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Proceedings of the National Academy of... Feb 2017A hallmark of muscle atrophy is the excessive degradation of myofibrillar proteins primarily by the ubiquitin proteasome system. In mice, during the rapid muscle atrophy...
A hallmark of muscle atrophy is the excessive degradation of myofibrillar proteins primarily by the ubiquitin proteasome system. In mice, during the rapid muscle atrophy induced by fasting, the desmin cytoskeleton and the attached Z-band-bound thin filaments are degraded after ubiquitination by the ubiquitin ligase tripartite motif-containing protein 32 (Trim32). To study the order of events leading to myofibril destruction, we investigated the slower atrophy induced by denervation (disuse). We show that myofibril breakdown is a two-phase process involving the initial disassembly of desmin filaments by Trim32, which leads to the later myofibril breakdown by enzymes, whose expression is increased by the paired box 4 (PAX4) transcription factor. After denervation of mouse tibialis anterior muscles, phosphorylation and Trim32-dependent ubiquitination of desmin filaments increased rapidly and stimulated their gradual depolymerization (unlike their rapid degradation during fasting). Trim32 down-regulation attenuated the loss of desmin and myofibrillar proteins and reduced atrophy. Although myofibrils and desmin filaments were intact at 7 d after denervation, inducing the dissociation of desmin filaments caused an accumulation of ubiquitinated proteins and rapid destruction of myofibrils. The myofibril breakdown normally observed at 14 d after denervation required not only dissociation of desmin filaments, but also gene induction by PAX4. Down-regulation of PAX4 or its target gene encoding the p97/VCP ATPase reduced myofibril disassembly and degradation on denervation or fasting. Thus, during atrophy, the initial loss of desmin is critical for the subsequent myofibril destruction, and over time, myofibrillar proteins become more susceptible to PAX4-induced enzymes that promote proteolysis.
Topics: Adenosine Triphosphatases; Animals; Cytoskeleton; Desmin; Down-Regulation; Homeodomain Proteins; Male; Mice; Muscle Denervation; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Myofibrils; Nuclear Proteins; Paired Box Transcription Factors; Ubiquitin; Ubiquitination
PubMed: 28096335
DOI: 10.1073/pnas.1612988114 -
Biophysical Journal Feb 2022Myocyte disarray is a hallmark of many cardiac disorders. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to...
Myocyte disarray is a hallmark of many cardiac disorders. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to disease progression has been largely underexplored. This oversight has predominantly been because of a paucity of methods for objective and quantitative analysis. Here, we introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near-physiological conditions and demonstrate its superiority as compared with conventional histological assessments. Using small-angle x-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wild-type mouse myocardium from the left ventricle by assessing the angular spread of the 1,0 equatorial reflection (angle σ). At a sarcomere length of 1.9 μm, the angle σ was 0.23 ± 0.01 rad, decreased to 0.19 ± 0.01 rad at a sarcomere length of 2.1 μm, and further decreased to 0.15 ± 0.01 rad at a sarcomere length of 2.3 μm (p < 0.0001). Angle σ was significantly larger in R403Q, a MYH7 hypertrophic cardiomyopathy model, porcine myocardium (0.24 ± 0.01 rad) compared with wild-type myocardium (0.14 ± 0.005 rad; p < 0.0001), as well as in human heart failure tissue (0.19 ± 0.006 rad) when compared with nonfailing samples (0.17 ± 0.007 rad; p = 0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared with healthy controls. Finally, we showed that conventional, histology-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Our method for directly assessing myofibrillar orientation avoids the artifacts introduced by conventional histological approaches that assess myocyte orientation and only indirectly evaluate myofibrillar orientation, and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent x-ray diffraction patterns from frozen human myocardium provides a new tool for investigating structural anomalies associated with cardiac diseases.
Topics: Animals; Cardiomyopathy, Hypertrophic; Heart Ventricles; Mice; Myocardial Contraction; Myocardium; Myofibrils; Sarcomeres; Swine
PubMed: 35032456
DOI: 10.1016/j.bpj.2022.01.009 -
The Journal of Physiology Jan 2016The contractile properties of human fetal cardiac muscle have not been previously studied. Small-scale approaches such as isolated myofibril and isolated contractile...
