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Life Science Alliance Apr 2022Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox...
Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type-specific gene and splice isoform expression, notably loss of an indirect flight muscle-specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. We further show that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins.
Topics: Animals; Drosophila; Drosophila Proteins; Female; Gene Knockdown Techniques; Male; Myofibrils; Protein Isoforms; RNA-Binding Proteins
PubMed: 34996845
DOI: 10.26508/lsa.202101342 -
Journal of Cell Science Oct 2022Myofibrils are the intracellular structures formed by actin and myosin filaments. They are paracrystalline contractile cables with unusually well-defined dimensions. The...
Myofibrils are the intracellular structures formed by actin and myosin filaments. They are paracrystalline contractile cables with unusually well-defined dimensions. The sliding of actin past myosin filaments powers contractions, and the entire system is held in place by a structure called the Z-disc, which anchors the actin filaments. Myosin filaments, in turn, are anchored to another structure called the M-line. Most of the complex architecture of myofibrils can be reduced to studying the Z-disc, and recently, important advances regarding the arrangement and function of Z-discs in insects have been published. On a very small scale, we have detailed protein structure information. At the medium scale, we have cryo-electron microscopy maps, super-resolution microscopy and protein-protein interaction networks, while at the functional scale, phenotypic data are available from precise genetic manipulations. All these data aim to answer how the Z-disc works and how it is assembled. Here, we summarize recent data from insects and explore how it fits into our view of the Z-disc, myofibrils and, ultimately, muscles.
Topics: Actins; Animals; Biology; Cryoelectron Microscopy; Insecta; Myofibrils; Myosins; Sarcomeres
PubMed: 36226637
DOI: 10.1242/jcs.260179 -
Cells Oct 2021Life-threatening ventricular arrhythmias are the main clinical burden in patients with hypertrophic cardiomyopathy (HCM), and frequently occur in young patients with... (Review)
Review
Life-threatening ventricular arrhythmias are the main clinical burden in patients with hypertrophic cardiomyopathy (HCM), and frequently occur in young patients with mild structural disease. While massive hypertrophy, fibrosis and microvascular ischemia are the main mechanisms underlying sustained reentry-based ventricular arrhythmias in advanced HCM, cardiomyocyte-based functional arrhythmogenic mechanisms are likely prevalent at earlier stages of the disease. In this review, we will describe studies conducted in human surgical samples from HCM patients, transgenic animal models and human cultured cell lines derived from induced pluripotent stem cells. Current pieces of evidence concur to attribute the increased risk of ventricular arrhythmias in early HCM to different cellular mechanisms. The increase of late sodium current and L-type calcium current is an early observation in HCM, which follows post-translation channel modifications and increases the occurrence of early and delayed afterdepolarizations. Increased myofilament Ca sensitivity, commonly observed in HCM, may promote afterdepolarizations and reentry arrhythmias with direct mechanisms. Decrease of K-currents due to transcriptional regulation occurs in the advanced disease and contributes to reducing the repolarization-reserve and increasing the early afterdepolarizations (EADs). The presented evidence supports the idea that patients with early-stage HCM should be considered and managed as subjects with an acquired channelopathy rather than with a structural cardiac disease.
Topics: Animals; Arrhythmias, Cardiac; Calcium; Cardiomyopathy, Hypertrophic; Humans; Ion Channels; Myocytes, Cardiac; Myofibrils
PubMed: 34685769
DOI: 10.3390/cells10102789 -
The Journal of General Physiology Jul 2021Stronczek et al. investigate the structure of titin–N2A and how it affects the binding of signaling proteins.
Stronczek et al. investigate the structure of titin–N2A and how it affects the binding of signaling proteins.
Topics: Connectin; Muscle Proteins; Sarcomeres
PubMed: 34081095
DOI: 10.1085/jgp.202112904 -
Annals of Anatomy = Anatomischer... Aug 2023The force a muscle exerts is partly determined by anatomical parameters, such as its physiological cross-section. The temporal muscle is structurally heterogeneous. To...
BACKGROUND
The force a muscle exerts is partly determined by anatomical parameters, such as its physiological cross-section. The temporal muscle is structurally heterogeneous. To the authors' knowledge, the ultrastructure of this muscle has been poorly specifically studied.
METHODS
Five adult Wistar rats weighting 350-400 g were used as temporal muscle donors. Tissues were specifically processed and studied under transmission electron microscope.
RESULTS
On ultrathin cuts, the general ultrastructural pattern of striated muscles was observed. Moreover, pennate sarcomeres were identified, sharing a one-end insertion on the same Z-disc. Bipennate morphologies resulted when two neighbor sarcomeres, attached on different neighbor Z-discs and separated at that end by a triad, converged to the same Z-disc at the opposite ends, thus building a thicker myofibril distinctively flanked by triads. Tripennate morphologies were identified when sarcomeres from three different Z-discs converged to the same Z-disc at the opposite ends.
