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PloS One 2024The sarcomere is the fundamental contractile unit in skeletal muscle, and the regularity of its structure is critical for function. Emerging data demonstrates that...
The sarcomere is the fundamental contractile unit in skeletal muscle, and the regularity of its structure is critical for function. Emerging data demonstrates that nanoscale changes to the regularity of sarcomere structure can affect the overall function of the protein dense ~2μm sarcomere. Further, sarcomere structure is implicated in many clinical conditions of muscle weakness. However, our understanding of how sarcomere structure changes in disease, especially at the nanoscale, has been limited in part due to the inability to robustly detect and measure at sub-sarcomere resolution. We optimized several methodological steps and developed a robust pipeline to analyze sarcomere structure using structured illumination super-resolution microscopy in conjunction with commercially-available and fluorescently-conjugated Variable Heavy-Chain only fragment secondary antibodies (nanobodies), and achieved a significant increase in resolution of z-disc width (353nm vs. 62nm) compared to confocal microscopy. The combination of these methods provides a unique approach to probe sarcomere protein localization at the nanoscale and may prove advantageous for analysis of other cellular structures.
Topics: Sarcomeres; Single-Domain Antibodies; Animals; Microscopy, Fluorescence; Mice; Microscopy, Confocal
PubMed: 38687705
DOI: 10.1371/journal.pone.0300348 -
PLoS Biology Apr 2024Muscles undergo developmental transitions in gene expression and alternative splicing that are necessary to refine sarcomere structure and contractility. CUG-BP and...
Muscles undergo developmental transitions in gene expression and alternative splicing that are necessary to refine sarcomere structure and contractility. CUG-BP and ETR-3-like (CELF) family RNA-binding proteins are important regulators of RNA processing during myogenesis that are misregulated in diseases such as Myotonic Dystrophy Type I (DM1). Here, we report a conserved function for Bruno 1 (Bru1, Arrest), a CELF1/2 family homolog in Drosophila, during early muscle myogenesis. Loss of Bru1 in flight muscles results in disorganization of the actin cytoskeleton leading to aberrant myofiber compaction and defects in pre-myofibril formation. Temporally restricted rescue and RNAi knockdown demonstrate that early cytoskeletal defects interfere with subsequent steps in sarcomere growth and maturation. Early defects are distinct from a later requirement for bru1 to regulate sarcomere assembly dynamics during myofiber maturation. We identify an imbalance in growth in sarcomere length and width during later stages of development as the mechanism driving abnormal radial growth, myofibril fusion, and the formation of hollow myofibrils in bru1 mutant muscle. Molecularly, we characterize a genome-wide transition from immature to mature sarcomere gene isoform expression in flight muscle development that is blocked in bru1 mutants. We further demonstrate that temporally restricted Bru1 rescue can partially alleviate hypercontraction in late pupal and adult stages, but it cannot restore myofiber function or correct structural deficits. Our results reveal the conserved nature of CELF function in regulating cytoskeletal dynamics in muscle development and demonstrate that defective RNA processing due to misexpression of CELF proteins causes wide-reaching structural defects and progressive malfunction of affected muscles that cannot be rescued by late-stage gene replacement.
