at lower temperatures compared with fast skeletal thin filaments on either type of myosin. Therefore, the mammalian striated muscle may be fine-tuned to contract efficiently via complementary regulation of myosin and tropomyosin-troponin within the body temperature range, depending on the physiological demands of various circumstances.

1. Changes in cytosolic [Ca2+] ([Ca2+]i) were measured in isolated rat trabeculae that had been micro-injected with fura-2 salt, in order to investigate the mechanism by which twitch force changes following an alteration of muscle length. 2. A step increase in length of the muscle produced a rapid potentiation of twitch force but not of the Ca2+ transient. The rapid rise of force was unaffected by inhibiting the sarcoplasmic reticulum (SR) with ryanodine and cyclopiazonic acid. 3. The force-[Ca2+]i relationship of the myofibrils in situ, determined from twitches and tetanic contractions in SR-inhibited muscles, showed that the rapid rise of force was due primarily to an increase in myofibrillar Ca2+ sensitivity, with a contribution from an increase in the maximum force production of the myofibrils. 4. After stretch of the muscle there was a further, slow increase of twitch force which was due entirely to a slow increase of the Ca2+ transient, since there was no change in the myofibrillar force-[Ca2+]i relationship. SR inhibition slowed down, but did not alter the magnitude of, the slow force response. 5. During the slow rise of force there was no slow increase of diastolic [Ca2+]i, whether or not the SR was inhibited. The same was true in unstimulated muscles. 6. We conclude that the rapid increase in twitch force after muscle stretch is due to the length-dependent properties of the myofibrils. The slow force increase is not explained by length dependence of the myofibrils or the SR, or by a rise in diastolic [Ca2+]i. Evidence from tetani suggests the slow force responses result from increased Ca2+ loading of the cell during the action potential.

The Xin actin-binding repeat-containing proteins Xin and XIRP2 are exclusively expressed in striated muscle cells, where they are believed to play an important role in development. In adult muscle, both proteins are concentrated at attachment sites of myofibrils to the membrane. In contrast, during development they are localized to immature myofibrils together with their binding partner, filamin C, indicating an involvement of both proteins in myofibril assembly. We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich regions of Xin and XIRP2. Precise binding motifs are mapped and shown to bind both SH3 domains with micromolar affinity. Cocrystallization of the nebulette SH3 domain with the interacting XIRP2 peptide PPPTLPKPKLPKH reveals selective interactions that conform to class II SH3 domain-binding peptides. Bimolecular fluorescence complementation experiments in cultured muscle cells indicate a temporally restricted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril development that is lost upon further maturation. In mature myofibrils, this interaction is limited to longitudinally oriented structures associated with myofibril development and remodeling. These data provide new insights into the role of Xin actin-binding repeat-containing proteins (together with their interaction partners) in myofibril assembly and after muscle damage.

During heartbeat, the repeated contractions of myocardium are induced by the oscillation of intracellular Ca(2+) concentration. On the other hand, when intermediately activated at a certain Ca(2+) concentration, cardiac myofibrils exhibit the spontaneous sarcomeric oscillation (Ca-SPOC) under steady ionic conditions. In the present study, we found that Ca-SPOC occurred over a wide range of Ca(2+) concentrations, including physiological contractile conditions, in skinned myocardium prepared from various animal species (rat, rabbit, pig, and cow). The period of sarcomeric oscillation fell within the same range as the period of heartbeat of each animal species. On the basis of these results we propose that the intrinsic auto-oscillatory property of sarcomeres (myofibrils) significantly contributes to myocardial beating in vivo.

Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. More efficient myofiber bundling accelerates the speed of sarcomerogenesis suggesting that tension generated by bundling promotes sarcomerogenesis. We tested this hypothesis by directly probing tension and found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.

Using deconvolved confocal microscopy of fluorescently labeled markers for z-disks, t-tubules and ryanodine receptors, we have examined sarcomere organization in cardiac myocytes from rat, rabbit and human. We show that sarcomeres exhibit dislocations in registration and occasionally more complex helicoidal topology. This organization was present at both slack ( approximately 1.8 microm) and long sarcomere lengths ( approximately 2.2 microm). Misregistrations in z-disks persisted over 15-20 sarcomere lengths and appeared to arise primarily from variations in fiber direction; particularly as myofibrils pass around nuclei. In addition, myofibrils twist along the cell length. T-tubules generally follow the sarcomere z-disks although additional elements bridging adjacent myofibrils and along the length of the myofibril are present to varying degrees in all cells. Ryanodine receptors (the sarcoplasmic reticulum Ca(2+) release channel) are generally located within 250 nm of the local plane containing t-tubules and z-disks, but a small fraction ( approximately 2%) is found on longitudinal elements of the t-system between z-disks. The results are discussed with respect to the possible role(s) of such complex z-disk organization and z-disk dislocations in the maintenance of cell structure and sarcomere assembly. In addition, the non-planar organization of z-disks may be important in the propagation of local Ca(2+) waves which may have a useful role in helping maintain the uniformity of sarcomere activation in the presence of t-tubule remodeling.