KEY POINTS
The contractile properties of human fetal cardiac muscle have not been previously studied. Small-scale approaches such as isolated myofibril and isolated contractile protein biomechanical assays allow study of activation and relaxation kinetics of human fetal cardiac muscle under well-controlled conditions. We have examined the contractile properties of human fetal cardiac myofibrils and myosin across gestational age 59-134 days. Human fetal cardiac myofibrils have low force and slow kinetics of activation and relaxation that increase during the time period studied, and kinetic changes may result from structural maturation and changes in protein isoform expression. Understanding the time course of human fetal cardiac muscle structure and contractile maturation can provide a framework to study development of contractile dysfunction with disease and evaluate the maturation state of cultured stem cell-derived cardiomyocytes.
ABSTRACT
Little is known about the contractile properties of human fetal cardiac muscle during development. Understanding these contractile properties, and how they change throughout development, can provide valuable insight into human heart development, and provide a framework to study the early stages of cardiac diseases that develop in utero. We characterized the contractile properties of isolated human fetal cardiac myofibrils across 8-19 weeks of gestation. Mechanical measurements revealed that in early stages of gestation there is low specific force and slow rates of force development and relaxation, with increases in force and the rates of activation and relaxation as gestation progresses. The duration and slope of the initial, slow phase of relaxation, related to myosin detachment and thin filament deactivation rates, decreased with gestation age. F-actin sliding on human fetal cardiac myosin-coated surfaces slowed significantly from 108 to 130 days of gestation. Electron micrographs showed human fetal muscle myofibrils elongate and widen with age, but features such as the M-line and Z-band are apparent even as early as day 52. Protein isoform analysis revealed that β-myosin is predominantly expressed even at the earliest time point studied, but there is a progressive increase in expression of cardiac troponin I (TnI), with a concurrent decrease in slow skeletal TnI. Together, our results suggest that cardiac myofibril force production and kinetics of activation and relaxation change significantly with gestation age and are influenced by the structural maturation of the sarcomere and changes in contractile filament protein isoforms.
Topics: Actins; Adult; Female; Fetal Heart; Humans; Male; Myocardial Contraction; Myofibrils; Myosins; Troponin I
PubMed: 26460603
DOI: 10.1113/JP271290 -
Quarterly Reviews of Biophysics Jan 2023The cardiac sarcomere is a cellular structure in the heart that enables muscle cells to contract. Dozens of proteins belong to the cardiac sarcomere, which work in... (Review)
Review
The cardiac sarcomere is a cellular structure in the heart that enables muscle cells to contract. Dozens of proteins belong to the cardiac sarcomere, which work in tandem to generate force and adapt to demands on cardiac output. Intriguingly, the majority of these proteins have significant intrinsic disorder that contributes to their functions, yet the biophysics of these intrinsically disordered regions (IDRs) have been characterized in limited detail. In this review, we first enumerate these myofilament-associated proteins with intrinsic disorder (MAPIDs) and recent biophysical studies to characterize their IDRs. We secondly summarize the biophysics governing IDR properties and the state-of-the-art in computational tools toward MAPID identification and characterization of their conformation ensembles. We conclude with an overview of future computational approaches toward broadening the understanding of intrinsic disorder in the cardiac sarcomere.
Topics: Myofibrils; Actin Cytoskeleton; Sarcomeres; Computer Simulation; Molecular Conformation
PubMed: 36628457
DOI: 10.1017/S003358352300001X -
Journal of Biomechanical Engineering Feb 2022Through a variety of mechanisms, a healthy heart is able to regulate its structure and dynamics across multiple length scales. Disruption of these mechanisms can have a...