Topics: Animals; Alternative Splicing; Cytoskeleton; Drosophila melanogaster; Drosophila Proteins; Flight, Animal; Gene Expression Regulation, Developmental; Muscle Development; Muscles; Myofibrils; RNA Splicing; RNA-Binding Proteins; Sarcomeres
PubMed: 38683844
DOI: 10.1371/journal.pbio.3002575 -
International Journal of Molecular... Apr 2024This review deals with the developmental origins of extraocular, jaw and laryngeal muscles, the expression, regulation and functional significance of sarcomeric myosin... (Review)
Review
This review deals with the developmental origins of extraocular, jaw and laryngeal muscles, the expression, regulation and functional significance of sarcomeric myosin heavy chains (MyHCs) that they express and changes in MyHC expression during phylogeny. Myogenic progenitors from the mesoderm in the prechordal plate and branchial arches specify craniofacial muscle allotypes with different repertoires for MyHC expression. To cope with very complex eye movements, extraocular muscles (EOMs) express 11 MyHCs, ranging from the superfast extraocular MyHC to the slowest, non-muscle MyHC IIB (nmMyH IIB). They have distinct global and orbital layers, singly- and multiply-innervated fibres, longitudinal MyHC variations, and palisade endings that mediate axon reflexes. Jaw-closing muscles express the high-force masticatory MyHC and cardiac or limb MyHCs depending on the appropriateness for the acquisition and mastication of food. Laryngeal muscles express extraocular and limb muscle MyHCs but shift toward expressing slower MyHCs in large animals. During postnatal development, MyHC expression of craniofacial muscles is subject to neural and hormonal modulation. The primary and secondary myotubes of developing EOMs are postulated to induce, via different retrogradely transported neurotrophins, the rich diversity of neural impulse patterns that regulate the specific MyHCs that they express. Thyroid hormone shifts MyHC 2A toward 2B in jaw muscles, laryngeal muscles and possibly extraocular muscles. This review highlights the fact that the pattern of myosin expression in mammalian craniofacial muscles is principally influenced by the complex interplay of cell lineages, neural impulse patterns, thyroid and other hormones, functional demands and body mass. In these respects, craniofacial muscles are similar to limb muscles, but they differ radically in the types of cell lineage and the nature of their functional demands.
Topics: Animals; Humans; Facial Muscles; Gene Expression Regulation, Developmental; Muscle Development; Myosin Heavy Chains; Oculomotor Muscles; Phylogeny
PubMed: 38674131
DOI: 10.3390/ijms25084546 -
Veterinary Sciences Apr 2024Quadriceps contracture is a condition where the muscle-tendon unit is abnormally shortened. The treatment prognosis is guarded to poor depending on the progress of the...
Quadriceps contracture is a condition where the muscle-tendon unit is abnormally shortened. The treatment prognosis is guarded to poor depending on the progress of the disease. To improve the prognosis, we investigated the effectiveness of therapeutic ultrasound and NMES in treating quadriceps contracture in an immobilized rat model. Thirty-six Wistar rats were randomized into control, immobilization alone, immobilization and spontaneous recovery, immobilization and therapeutic ultrasound, immobilization and NMES, and immobilization and therapeutic ultrasound and NMES combination groups. The continuous therapeutic ultrasound (frequency, 3 MHz, intensity 1 W/cm) and NMES (TENS mode, frequency 50 Hz; intensity 5.0 ± 0.8 mA) were performed on the quadriceps muscle. On Day 15, immobilization-induced quadriceps contracture resulted in a decreased ROM of the stifle joint, reduction in the sarcomere length, muscle atrophy, and muscle fibrosis. On Day 43, therapeutic ultrasound, NMES, and combining both methods improved muscle atrophy and shortening and decreased collagen type I and III and α-SMA protein. The combination of therapeutic ultrasound and NMES significantly reduced the mRNA expression of , , and and increased . Therefore, the combination of therapeutic ultrasound and NMES is the most potent rehabilitation program for treating quadriceps contracture.
PubMed: 38668425
DOI: 10.3390/vetsci11040158 -
Journal of Cardiovascular Development... Mar 2024Any difference in biomarkers between genotype-positive individuals with overt hypertrophic cardiomyopathy (HCM), and genotype-positive but phenotype-negative...
