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Cardiovascular Research Apr 2015Over the past decade, our understanding of cardiomyopathies has improved dramatically, due to improvements in screening and detection of gene defects in the human genome... (Review)
Review
Over the past decade, our understanding of cardiomyopathies has improved dramatically, due to improvements in screening and detection of gene defects in the human genome as well as a variety of novel animal models (mouse, zebrafish, and drosophila) and in silico computational models. These novel experimental tools have created a platform that is highly complementary to the naturally occurring cardiomyopathies in cats and dogs that had been available for some time. A fully integrative approach, which incorporates all these modalities, is likely required for significant steps forward in understanding the molecular underpinnings and pathogenesis of cardiomyopathies. Finally, novel technologies, including CRISPR/Cas9, which have already been proved to work in zebrafish, are currently being employed to engineer sarcomeric cardiomyopathy in larger animals, including pigs and non-human primates. In the mouse, the increased speed with which these techniques can be employed to engineer precise 'knock-in' models that previously took years to make via multiple rounds of homologous recombination-based gene targeting promises multiple and precise models of human cardiac disease for future study. Such novel genetically engineered animal models recapitulating human sarcomeric protein defects will help bridging the gap to translate therapeutic targets from small animal and in silico models to the human patient with sarcomeric cardiomyopathy.
Topics: Animals; Cardiomyopathies; Cats; Computer Simulation; Dogs; Drosophila; Genetic Markers; Genetic Predisposition to Disease; Humans; Mice, Transgenic; Models, Animal; Models, Cardiovascular; Mutation; Phenotype; Sarcomeres; Zebrafish
PubMed: 25600962
DOI: 10.1093/cvr/cvv006 -
PLoS Computational Biology Mar 2020As sarcomeres produce the force necessary for contraction, assessment of sarcomere order is paramount in evaluation of cardiac and skeletal myocytes. The uniaxial force...
As sarcomeres produce the force necessary for contraction, assessment of sarcomere order is paramount in evaluation of cardiac and skeletal myocytes. The uniaxial force produced by sarcomeres is ideally perpendicular to their z-lines, which couple parallel myofibrils and give cardiac and skeletal myocytes their distinct striated appearance. Accordingly, sarcomere structure is often evaluated by staining for z-line proteins such as α-actinin. However, due to limitations of current analysis methods, which require manual or semi-manual handling of images, the mechanism by which sarcomere and by extension z-line architecture can impact contraction and which characteristics of z-line architecture should be used to assess striated myocytes has not been fully explored. Challenges such as isolating z-lines from regions of off-target staining that occur along immature stress fibers and cell boundaries and choosing metrics to summarize overall z-line architecture have gone largely unaddressed in previous work. While an expert can qualitatively appraise tissues, these challenges leave researchers without robust, repeatable tools to assess z-line architecture across different labs and experiments. Additionally, the criteria used by experts to evaluate sarcomeric architecture have not been well-defined. We address these challenges by providing metrics that summarize different aspects of z-line architecture that correspond to expert tissue quality assessment and demonstrate their efficacy through an examination of engineered tissues and single cells. In doing so, we have elucidated a mechanism by which highly elongated cardiomyocytes become inefficient at producing force. Unlike previous manual or semi-manual methods, characterization of z-line architecture using the metrics discussed and implemented in this work can quantitatively evaluate engineered tissues and contribute to a robust understanding of the development and mechanics of striated muscles.
Topics: Algorithms; Animals; Cells, Cultured; Humans; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Muscle Fibers, Skeletal; Muscle, Skeletal; Myocytes, Cardiac; Myofibrils; Rats; Rats, Sprague-Dawley; Sarcomeres
PubMed: 32130207
DOI: 10.1371/journal.pcbi.1007676 -
American Journal of Physiology. Cell... Jul 2022When muscle fibers from limb muscles are stretched while activated, the force increases to a steady-state level that is higher than that produced during isometric...
