RESUMEN
Obscurin is a giant sarcomeric protein expressed in striated muscles known to establish several interactions with other proteins of the sarcomere, but also with proteins of the sarcoplasmic reticulum and costameres. Here, we report experiments aiming to better understand the contribution of obscurin to skeletal muscle fibers, starting with a detailed characterization of the diaphragm muscle function, which we previously reported to be the most affected muscle in obscurin (Obscn) KO mice. Twitch and tetanus tension were not significantly different in the diaphragm of WT and Obscn KO mice, while the time to peak (TTP) and half relaxation time (HRT) were prolonged. Differences in force-frequency and force-velocity relationships and an enhanced fatigability are observed in an Obscn KO diaphragm with respect to WT controls. Voltage clamp experiments show that a sarcoplasmic reticulum's Ca2+ release and SERCA reuptake rates were decreased in muscle fibers from Obscn KO mice, suggesting that an impairment in intracellular Ca2+ dynamics could explain the observed differences in the TTP and HRT in the diaphragm. In partial contrast with previous observations, Obscn KO mice show a normal exercise tolerance, but fiber damage, the altered sarcomere ultrastructure and M-band disarray are still observed after intense exercise.
Asunto(s)
Calcio/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Sarcómeros/metabolismo , Animales , Ancirinas/metabolismo , Conectina/metabolismo , Conectina/fisiología , Masculino , Ratones , Ratones Noqueados , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Condicionamiento Físico Animal , Proteínas Serina-Treonina Quinasas/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Sarcómeros/fisiología , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
KEY POINTS: Fast sarcomere-level mechanics in contracting intact fibres from frog skeletal muscle reveal an I-band spring with an undamped stiffness 100 times larger than the known static stiffness. This undamped stiffness remains constant in the range of sarcomere length 2.7-3.1 µm, showing the ability of the I-band spring to adapt its length to the width of the I-band. The stiffness and tunability of the I-band spring implicate titin as a force contributor that, during contraction, allows weaker half-sarcomeres to equilibrate with in-series stronger half-sarcomeres, preventing the development of sarcomere length inhomogeneity. This work opens new possibilities for the detailed in situ description of the structural-functional basis of muscle dysfunctions related to mutations or site-directed mutagenesis in titin that alter the I-band stiffness. ABSTRACT: Force and shortening in the muscle sarcomere are due to myosin motors from thick filaments pulling nearby actin filaments toward the sarcomere centre. Thousands of serially linked sarcomeres in muscle make the shortening (and the shortening speed) macroscopic, while the intrinsic instability of in-series force generators is likely prevented by the cytoskeletal protein titin that connects the thick filament with the sarcomere end, working as an I-band spring that accounts for the rise of passive force with sarcomere length (SL). However, current estimates of titin stiffness, deduced from the passive force-SL relation and single molecule mechanics, are much smaller than what is required to avoid the development of large inhomogeneities among sarcomeres. In this work, using 4 kHz stiffness measurements on a population of sarcomeres selected along an intact fibre isolated from frog skeletal muscle contracting at different SLs (temperature 4°C), we measure the undamped stiffness of an I-band spring that at SL > 2.7 µm attains a maximum constant value of â¼6 pN nm-1 per half-thick filament, two orders of magnitude larger than expected from titin-related passive force. We conclude that a titin-like dynamic spring in the I-band, made by an undamped elastic element in-series with damped elastic elements, adapts its length to the SL with kinetics that provide force balancing among serially linked sarcomeres during contraction. In this way, the I-band spring plays a fundamental role in preventing the development of SL inhomogeneity.
Asunto(s)
Conectina/fisiología , Contracción Muscular , Músculo Esquelético/fisiología , Sarcómeros/fisiología , Animales , Anuros , Técnicas In VitroRESUMEN
Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a new methodology using Monte Carlo simulations of muscle contraction in an explicit 3D sarcomere lattice to predict the fiber deformations and length changes along thin filaments during contraction. Comparison of predicted diffraction patterns to experimental meridional X-ray reflection profiles allows assessment of the stepwise changes in intermonomer spacings and forces in the myofilaments within living muscle cells. These changes along the filament length reflect the effect of forces from randomly attached crossbridges. This approach enables correlation of the molecular events, such as the current number of attached crossbridges and the distributions of crossbridge forces to macroscopic measurements of force and length changes during muscle contraction. In addition, assessments of fluctuations in local forces in the myofilaments may reveal how variations in the filament forces acting on signaling proteins in the sarcomere M-bands and Z-discs modulate gene expression, protein synthesis and degradation, and as well to mechanisms of adaptation of muscle in response to changes in mechanical loading.