Any difference in biomarkers between genotype-positive individuals with overt hypertrophic cardiomyopathy (HCM), and genotype-positive but phenotype-negative individuals (G+P-) in HCM-associated pathways might shed light on pathophysiological mechanisms. We studied this in young HCM patients. 29 HCM patients, 17 G+P--individuals, and age- and sex-matched controls were prospectively included. We analyzed 184 cardiovascular disease-associated proteins by two proximity extension assays, categorized into biological pathways, and analyzed with multivariate logistic regression analysis. Significant proteins were dichotomized into groups above/below median concentration in control group. Dichotomized values of significant proteins showed high odds ratio (OR) in overt HCMphenotype for Fibroblast growth factor-21 (FGF-21) 10 ( = 0.001), P-selectin glycoprotein ligand-1 (PSGL-1) OR 8.6 ( = 0.005), and Galectin-9 (Gal-9) OR 5.91 ( = 0.004). For G+P-, however, angiopoietin-1 receptor (TIE2) was notably raised, OR 65.5 ( = 0.004), whereas metalloproteinase inhibitor 4 (TIMP4) involved in proteolysis, in contrast, had reduced OR 0.06 ( = 0.013). This study is one of the first in young HCM patients and G+P- individuals. We found significantly increased OR for HCM in FGF-21 involved in RAS-MAPK pathway, associated with cardiomyocyte hypertrophy. Upregulation of FGF-21 indicates involvement of the RAS-MAPK pathway in HCM regardless of genetic background, which is a novel finding.
PubMed: 38667723
DOI: 10.3390/jcdd11040105 -
Frontiers in Physiology 2024The giant protein titin is an essential component of muscle sarcomeres. A single titin molecule spans half a sarcomere and mediates diverse functions along its length by... (Review)
Review
The giant protein titin is an essential component of muscle sarcomeres. A single titin molecule spans half a sarcomere and mediates diverse functions along its length by virtue of its unique domains. The A-band of titin functions as a molecular blueprint that defines the length of the thick filaments, the I-band constitutes a molecular spring that determines cell-based passive stiffness, and various domains, including the Z-disk, I-band, and M-line, serve as scaffolds for stretch-sensing signaling pathways that mediate mechanotransduction. This review aims to discuss recent insights into titin's functional roles and their relationship to cardiac function. The role of titin in heart diseases, such as dilated cardiomyopathy and heart failure with preserved ejection fraction, as well as its potential as a therapeutic target, is also discussed.
PubMed: 38660537
DOI: 10.3389/fphys.2024.1385821 -
BioRxiv : the Preprint Server For... Apr 2024Cardiometabolic syndromes including diabetes and obesity are associated with occurrence of heart failure with diastolic dysfunction. There are no specific treatments for...
UNLABELLED
Cardiometabolic syndromes including diabetes and obesity are associated with occurrence of heart failure with diastolic dysfunction. There are no specific treatments for diastolic dysfunction and therapies to manage symptoms have limited efficacy. Understanding of the cardiomyocyte origins of diastolic dysfunction is an important priority to identify new therapeutics. The investigative goal was to experimentally define stiffness (stress/strain) properties of isolated cardiomyocytes derived from rodent hearts exhibiting diastolic dysfunction in response to dietary induction of cardiometabolic disease. Mice fed a High Fat/Sugar Diet (HFSD vs control) for at least 25 weeks exhibited glucose intolerance, obesity and diastolic dysfunction (echo E/e'). Intact paced cardiomyocytes were functionally investigated in three conditions: non-loaded, loaded and stretched. Mean stiffness of HFSD cardiomyocytes was 70% higher than control. The E/e' doppler ratio for the origin hearts was elevated by 35%. A significant relationship was identified between cardiomyocyte stiffness and dysfunction severity. With conversion from non-loaded to loaded condition, the decrement in maximal sarcomere lengthening rate was more accentuated in HFSD cardiomyocytes (vs control). With stretch, the Ca transient decay time course was prolonged. With transition from 2-4Hz pacing, HFSD cardiomyocyte stiffness was further increased, yet diastolic Ca rise was 50% less than control. Collectively, these findings demonstrate that a component of cardiac diastolic dysfunction in cardiometabolic disease is derived from intrinsic cardiomyocyte mechanical abnormality. Differential responses to load, stretch and pacing suggest that a previously undescribed alteration in myofilament-Ca interaction contributes to cardiomyocyte stiffness in cardiometabolic disease.