CONCLUSIONS
These results support recent evidence of sarcomeres branching gathered in mice. Adequate identification of the sites of excitation-contraction coupling should be on both sides of a myofibril, on bidimensional ultrathin cuts, to avoid false positive results due to putative longitudinal folds of myofibrils.
Topics: Animals; Rats; Mice; Myofibrils; Temporal Muscle; Rats, Wistar; Sarcomeres; Muscle, Skeletal; Muscle Contraction
PubMed: 37011827
DOI: 10.1016/j.aanat.2023.152096 -
BMC Developmental Biology Aug 2021Flying is an essential function for mosquitoes, required for mating and, in the case of females, to get a blood meal and consequently function as a vector. Flight...
BACKGROUND
Flying is an essential function for mosquitoes, required for mating and, in the case of females, to get a blood meal and consequently function as a vector. Flight depends on the action of the indirect flight muscles (IFMs), which power the wings beat. No description of the development of IFMs in mosquitoes, including Aedes aegypti, is available.
METHODS
A. aegypti thoraces of larvae 3 and larvae 4 (L3 and L4) instars were analyzed using histochemistry and bright field microscopy. IFM primordia from L3 and L4 and IFMs from pupal and adult stages were dissected and processed to detect F-actin labelling with phalloidin-rhodamine or TRITC, or to immunodetection of myosin and tubulin using specific antibodies, these samples were analyzed by confocal microscopy. Other samples were studied using transmission electron microscopy.
RESULTS
At L3-L4, IFM primordia for dorsal-longitudinal muscles (DLM) and dorsal-ventral muscles (DVM) were identified in the expected locations in the thoracic region: three primordia per hemithorax corresponding to DLM with anterior to posterior orientation were present. Other three primordia per hemithorax, corresponding to DVM, had lateral position and dorsal to ventral orientation. During L3 to L4 myoblast fusion led to syncytial myotubes formation, followed by myotendon junctions (MTJ) creation, myofibrils assembly and sarcomere maturation. The formation of Z-discs and M-line during sarcomere maturation was observed in pupal stage and, the structure reached in teneral insects a classical myosin thick, and actin thin filaments arranged in a hexagonal lattice structure.
CONCLUSIONS
A general description of A. aegypti IFM development is presented, from the myoblast fusion at L3 to form myotubes, to sarcomere maturation at adult stage. Several differences during IFM development were observed between A. aegypti (Nematoceran) and Drosophila melanogaster (Brachyceran) and, similitudes with Chironomus sp. were observed as this insect is a Nematoceran, which is taxonomically closer to A. aegypti and share the same number of larval stages.
Topics: Aedes; Animals; Arboviruses; Drosophila melanogaster; Mosquito Vectors; Sarcomeres
PubMed: 34445959
DOI: 10.1186/s12861-021-00242-8 -
Cell and Tissue Research Jul 2023In vertebrate skeletal muscles, the architecture of myofibrils is particularly well conserved throughout the taxa. It is composed of suites of repeating functional units...
In vertebrate skeletal muscles, the architecture of myofibrils is particularly well conserved throughout the taxa. It is composed of suites of repeating functional units called sarcomeres which give the muscle its striated structure. Here, we show that the skeletal sound producing muscles of the cusk eel Parophidion vassali have a different organisation, distinct from the classical type found in textbooks. Within sarcomeres, filaments are not straight lines but have a Y-shaped structure. This looks like chicken wire, with one branch connecting to a branch from the myofibril above and the other connecting to a branch from the myofibril below. This organisation seems to be an adaptation to counteract a trade-off between the speed and force. The low ratio of myofibrils within cell muscles and the high volume of sarcoplasmic reticulum strongly suggest that these muscles are capable of fast contractions. In parallel, the Z-bands are quite wide about 30% of the sarcomere length. This extraordinary long Z-band could smooth out the tension variations found in high-speed muscle contraction, helping to produce sounds with low variabilities in the sound features. Simultaneously, the Y-shaped structure allows having more cross-bridges, increasing the force in this high-speed muscle.
Topics: Myofibrils; Sarcomeres; Muscle Fibers, Skeletal; Muscle Contraction; Muscle, Skeletal
PubMed: 37129618
DOI: 10.1007/s00441-023-03775-5 -
Circulation Jan 2022Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The...
BACKGROUND
Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain-binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, titin (TTN) functions, and therapeutic targets.