When muscle fibers from limb muscles are stretched while activated, the force increases to a steady-state level that is higher than that produced during isometric contractions at a corresponding sarcomere length, a phenomenon known as residual force enhancement (RFE). The mechanisms responsible for the RFE are an increased stiffness of titin molecules that may lead to an increased Ca sensitivity of the contractile apparatus, and the development of sarcomere length nonuniformities. RFE is not observed in cardiac myofibrils, which makes this phenomenon specific to certain preparations. The aim of this study was to investigate whether the RFE is present in the diaphragm, and its potential association with an increased Ca sensitivity and the development of sarcomere length nonuniformities. We used two preparations: single intact fibers and myofibrils isolated from the diaphragm of mice. We investigated RFE in a variety of lengths across the force-length relationship. RFE was observed in both preparations at all lengths investigated and was larger with increasing magnitudes of stretch. RFE was accompanied by an increased Ca sensitivity as shown by a change in the force-pCa curve, and increased sarcomere length nonuniformities. Therefore, RFE is a phenomenon commonly observed in skeletal muscles, with mechanisms that are similar across preparations.
Topics: Animals; Diaphragm; Isometric Contraction; Mice; Muscle Contraction; Muscle Fibers, Skeletal; Myofibrils; Sarcomeres
PubMed: 35613356
DOI: 10.1152/ajpcell.00394.2021 -
Circulation. Heart Failure Jun 2023Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiomyopathy. Pathogenic germline variation in genes encoding the sarcomere is the predominant cause of...
BACKGROUND
Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiomyopathy. Pathogenic germline variation in genes encoding the sarcomere is the predominant cause of disease. However diagnostic features, including unexplained left ventricular hypertrophy, typically do not develop until late adolescence or after. The early stages of disease pathogenesis and the mechanisms underlying the transition to a clinically overt phenotype are not well understood. In this study, we investigated if circulating microRNAs (miRNAs) could stratify disease stage in sarcomeric HCM.
METHODS
We performed arrays for 381 miRNAs using serum from HCM sarcomere variant carriers with and without a diagnosis of HCM and healthy controls. To identify differentially expressed circulating miRNAs between groups, multiple approaches were used including random forest, Wilcoxon rank sum test, and logistic regression. The abundance of all miRNAs was normalized to miRNA-320.
RESULTS
Of 57 sarcomere variant carriers, 25 had clinical HCM and 32 had subclinical HCM with normal left ventricular wall thickness (21 with early phenotypic manifestations and 11 with no discernible phenotypic manifestations). Circulating miRNA profile differentiated healthy controls from sarcomere variant carriers with subclinical and clinical disease. Additionally, circulating miRNAs differentiated clinical HCM from subclinical HCM without early phenotypic changes; and subclinical HCM with and without early phenotypic changes. Circulating miRNA profiles did not differentiate clinical HCM from subclinical HCM with early phenotypic changes, suggesting biologic similarity between these groups.
CONCLUSIONS
Circulating miRNAs may augment the clinical stratification of HCM and improve understanding of the transition from health to disease in sarcomere gene variant carriers.
Topics: Humans; Sarcomeres; Circulating MicroRNA; Mutation; Heart Failure; Cardiomyopathy, Hypertrophic; Phenotype; MicroRNAs
PubMed: 36880380
DOI: 10.1161/CIRCHEARTFAILURE.122.010291 -
Scientific Reports Jan 2020The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. We previously reported...
The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. We previously reported that induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with a dilated cardiomyopathy (DCM) mutation, troponin T (TnT)-R173W, display sarcomere protein misalignment and impaired contractility. Yet it is not known how TnT mutation causes dysfunction of sarcomere microdomains and how these events contribute to misalignment of sarcomeric proteins in presence of DCM TnT-R173W. Using a human iPSC-CM model combined with CRISPR/Cas9-engineered isogenic controls, we uncovered that TnT-R173W destabilizes molecular interactions of troponin with tropomyosin, and limits binding of PKA to local sarcomere microdomains. This attenuates troponin phosphorylation and dysregulates local sarcomeric microdomains in DCM iPSC-CMs. Disrupted microdomain signaling impairs MYH7-mediated, AMPK-dependent sarcomere-cytoskeleton filament interactions and plasma membrane attachment. Small molecule-based activation of AMPK can restore TnT microdomain interactions, and partially recovers sarcomere protein misalignment as well as impaired contractility in DCM TnT-R173W iPSC-CMs. Our findings suggest a novel therapeutic direction targeting sarcomere- cytoskeleton interactions to induce sarcomere re-organization and contractile recovery in DCM.