Sarcomeres are the basic contractile units within cardiac myocytes, and the collective shortening of sarcomeres aligned along myofibrils generates the force driving the heartbeat. The alignment of the individual sarcomeres is important for proper force generation, and misaligned sarcomeres are associated with diseases including cardiomyopathies and COVID-19. The actin bundling protein, α-actinin-2, localizes to the "Z-Bodies" of sarcomere precursors and the "Z-Lines" of sarcomeres, and has been used previously to assess sarcomere assembly and maintenance. Previous measurements of α-actinin-2 organization have been largely accomplished manually, which is time-consuming and has hampered research progress. Here, we introduce sarcApp, an image analysis tool that quantifies several components of the cardiac sarcomere and their alignment in muscle cells and tissue. We first developed sarcApp to utilize deep learning-based segmentation and real space quantification to measure α-actinin-2 structures and determine the organization of both precursors and sarcomeres/myofibrils. We then expanded sarcApp to analyze "M-Lines" using the localization of myomesin and a protein that connects the Z-Lines to the M-Line (titin). sarcApp produces 33 distinct measurements per cell and 24 per myofibril that allow for precise quantification of changes in sarcomeres, myofibrils, and their precursors. We validated this system with perturbations to sarcomere assembly. Surprisingly, we found perturbations that affected Z-Lines and M-Lines differently, suggesting that they may be regulated independently during sarcomere assembly.

Monospecific antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a BALB/c mouse immunized with rat skeletal myofibrils. After cloning 3 times on agarose, two stable clones were obtained and chosen for further characterization. The first clone, JLB1, produced an antibody that recognizing an antigen distributed in the M-line region and on either site of the Z line of myofibrils. The second clone, JLB7, produced an antibody reacting only with an antigen located at the M-line region of myofibrils. Both JLB1 and JLB7 antibodies decorate the typical intermediate filaments of a variety of cultured cells. Colcemid treatment of cells before reaction with both antibodies resulted in the coiling or capping (or both) of the fibers around the nucleus. Brief treatment of cells with cytochalasin B did not affect the integrity of the fibers stained by both antibodies whereas, under the same conditions, microfilament bundles visualized by another monoclonal antibody (JLA20) against actin were disassembled into many aggregates in the cytoplasm. Identical staining patterns of the intermediate filaments are obtained by double-label immunofluorescence microscopy of the same cell stained with these monoclonal antibodies and rabbit autoimmune serum (which has been shown to react with the components of the intermediate filaments). By using immunoprecipitation, protein bands at 210,000 and 95,000 daltons from chicken embryo fibroblasts were identified as the potential antigens recognized by JLB1 and JLB7 monoclonal antibodies, respectively. The widespread occurrence of these antigenic determinants in different cultured cells suggests the highly conservative property of these intermediate-filament components.

Sarcomeres are the basic contractile units within cardiac myocytes, and the collective shortening of sarcomeres aligned along myofibrils generates the force driving the heartbeat. The alignment of the individual sarcomeres is important for proper force generation, and misaligned sarcomeres are associated with diseases, including cardiomyopathies and COVID-19. The actin bundling protein, α-actinin-2, localizes to the 'Z-Bodies" of sarcomere precursors and the 'Z-Lines' of sarcomeres, and has been used previously to assess sarcomere assembly and maintenance. Previous measurements of α-actinin-2 organization have been largely accomplished manually, which is time-consuming and has hampered research progress. Here, we introduce sarcApp, an image analysis tool that quantifies several components of the cardiac sarcomere and their alignment in muscle cells and tissue. We first developed sarcApp to utilize deep learning-based segmentation and real space quantification to measure α-actinin-2 structures and determine the organization of both precursors and sarcomeres/myofibrils. We then expanded sarcApp to analyze 'M-Lines' using the localization of myomesin and a protein that connects the Z-Lines to the M-Line (titin). sarcApp produces 33 distinct measurements per cell and 24 per myofibril that allow for precise quantification of changes in sarcomeres, myofibrils, and their precursors. We validated this system with perturbations to sarcomere assembly. We found perturbations that affected Z-Lines and M-Lines differently, suggesting that they may be regulated independently during sarcomere assembly.

Nuclear movements are important for multiple cellular functions, and are driven by polarized forces generated by motor proteins and the cytoskeleton. During skeletal myofibre formation or regeneration, nuclei move from the centre to the periphery of the myofibre for proper muscle function. Centrally located nuclei are also found in different muscle disorders. Using theoretical and experimental approaches, we demonstrate that nuclear movement to the periphery of myofibres is mediated by centripetal forces around the nucleus. These forces arise from myofibril contraction and crosslinking that 'zip' around the nucleus in combination with tight regulation of nuclear stiffness by lamin A/C. In addition, an Arp2/3 complex containing Arpc5L together with γ-actin is required to organize desmin to crosslink myofibrils for nuclear movement. Our work reveals that centripetal forces exerted by myofibrils squeeze the nucleus to the periphery of myofibres.