Asunto(s)
Citoesqueleto de Actina/fisiología , Actinas/fisiología , Contracción Isométrica/fisiología , Músculo Estriado/fisiología , Miosinas/fisiología , Sarcómeros/fisiología , Citoesqueleto de Actina/ultraestructura , Actinas/ultraestructura , Animales , Simulación por Computador , Conectina/fisiología , Conectina/ultraestructura , Modelos Biológicos , Método de Montecarlo , Músculo Estriado/diagnóstico por imagen , Miosinas/ultraestructura , Rana catesbeiana/fisiología , Sarcómeros/ultraestructura , Dispersión del Ángulo Pequeño , Técnicas de Cultivo de Tejidos , Difracción de Rayos XRESUMEN
Hypertrophic cardiomyopathy (HCM) is a hereditary disease characterized by cardiac hypertrophy with diastolic dysfunction. Gene mutations causing HCM have been found in about half of HCM patients, while the genetic etiology and pathogenesis remain unknown for many cases of HCM. To identify novel mechanisms underlying HCM pathogenesis, we generated a cardiovascular-mutant medaka fish, non-spring heart (nsh), which showed diastolic dysfunction and hypertrophic myocardium. The nsh homozygotes had fewer myofibrils, disrupted sarcomeres and expressed pathologically stiffer titin isoforms. In addition, the nsh heterozygotes showed M-line disassembly that is similar to the pathological changes found in HCM. Positional cloning revealed a missense mutation in an immunoglobulin (Ig) domain located in the M-line-A-band transition zone of titin. Screening of mutations in 96 unrelated patients with familial HCM, who had no previously implicated mutations in known sarcomeric gene candidates, identified two mutations in Ig domains close to the M-line region of titin. In vitro studies revealed that the mutations found both in medaka fish and in familial HCM increased binding of titin to muscle-specific ring finger protein 1 (MURF1) and enhanced titin degradation by ubiquitination. These findings implicate an impaired interaction between titin and MURF1 as a novel mechanism underlying the pathogenesis of HCM.
Asunto(s)
Cardiomiopatía Hipertrófica/etiología , Conectina/genética , Modelos Animales de Enfermedad , Proteínas Musculares/fisiología , Mutación , Proteínas de Motivos Tripartitos/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Conectina/fisiología , Humanos , Proteínas Musculares/genética , Oryzias , Transducción de Señal/fisiología , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Differences in passive muscle mechanical properties between amphibians and mammals have led to differing hypotheses on the functional role of titin in skeletal muscle. Early studies of frog muscle clearly demonstrated intracellular load bearing by titin, but more recent structural and biological studies in mice have shown that titin may serve other functions. Here, we present biomechanical studies of isolated frog and mouse fibers, and fiber bundles to compare the relative importance of intracellular versus extracellular load bearing in these species. Mouse bundles exhibited increased modulus compared with fibers on the descending limb of the length-tension curve, reaching a 2.4-fold elevation at the longest sarcomere lengths. By contrast, frog fibers and bundles had approximately the same modulus at all sarcomere lengths tested. These findings suggest that in the mouse, both muscle fibers and the ECM are involved in bearing whole muscle passive tension, which is distinct from the load bearing process in frog muscle, where titin bears the majority of whole muscle passive tension.
Asunto(s)
Ratones/fisiología , Fibras Musculares Esqueléticas/fisiología , Rana pipiens/fisiología , Animales , Fenómenos Biomecánicos , Conectina/fisiología , Módulo de ElasticidadRESUMEN
Gaps in our understanding of muscle contraction at the molecular level limit the ability to predict in vivo muscle forces in humans and animals during natural movements. Because muscles function as motors, springs, brakes, or struts, it is not surprising that uncertainties remain as to how sarcomeres produce these different behaviors. Current theories fail to explain why a single extra stimulus, added shortly after the onset of a train of stimuli, doubles the rate of force development. When stretch and doublet stimulation are combined in a work loop, muscle force doubles and work increases by 50% per cycle, yet no theory explains why this occurs. Current theories also fail to predict persistent increases in force after stretch and decreases in force after shortening. Early studies suggested that all of the instantaneous elasticity of muscle resides in the cross-bridges. Subsequent cross-bridge models explained the increase in force during active stretch, but required ad hoc assumptions that are now thought to be unreasonable. Recent estimates suggest that cross-bridges account for only â¼12% of the energy stored by muscles during active stretch. The inability of cross-bridges to account for the increase in force that persists after active stretching led to development of the sarcomere inhomogeneity theory. Nearly all predictions of this theory fail, yet the theory persists. In stretch-shortening cycles, muscles with similar activation and contractile properties function as motors or brakes. A change in the phase of activation relative to the phase of length changes can convert a muscle from a motor into a spring or brake. Based on these considerations, it is apparent that the current paradigm of muscle mechanics is incomplete. Recent advances in our understanding of giant muscle proteins, including twitchin and titin, allow us to expand our vision beyond cross-bridges to understand how muscles contribute to the biomechanics and control of movement.