KEY POINTS
Understanding cardiomyocyte stiffness components is an important priority for identifying new therapeutics for diastolic dysfunction, a key feature of cardiometabolic disease. In this study cardiac function was measured (echocardiography) for mice fed a high-fat/sugar diet (HFSD, ≥25weeks) and performance of intact isolated cardiomyocytes derived from the same hearts was measured during pacing under non-loaded, loaded and stretched conditions . Using a calibrated cardiomyocyte stretch protocol, stiffness (stress/strain) was elevated in HFSD cardiomyocytes and correlated with diastolic dysfunction (E/e') . The HFSD cardiomyocyte Ca transient decay was prolonged in response to stretch, and stiffness was accentuated in response to pacing increase while the rise in diastolic Ca was attenuated. These findings suggest that stretch-dependent augmentation of the myofilament-Ca response during diastole partially underlies elevated cardiomyocyte stiffness and diastolic dysfunction of hearts of animals with cardiometabolic disease.
PubMed: 38659933
DOI: 10.1101/2024.02.21.581448 -
BioRxiv : the Preprint Server For... Apr 2024Phospholamban (PLN) is a key regulator of cardiac function connecting adrenergic signaling and calcium homeostasis. The R9C mutation of PLN is known to cause early onset...
BACKGROUND
Phospholamban (PLN) is a key regulator of cardiac function connecting adrenergic signaling and calcium homeostasis. The R9C mutation of PLN is known to cause early onset dilated cardiomyopathy (DCM) and premature death, yet the detailed mechanisms underlie the pathologic remodeling process are not well defined in human cardiomyocytes. The aim of this study is to unravel the role of PLN R9C in DCM and identify potential therapeutic targets.
METHODS
PLN R9C knock-in (KI) and patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated and comprehensively examined for their expression profile, contractile function, and cellular signaling under both baseline conditions and following functional challenges.
RESULTS
PLN R9C KI iPSC-CMs exhibited near-normal morphology and calcium handling, slightly increased contractility, and an attenuated response to β-adrenergic activation compared to wild-type (WT) cells. However, treatment with a maturation medium (MM) has induced fundamentally different remodeling in the two groups: while it improved the structural integrity and functional performance of WT cells, the same treatment result in sarcomere disarrangement, calcium handling deficiency, and further disrupted adrenergic signaling in PLN R9C KI cells. To understand the mechanism, transcriptomic analysis showed the enrichment of protein homeostasis signaling pathways specifically in PLN R9C KI cells in response to the MM treatment and increased contractile demands. Further studies also indicated elevated ROS levels, interrupted autophagic flux, and increased pentamer PLN aggregation in functionally challenged KI cells. These results were further confirmed in patient-specific iPSC-CM models, suggesting that functional stresses exacerbate the deficiencies in PLN R9C cells through disrupting protein homeostasis. Indeed, treating stressed patient cells with autophagy-accelerating reagents, such as metformin and rapamycin, has restored autophagic flux, mitigated sarcomere disarrangement, and partially rescued β-adrenergic signaling and cardiac function.
CONCLUSIONS
PLN R9C leads to a mild increase of calcium recycling and contractility. Functional challenges further enhanced contractile and proteostasis stress, leading to autophagic overload, structural remodeling, and functional deficiencies in PLN R9C cardiomyocytes. Activation of autophagy signaling partially rescues these effects, revealing a potential therapeutic target for DCM patients with the PLN R9C mutation.
GRAPHIC ABSTRACTS
A graphic abstract is available for this article.
PubMed: 38659742
DOI: 10.1101/2024.04.17.589921 -
Journal of the American College of... Apr 2024Disease penetrance in genotype-positive (G+) relatives of families with dilated cardiomyopathy (DCM) and the characteristics associated with DCM onset in these...
BACKGROUND
Disease penetrance in genotype-positive (G+) relatives of families with dilated cardiomyopathy (DCM) and the characteristics associated with DCM onset in these individuals are unknown.