Topics: Calcium; Cardiomyopathy, Dilated; Cell Differentiation; Cytoskeleton; Excitation Contraction Coupling; Humans; Induced Pluripotent Stem Cells; Mutation; Myocardial Contraction; Myocytes, Cardiac; Sarcomeres; Troponin
PubMed: 31937807
DOI: 10.1038/s41598-019-56597-3 -
Proceedings of the National Academy of... Nov 2019Atrial fibrillation (AF) is the most common type of cardiac arrhythmia. The major AF susceptibility locus 4q25 establishes long-range interactions with the promoter of ,...
Atrial fibrillation (AF) is the most common type of cardiac arrhythmia. The major AF susceptibility locus 4q25 establishes long-range interactions with the promoter of , a transcription factor gene with critical functions during cardiac development. While many AF-linked loci have been identified in genome-wide association studies, mechanistic understanding into how genetic variants, including those at the 4q25 locus, increase vulnerability to AF is mostly lacking. Here, we show that loss of in zebrafish leads to adult cardiac phenotypes with substantial similarities to pathologies observed in AF patients, including arrhythmia, atrial conduction defects, sarcomere disassembly, and altered cardiac metabolism. These phenotypes are also observed in a subset of fish, mimicking the situation in humans. Most notably, the onset of these phenotypes occurs at an early developmental stage. Detailed analyses of loss- and gain-of-function embryonic hearts reveal changes in sarcomeric and metabolic gene expression and function that precede the onset of cardiac arrhythmia first observed at larval stages. We further find that antioxidant treatment of larvae significantly reduces the incidence and severity of cardiac arrhythmia, suggesting that metabolic dysfunction is an important driver of conduction defects. We propose that these early sarcomere and metabolic defects alter cardiac function and contribute to the electrical instability and structural remodeling observed in adult fish. Overall, these data provide insight into the mechanisms underlying the development and pathophysiology of some cardiac arrhythmias and importantly, increase our understanding of how developmental perturbations can predispose to functional defects in the adult heart.
Topics: Acetylcysteine; Animals; Animals, Genetically Modified; Antioxidants; Arrhythmias, Cardiac; Cardiac Conduction System Disease; Cardiomyopathies; Disease Models, Animal; Electrocardiography; Gene Expression Regulation; Homeodomain Proteins; Larva; Mitochondria, Heart; Sarcomeres; Stress, Physiological; Transcription Factors; Zebrafish; Zebrafish Proteins
PubMed: 31704768
DOI: 10.1073/pnas.1913905116 -
Journal of Biomedical Science Mar 2022Transversal structural elements in cross-striated muscles, such as the M-band or the Z-disc, anchor and mechanically stabilize the contractile apparatus and its minimal... (Review)
Review
Transversal structural elements in cross-striated muscles, such as the M-band or the Z-disc, anchor and mechanically stabilize the contractile apparatus and its minimal unit-the sarcomere. The ability of proteins to target and interact with these structural sarcomeric elements is an inevitable necessity for the correct assembly and functionality of the myofibrillar apparatus. Specifically, the M-band is a well-recognized mechanical and signaling hub dealing with active forces during contraction, while impairment of its function leads to disease and death. Research on the M-band architecture is focusing on the assembly and interactions of the three major filamentous proteins in the region, mainly the three myomesin proteins including their embryonic heart (EH) isoform, titin and obscurin. These proteins form the basic filamentous network of the M-band, interacting with each other as also with additional proteins in the region that are involved in signaling, energetic or mechanosensitive processes. While myomesin-1, titin and obscurin are found in every muscle, the expression levels of myomesin-2 (also known as M-protein) and myomesin-3 are tissue specific: myomesin-2 is mainly expressed in the cardiac and fast skeletal muscles, while myomesin-3 is mainly expressed in intermediate muscles and specific regions of the cardiac muscle. Furthermore, EH-myomesin apart from its role during embryonic stages, is present in adults with specific cardiac diseases. The current work in structural, molecular, and cellular biology as well as in animal models, provides important details about the assembly of myomesin-1, obscurin and titin, the information however about the myomesin-2 and -3, such as their interactions, localization and structural details remain very limited. Remarkably, an increasing number of reports is linking all three myomesin proteins and particularly myomesin-2 to serious cardiovascular diseases suggesting that this protein family could be more important than originally thought. In this review we will focus on the myomesin protein family, the myomesin interactions and structural differences between isoforms and we will provide the most recent evidence why the structurally and biophysically unexplored myomesin-2 and myomesin-3 are emerging as hot targets for understanding muscle function and disease.