Through a variety of mechanisms, a healthy heart is able to regulate its structure and dynamics across multiple length scales. Disruption of these mechanisms can have a cascading effect, resulting in severe structural and/or functional changes that permeate across different length scales. Due to this hierarchical structure, there is interest in understanding how the components at the various scales coordinate and influence each other. However, much is unknown regarding how myofibril bundles are organized within a densely packed cell and the influence of the subcellular components on the architecture that is formed. To elucidate potential factors influencing cytoskeletal development, we proposed a computational model that integrated interactions at both the cellular and subcellular scale to predict the location of individual myofibril bundles that contributed to the formation of an energetically favorable cytoskeletal network. Our model was tested and validated using experimental metrics derived from analyzing single-cell cardiomyocytes. We demonstrated that our model-generated networks were capable of reproducing the variation observed in experimental cells at different length scales as a result of the stochasticity inherent in the different interactions between the various cellular components. Additionally, we showed that incorporating length-scale parameters resulted in physical constraints that directed cytoskeletal architecture toward a structurally consistent motif. Understanding the mechanisms guiding the formation and organization of the cytoskeleton in individual cardiomyocytes can aid tissue engineers toward developing functional cardiac tissue.
Topics: Cytoskeleton; Microtubules; Myocytes, Cardiac; Myofibrils; Tissue Engineering
PubMed: 34382649
DOI: 10.1115/1.4052112 -
Journal of Molecular and Cellular... Feb 2020Cardiac troponin I (cTnI) is an essential physiological and pathological regulator of cardiac relaxation. Significant to this regulation, the post-translational...
OBJECTIVE
Cardiac troponin I (cTnI) is an essential physiological and pathological regulator of cardiac relaxation. Significant to this regulation, the post-translational modification of cTnI through phosphorylation functions as a key mechanism to accelerate myofibril relaxation. Similar to phosphorylation, post-translational modification by acetylation alters amino acid charge and protein function. Recent studies have demonstrated that the acetylation of cardiac myofibril proteins accelerates relaxation and that cTnI is acetylated in the heart. These findings highlight the potential significance of myofilament acetylation; however, it is not known if site-specific acetylation of cTnI can lead to changes in myofilament, myofibril, and/or cellular mechanics. The objective of this study was to determine the effects of mimicking acetylation at a single site of cTnI (lysine-132; K132) on myofilament, myofibril, and cellular mechanics and elucidate its influence on molecular function.
METHODS
To determine if pseudo-acetylation of cTnI at 132 modulates thin filament regulation of the acto-myosin interaction, we reconstituted thin filaments containing WT or K132Q (to mimic acetylation) cTnI and assessed in vitro motility. To test if mimicking acetylation at K132 alters cellular relaxation, adult rat ventricular cardiomyocytes were infected with adenoviral constructs expressing either cTnI K132Q or K132 replaced with arginine (K132R; to prevent acetylation) and cell shortening and isolated myofibril mechanics were measured. Finally, to confirm that changes in cell shortening and myofibril mechanics were directly due to pseudo-acetylation of cTnI at K132, we exchanged troponin containing WT or K132Q cTnI into isolated myofibrils and measured myofibril mechanical properties.
RESULTS
Reconstituted thin filaments containing K132Q cTnI exhibited decreased calcium sensitivity compared to thin filaments reconstituted with WT cTnI. Cardiomyocytes expressing K132Q cTnI had faster relengthening and myofibrils isolated from these cells had faster relaxation along with decreased calcium sensitivity compared to cardiomyocytes expressing WT or K132R cTnI. Myofibrils exchanged with K132Q cTnI ex vivo demonstrated faster relaxation and decreased calcium sensitivity.
CONCLUSIONS
Our results indicate for the first time that mimicking acetylation of a specific cTnI lysine accelerates myofilament, myofibril, and myocyte relaxation. This work underscores the importance of understanding how acetylation of specific sarcomeric proteins affects cardiac homeostasis and disease and suggests that modulation of myofilament lysine acetylation may represent a novel therapeutic target to alter cardiac relaxation.
Topics: Acetylation; Animals; Calcium; Female; Heart Ventricles; Lysine; Myocardium; Myocytes, Cardiac; Myofibrils; Rats, Inbred Dahl; Rats, Sprague-Dawley; Troponin I
PubMed: 31981571
DOI: 10.1016/j.yjmcc.2020.01.007 -
Proceedings of the National Academy of... Jun 2018Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac...