OBJECTIVES
This study sought to determine the penetrance of new DCM diagnosis in G+ relatives and to identify factors associated with DCM development.
METHODS
The authors evaluated 779 G+ patients (age 35.8 ± 17.3 years; 459 [59%] females; 367 [47%] with variants in TTN) without DCM followed at 25 Spanish centers.
RESULTS
After a median follow-up of 37.1 months (Q1-Q3: 16.3-63.8 months), 85 individuals (10.9%) developed DCM (incidence rate of 2.9 per 100 person-years; 95% CI: 2.3-3.5 per 100 person-years). DCM penetrance and age at DCM onset was different according to underlying gene group (log-rank P = 0.015 and P <0.01, respectively). In a multivariable model excluding CMR parameters, independent predictors of DCM development were: older age (HR per 1-year increase: 1.02; 95% CI: 1.0-1.04), an abnormal electrocardiogram (HR: 2.13; 95% CI: 1.38-3.29); presence of variants in motor sarcomeric genes (HR: 1.92; 95% CI: 1.05-3.50); lower left ventricular ejection fraction (HR per 1% increase: 0.86; 95% CI: 0.82-0.90) and larger left ventricular end-diastolic diameter (HR per 1-mm increase: 1.10; 95% CI: 1.06-1.13). Multivariable analysis in individuals with cardiac magnetic resonance and late gadolinium enhancement assessment (n = 360, 45%) identified late gadolinium enhancement as an additional independent predictor of DCM development (HR: 2.52; 95% CI: 1.43-4.45).
CONCLUSIONS
Following a first negative screening, approximately 11% of G+ relatives developed DCM during a median follow-up of 3 years. Older age, an abnormal electrocardiogram, lower left ventricular ejection fraction, increased left ventricular end-diastolic diameter, motor sarcomeric genetic variants, and late gadolinium enhancement are associated with a higher risk of developing DCM.
Topics: Adult; Female; Humans; Male; Middle Aged; Young Adult; Cardiomyopathy, Dilated; Connectin; Electrocardiography; Follow-Up Studies; Genotype; Penetrance; Spain; Retrospective Studies
PubMed: 38658103
DOI: 10.1016/j.jacc.2024.02.036 -
BioRxiv : the Preprint Server For... Apr 2024Familial dilated cardiomyopathy (DCM) is frequently caused by autosomal dominant point mutations in genes involved in diverse cellular processes, including sarcomeric...
Familial dilated cardiomyopathy (DCM) is frequently caused by autosomal dominant point mutations in genes involved in diverse cellular processes, including sarcomeric contraction. While patient studies have defined the genetic landscape of DCM, genetics are not currently used in patient care, and patients receive similar treatments regardless of the underlying mutation. It has been suggested that a precision medicine approach based on the molecular mechanism of the underlying mutation could improve outcomes; however, realizing this approach has been challenging due to difficulties linking genotype and phenotype and then leveraging this information to identify therapeutic approaches. Here, we used multiscale experimental and computational approaches to test whether knowledge of molecular mechanism could be harnessed to connect genotype, phenotype, and drug response for a DCM mutation in troponin T, deletion of K210. Previously, we showed that at the molecular scale, the mutation reduces thin filament activation. Here, we used computational modeling of this molecular defect to predict that the mutant will reduce cellular and tissue contractility, and we validated this prediction in human cardiomyocytes and engineered heart tissues. We then used our knowledge of molecular mechanism to computationally model the effects of a small molecule that can activate the thin filament. We demonstrate experimentally that the modeling correctly predicts that the small molecule can partially rescue systolic dysfunction at the expense of diastolic function. Taken together, our results demonstrate how molecular mechanism can be harnessed to connect genotype and phenotype and inspire strategies to optimize mechanism-based therapeutics for DCM.
PubMed: 38645235
DOI: 10.1101/2024.04.05.588306