Topics: Animals; Connectin; Heart Diseases; Muscle Proteins; Muscle, Skeletal; Sarcomeres
PubMed: 35255917
DOI: 10.1186/s12929-022-00801-6 -
Journal of Muscle Research and Cell... Mar 2021Full muscle relaxation happens when [Ca] falls below the threshold for force activation. Several experimental models, from whole muscle organs and intact muscle down to...
Full muscle relaxation happens when [Ca] falls below the threshold for force activation. Several experimental models, from whole muscle organs and intact muscle down to skinned fibers, have been used to explore the cascade of kinetic events leading to mechanical relaxation. The use of single myofibrils together with fast solution switching techniques, has provided new information about the role of cross-bridge (CB) dissociation in the time course of isometric force decay. Myofibril's relaxation is biphasic starting with a slow seemingly linear phase, with a rate constant, slow k, followed by a fast mono-exponential phase. Sarcomeres remain isometric during the slow force decay that reflects CB detachment under isometric conditions while the final fast relaxation phase begins with a sudden give of few sarcomeres and is then dominated by intersarcomere dynamics. Based on a simple two-state model of the CB cycle, myofibril slow k represents the apparent forward rate with which CBs leave force generating states (g) under isometric conditions and correlates with the energy cost of tension generation (ATPase/tension ratio); in short slow k ~ g ~ tension cost. The validation of this relationship is obtained by simultaneously measuring maximal isometric force and ATP consumption in skinned myocardial strips that provide an unambiguous determination of the relation between contractile and energetic properties of the sarcomere. Thus, combining kinetic experiments in isolated myofibrils and mechanical and energetic measurements in multicellular cardiac strips, we are able to provide direct evidence for a positive linear correlation between myofibril isometric relaxation kinetics (slow k) and the energy cost of force production both measured in preparations from the same cardiac sample. This correlation remains true among different types of muscles with different ATPase activities and also when CB kinetics are altered by cardiomyopathy-related mutations. Sarcomeric mutations associated to hypertrophic cardiomyopathy (HCM), a primary cardiac disorder caused by mutations in genes encoding sarcomeric proteins, have been often found to accelerate CB turnover rate and increase the energy cost of myocardial contraction. Here we review data showing that faster CB detachment results in a proportional increase in the energetic cost of tension generation in heart samples from both HCM patients and mouse models of the disease.
Topics: Animals; Humans; Mice; Myocardial Contraction; Myocardium; Sarcomeres
PubMed: 31745760
DOI: 10.1007/s10974-019-09566-2 -
The Journal of General Physiology Aug 2010
Topics: Computer Simulation; Heart; Humans; Models, Cardiovascular; Sarcomeres
PubMed: 20660659
DOI: 10.1085/jgp.201010497 -
The Journal of General Physiology Apr 2023Sarcomere length (SL) and its variation along the myofibril strongly regulate integrated coordinated myocyte contraction. It is therefore important to obtain individual... (Comparative Study)
Comparative Study
Sarcomere length (SL) and its variation along the myofibril strongly regulate integrated coordinated myocyte contraction. It is therefore important to obtain individual SL properties. Optical imaging by confocal fluorescence (for example, using ANEPPS) or transmitted light microscopy is often used for this purpose. However, this allows for the visualization of structures related to Z-disks only. In contrast, second-harmonic generation (SHG) microscopy visualizes A-band sarcomeric structures directly. Here, we compared averaged SL and its variability in isolated relaxed rat cardiomyocytes by imaging with ANEPPS and SHG. We found that SL variability, evaluated by several absolute and relative measures, is two times smaller using SHG vs. ANEPPS, while both optical methods give the same average (median) SL. We conclude that optical methods with similar optical spatial resolution provide valid estimations of average SL, but the use of SHG microscopy for visualization of sarcomeric A-bands may be the "gold standard" for evaluation of SL variability due to the absence of optical interference between the sarcomere center and non-sarcomeric structures. This contrasts with sarcomere edges where t-tubules may not consistently colocalize to Z-disks. The use of SHG microscopy instead of fluorescent imaging can be a prospective tool to map sarcomere variability both in vitro and in vivo conditions and to reveal its role in the functional behavior of living myocardium.
Topics: Animals; Rats; Myocytes, Cardiac; Myofibrils; Myosins; Sarcomeres; Second Harmonic Generation Microscopy
PubMed: 36695814
DOI: 10.1085/jgp.202213289