The formation of contractile myofibrils requires the stepwise onset of expression of muscle specific proteins. It is likely that elucidation of the expression patterns of muscle-specific sarcomeric proteins is important to understand muscle disorders originating from defects in contractile sarcomeric proteins.

High resolution information about the three-dimensional (3D) structure of myosin filaments has always been hard to obtain. Solving the 3D structure of myosin filaments is very important because mutations in human cardiac muscle myosin and its associated proteins (e.g. titin and myosin binding protein C) are known to be associated with a number of familial human cardiomyopathies (e.g. hypertrophic cardiomyopathy and dilated cardiomyopathy). In order to understand how normal heart muscle works and how it fails, as well as the effects of the known mutations on muscle contractility, it is essential to properly understand myosin filament 3D structure and properties in both healthy and diseased hearts. The aim of this review is firstly to provide a general overview of the 3D structure of myosin thick filaments, as studied so far in both vertebrates and invertebrate striated muscles. Knowledge of this 3D structure is the starting point from which myosin filaments isolated from human cardiomyopathic samples, with known mutations in either myosin or its associated proteins (titin or C-protein), can be studied in detail. This should, in turn, enable us to relate the structure of myosin thick filament to its function and to understanding the disease process. A long term objective of this research would be to assist the design of possible therapeutic solutions to genetic myosin-related human cardiomyopathies.

Myofibrillogenesis is an essential process for cardiogenesis and is closely related to excitation-contraction coupling and the maintenance of heartbeat. It remains unclear whether the formation of myofibrils and sarcomeres is associated with heartbeat initiation in the early embryonic heart development. Here, we investigated the association between the ultrastructure of myofibrils assessed by transmission electron microscopy and their proteomic profiling assessed by data-independent acquisition mass spectrometry (DIA-MS) in the rat heart primordia before and after heartbeat initiation at embryonic day 10.0, when heartbeat begins in rats, and in the primitive heart tube at embryonic day 11.0. Bundles of myofilaments were scattered in a few cells of the heart primordium after heartbeat initiation, whereas there were no typical sarcomeres in the heart primordia both before and after heartbeat initiation. Sarcomeres with Z-lines were identified in cells of the primitive heart tube, though myofilaments were not aligned. DIA-MS proteome analysis revealed that only 43 proteins were significantly upregulated by more than 2.0 fold among a total of 7,762 detected proteins in the heart primordium after heartbeat initiation compared with that before heartbeat initiation. Indeed, of those upregulated proteins, 12 (27.9%) were constituent proteins of myofibrils and 10 (23.3%) were proteins that were accessories and regulators for myofibrillogenesis, suggesting that upregulated proteins that are associated with heartbeat initiation were enriched in myofibrillogenesis. Collectively, our results suggest that the establishment of heartbeat is induced by development of bundles of myofilaments with upregulated proteins associated with myofibrillogensis, whereas sarcomeres are not required for the initial heartbeat.

Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.

Formation of oriented myofibrils is a key event in musculoskeletal development. However, the mechanisms that drive myocyte orientation and fusion to control muscle directionality in adults remain enigmatic. Here, we demonstrate that the developing skeleton instructs the directional outgrowth of skeletal muscle and other soft tissues during limb and facial morphogenesis in zebrafish and mouse. Time-lapse live imaging reveals that during early craniofacial development, myoblasts condense into round clusters corresponding to future muscle groups. These clusters undergo oriented stretch and alignment during embryonic growth. Genetic perturbation of cartilage patterning or size disrupts the directionality and number of myofibrils in vivo. Laser ablation of musculoskeletal attachment points reveals tension imposed by cartilage expansion on the forming myofibers. Application of continuous tension using artificial attachment points, or stretchable membrane substrates, is sufficient to drive polarization of myocyte populations in vitro. Overall, this work outlines a biomechanical guidance mechanism that is potentially useful for engineering functional skeletal muscle.

Heart development requires organized integration of actin filaments into the sarcomere, the contractile unit of myofibrils, although it remains largely unknown how actin filaments are assembled during myofibrillogenesis. Here we show that Fhod3, a member of the formin family of proteins that play pivotal roles in actin filament assembly, is essential for myofibrillogenesis at an early stage of heart development. Fhod3(-/-) mice appear normal up to embryonic day (E) 8.5, when the developing heart, composed of premyofibrils, initiates spontaneous contraction. However, these premyofibrils fail to mature and myocardial development does not continue, leading to embryonic lethality by E11.5. Transgenic expression of wild-type Fhod3 in the heart restores myofibril maturation and cardiomyogenesis, which allow Fhod3(-/-) embryos to develop further. Moreover, cardiomyopathic changes with immature myofibrils are caused in mice overexpressing a mutant Fhod3, defective in binding to actin. These findings indicate that actin dynamics, regulated by Fhod3, participate in sarcomere organization during myofibrillogenesis and thus play a crucial role in heart development.