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.
Topics: Animals; DNA Helicases; Female; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Genes, Lethal; Heart; Heart Defects, Congenital; Male; Mi-2 Nucleosome Remodeling and Deacetylase Complex; Mice; Muscle Proteins; Myocytes, Cardiac; Myofibrils; Nucleosomes; Sarcomeres; Transcription, Genetic; Ultrasonography, Prenatal
PubMed: 29891665
DOI: 10.1073/pnas.1722219115 -
PLoS Genetics 2013The Drosophila Alp/Enigma family protein Zasp52 localizes to myotendinous junctions and Z-discs. It is required for terminal muscle differentiation and muscle...
The Drosophila Alp/Enigma family protein Zasp52 localizes to myotendinous junctions and Z-discs. It is required for terminal muscle differentiation and muscle attachment. Its vertebrate ortholog ZASP/Cypher also localizes to Z-discs, interacts with α-actinin through its PDZ domain, and is involved in Z-disc maintenance. Human mutations in ZASP cause myopathies and cardiomyopathies. Here we show that Drosophila Zasp52 is one of the earliest markers of Z-disc assembly, and we use a Zasp52-GFP fusion to document myofibril assembly by live imaging. We demonstrate that Zasp52 is required for adult Z-disc stability and pupal myofibril assembly. In addition, we show that two closely related proteins, Zasp66 and the newly identified Zasp67, are also required for adult Z-disc stability and are participating with Zasp52 in Z-disc assembly resulting in more severe, synergistic myofibril defects in double mutants. Zasp52 and Zasp66 directly bind to α-actinin, and they can also form a ternary complex. Our results indicate that Alp/Enigma family members cooperate in Z-disc assembly and myofibril formation; and we propose, based on sequence analysis, a novel class of PDZ domain likely involved in α-actinin binding.
Topics: Actinin; Animals; Carrier Proteins; Cell Differentiation; Drosophila Proteins; Drosophila melanogaster; LIM Domain Proteins; Muscle Proteins; Muscles; Myofibrils; PDZ Domains; Protein Binding; Sarcomeres
PubMed: 23505387
DOI: 10.1371/journal.pgen.1003342 -
Development (Cambridge, England) Mar 2023Proper muscle contraction requires the assembly and maintenance of sarcomeres and myofibrils. Although the protein components of myofibrils are generally known, less is...
Proper muscle contraction requires the assembly and maintenance of sarcomeres and myofibrils. Although the protein components of myofibrils are generally known, less is known about the mechanisms by which they individually function and together synergize for myofibril assembly and maintenance. For example, it is unclear how the disruption of actin filament (F-actin) regulatory proteins leads to the muscle weakness observed in myopathies. Here, we show that knockdown of Drosophila Tropomodulin (Tmod), results in several myopathy-related phenotypes, including reduction of muscle cell (myofiber) size, increased sarcomere length, disorganization and misorientation of myofibrils, ectopic F-actin accumulation, loss of tension-mediating proteins at the myotendinous junction, and misshaped and internalized nuclei. Our findings support and extend the tension-driven self-organizing myofibrillogenesis model. We show that, like its mammalian counterpart, Drosophila Tmod caps F-actin pointed-ends, and we propose that this activity is crucial for cellular processes in different locations within the myofiber that directly and indirectly contribute to the maintenance of muscle function. Our findings provide significant insights to the role of Tmod in muscle development, maintenance and disease.
Topics: Animals; Actins; Tropomodulin; Microfilament Proteins; Drosophila; Myofibrils; Actin Cytoskeleton; Sarcomeres; Mammals
PubMed: 36806912
DOI: 10.1242/dev.201194 -
ELife Aug 2022Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic...
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.
Topics: Humans; Induced Pluripotent Stem Cells; Muscle Development; Muscle Fibers, Skeletal; Myofibrils; Sarcomeres
PubMed: 35920628
DOI: 10.7554/eLife.76649