Asunto(s)
Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Animales , Fenómenos Biomecánicos , Conectina/fisiología , Elasticidad , Humanos , Modelos Biológicos , Sarcómeros/fisiologíaRESUMEN
In striated muscle, the giant protein titin spans the entire length of a half-sarcomere and extends from the backbone of the thick filament, reversibly attaches to the thin filaments, and anchors to the dense protein network of the z-disk capping the end of the half-sarcomere. However, little is known about the relationship between the basic mechanical properties of titin and muscle contractility. Here, we build upon our previous multi-filament, spatially explicit computational model of the half-sarcomere by incorporating the nonlinear mechanics of titin filaments in the I-band. We vary parameters of the nonlinearity to understand the effects of titin stiffness on contraction dynamics and efficiency. We do so by simulating isometric contraction for a range of sarcomere lengths (SLs; 1.6-3.25 µm). Intermediate values of titin stiffness accurately reproduce the passive force-SL relation for skeletal muscle. The maximum force-SL relation is not affected by titin for SL≤2.5 µm. However, as titin stiffness increases, maximum force for the four thick filament system at SL = 3.0 µm significantly decreases from 103.2 ± 2 to 58.8 ± 1 pN. Additionally, by monitoring ATP consumption, we measure contraction efficiency as a function of titin stiffness. We find that at SL = 3.0 µm, efficiency significantly decreases from 13.9 ± 0.4 to 7.0 ± 0.3 pN/ATP when increasing titin stiffness, with little or no effect below 2.5 µm. Taken together, our results suggest that, despite an increase in the fraction of motors bound to actin along the descending limb when titin is stiffer, the force-generating capacity of the motors is reduced. These results suggest that titin stiffness has the potential to affect contractile efficiency.
Asunto(s)
Conectina/fisiología , Metabolismo Energético , Músculo Estriado/fisiología , Animales , Fenómenos Biomecánicos , Simulación por Computador , Humanos , Modelos BiológicosRESUMEN
RNA binding motif 20 (RBM20) regulates pre-mRNA splicing of over thirty genes, among which titin is a major target. With RBM20 expression, titin expresses a larger isoform at fetal stage to a smaller isoform at adult resulting from alternative splicing, while, without RBM20, titin expresses exclusively a larger isoform throughout all ages. In addition to splicing regulation, it is unknown whether RBM20 also regulates gene expression. In this study, we employed Rbm20 knockout rats to investigate gene expression profile using Affymetrix expression array. We compared wild type to Rbm20 knockout at day1, 20 and 49. Bioinformatics analysis showed RBM20 regulates fewer genes expression at younger age and more at older age and commonly expressed genes have the same trends. GSEA indicated up-regulated genes are associated with heart failure. We examined titin binding partners. All titin direct binding partners are up-regulated and their increased expression is associated with dilated cardiomyopathy. Particularly, we found that genes involving calcium handling and muscle contraction are changed by RBM20. Intracellular calcium level measurement with individual cardiomyocytes further confirmed that changes of these proteins impact calcium handling. Selected genes from titin binding partners and calcium handling were validated with QPCR and western blotting. These data demonstrate that RBM20 regulates gene splicing as well as gene expression. Altered gene expression by RBM20 influences protein-protein interaction, calcium releasing and thus muscle contraction. Our results first reported gene expression impacted by RBM20 with heart maturation, and provided new insights into the role of RBM20 in the progression of heart failure.
Asunto(s)
Conectina/metabolismo , Redes Reguladoras de Genes , Miocardio/metabolismo , Proteínas de Unión al ARN/fisiología , Animales , Conectina/genética , Conectina/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Miocitos Cardíacos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratas Sprague-DawleyRESUMEN
Muscle cells are designed to generate force and movement. There are three types of mammalian muscles-skeletal, cardiac, and smooth. Skeletal muscles are attached to bones and move them relative to each other. Cardiac muscle comprises the heart, which pumps blood through the vasculature. Skeletal and cardiac muscles are known as striated muscles, because the filaments of actin and myosin that power their contraction are organized into repeating arrays, called sarcomeres, that have a striated microscopic appearance. Smooth muscle does not contain sarcomeres but uses the contraction of filaments of actin and myosin to constrict blood vessels and move the contents of hollow organs in the body. Here, we review the principal molecular organization of the three types of muscle and their contractile regulation through signaling mechanisms and discuss their major structural and functional similarities that hint at the possible evolutionary relationships between the cell types.