METHODS
We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant-specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Last, we engineered a full-length TTN protein reporter assay and used next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs.
RESULTS
An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome editing using SpCas9 and a TTNtv-specific guide RNA restored the TTN protein reading frame, which increased full-length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays.
CONCLUSIONS
An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. Although both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a one-and-done genome editing strategy to target ≈30% of DCM-associated TTNtvs.
Topics: Cardiomyopathy, Dilated; Connectin; Gene Editing; Genetic Variation; Humans; Induced Pluripotent Stem Cells; Myocytes, Cardiac; Myofibrils; Reading Frames
PubMed: 34905694
DOI: 10.1161/CIRCULATIONAHA.120.049997 -
International Journal of Molecular... Aug 2020Deficits in plasma membrane repair have been identified in dysferlinopathy and Duchenne Muscular Dystrophy, and contribute to progressive myopathy. Although...
Deficits in plasma membrane repair have been identified in dysferlinopathy and Duchenne Muscular Dystrophy, and contribute to progressive myopathy. Although Facioscapulohumeral Muscular Dystrophy (FSHD) shares clinicopathological features with these muscular dystrophies, it is unknown if FSHD is characterized by plasma membrane repair deficits. Therefore, we exposed immortalized human FSHD myoblasts, immortalized myoblasts from unaffected siblings, and myofibers from a murine model of FSHD () to focal, pulsed laser ablation of the sarcolemma. Repair kinetics and success were determined from the accumulation of intracellular FM1-43 dye post-injury. We subsequently treated FSHD myoblasts with a -targeting antisense oligonucleotide (AON) to reduce expression, and with the antioxidant Trolox to determine the role of expression and oxidative stress in membrane repair. Compared to unaffected myoblasts, FSHD myoblasts demonstrate poor repair and a greater percentage of cells that failed to repair, which was mitigated by AON and Trolox treatments. Similar repair deficits were identified in myofibers. This is the first study to identify plasma membrane repair deficits in myoblasts from individuals with FSHD, and in myofibers from a murine model of FSHD. Our results suggest that expression and oxidative stress may be important targets for future membrane-repair therapies.
Topics: Adult; Aged; Animals; Antioxidants; Cell Membrane; Cells, Cultured; Female; Gene Expression Regulation; Homeodomain Proteins; Humans; Male; Mice; Middle Aged; Muscle Fibers, Skeletal; Muscular Dystrophy, Facioscapulohumeral; Myoblasts; Myofibrils; Oligonucleotides, Antisense; Oxidative Stress
PubMed: 32759720
DOI: 10.3390/ijms21155575 -
International Journal of Molecular... Mar 2020Although the presence of cardiac dysfunction and cardiomyopathy in chronic diabetes has been recognized, the pathophysiology of diabetes-induced metabolic and... (Review)
Review
Although the presence of cardiac dysfunction and cardiomyopathy in chronic diabetes has been recognized, the pathophysiology of diabetes-induced metabolic and subcellular changes as well as the therapeutic approaches for the prevention of diabetic cardiomyopathy are not fully understood. Cardiac dysfunction in chronic diabetes has been shown to be associated with Ca-handling abnormalities, increase in the availability of intracellular free Ca and impaired sensitivity of myofibrils to Ca. Metabolic derangements, including depressed high-energy phosphate stores due to insulin deficiency or insulin resistance, as well as hormone imbalance and ultrastructural alterations, are also known to occur in the diabetic heart. It is pointed out that the activation of the sympathetic nervous system and renin-angiotensin system generates oxidative stress, which produces defects in subcellular organelles including sarcolemma, sarcoplasmic reticulum and myofibrils. Such subcellular remodeling plays a critical role in the pathogenesis of diabetic cardiomyopathy. In fact, blockade of the effects of neurohormonal systems has been observed to attenuate oxidative stress and occurrence of subcellular remodeling as well as metabolic abnormalities in the diabetic heart. This review is intended to describe some of the subcellular and metabolic changes that result in cardiac dysfunction in chronic diabetes. In addition, the therapeutic values of some pharmacological, metabolic and antioxidant interventions will be discussed. It is proposed that a combination therapy employing some metabolic agents or antioxidants with insulin may constitute an efficacious approach for the prevention of diabetic cardiomyopathy.
Topics: Animals; Antioxidants; Calcium; Diabetes Complications; Diabetes Mellitus; Diabetic Cardiomyopathies; Heart; Heart Failure; Humans; Insulin; Insulin Resistance; Myofibrils; Oxidative Stress; Renin-Angiotensin System; Sarcolemma; Sarcoplasmic Reticulum; Sympathetic Nervous System
PubMed: 32244448
DOI: 10.3390/ijms21072413