Asunto(s)
Contracción Muscular/fisiología , Citoesqueleto de Actina/fisiología , Animales , Calcio/metabolismo , Conectina/fisiología , Humanos , Músculo Esquelético/fisiología , Músculo Estriado/fisiología , Miocitos del Músculo Liso/fisiología , Miosinas/fisiología , Sarcómeros/fisiología , Sarcómeros/ultraestructuraRESUMEN
Aims: Truncating titin variants (TTNtv) are the most prevalent genetic cause of dilated cardiomyopathy (DCM). We aim to study clinical parameters and long-term outcomes related to the TTNtv genotype and determine the related molecular changes at tissue level in TTNtv DCM patients. Methods and results: A total of 303 consecutive and extensively phenotyped DCM patients (including cardiac imaging, Holter monitoring, and endomyocardial biopsy) underwent DNA sequencing of 47 cardiomyopathy-associated genes including TTN, yielding 38 TTNtv positive (13%) patients. At long-term follow-up (median of 45 months, up to 12 years), TTNtv DCM patients had increased ventricular arrhythmias compared to other DCM, but a similar survival. Arrhythmias are especially prominent in TTNtv patients with an additional environmental trigger (i.e. virus infection, cardiac inflammation, systemic disease, toxic exposure). Importantly, cardiac mass is reduced in TTNtv patients, despite similar cardiac function and dimensions at cardiac magnetic resonance. These enhanced life-threatening arrhythmias and decreased cardiac mass in TTNtv DCM patients go along with significant cardiac energetic and matrix alterations. All components of the mitochondrial electron transport chain are significantly upregulated in TTNtv hearts at RNA-sequencing. Also, interstitial fibrosis was augmented in TTNtv patients at histological and transcript level. Conclusion: Truncating titin variants lead to pronounced cardiac alterations in mitochondrial function, with increased interstitial fibrosis and reduced hypertrophy. Those structural and metabolic alterations in TTNtv hearts go along with increased ventricular arrhythmias at long-term follow-up, with a similar survival and overall cardiac function.
Asunto(s)
Cardiomiopatías , Conectina , Arritmias Cardíacas/metabolismo , Cardiomiopatías/metabolismo , Cardiomiopatías/fisiopatología , Conectina/genética , Conectina/metabolismo , Conectina/fisiología , Fibrosis/metabolismo , Humanos , Mitocondrias/metabolismoRESUMEN
Titin-related diseases of the skeletal and cardiac muscles open a new, fruitful chapter of myology. Confined for a long time to a limited number of clinical entities, the phenotypic spectrum of titinopthies is nowadays expanding rapidly together with the discovery of many pathogenic mutations of the TTN gene. Like for many genes of large size, the fine tuning and use of high-throughput sequencing (NGS) constitutes a little revolution in the field. This powerful tool allows, although with real technical hurdles, the establishment of the definite diagnosis of titinopathy. A better knowledge of the natural history of each subtype of titinopathy enables as of now an optimized management of patients, notably when a cardiac or respiratory risk factor is identified. Research efforts in the titin-related conditions are gradually getting organized. Interactions between clinicians and geneticists are an absolute necessity. The still fragmentary knowledge of the pathogenesis of each titinopathy prevents to date to figure out any curative therapy in the very near future.
Asunto(s)
Conectina , Enfermedades Musculares , Conectina/genética , Conectina/fisiología , Diagnóstico Diferencial , Electromiografía , Humanos , Músculo Esquelético/patología , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/genética , Enfermedades Musculares/terapia , Mutación , Miocardio/patología , FenotipoRESUMEN
The contractile machinery of heart and skeletal muscles has as an essential component the thick filament, comprised of the molecular motor myosin. The thick filament is of a precisely controlled length, defining thereby the force level that muscles generate and how this force varies with muscle length. It has been speculated that the mechanism by which thick filament length is controlled involves the giant protein titin, but no conclusive support for this hypothesis exists. Here we show that in a mouse model in which we deleted two of titin's C-zone super-repeats, thick filament length is reduced in cardiac and skeletal muscles. In addition, functional studies reveal reduced force generation and a dilated cardiomyopathy (DCM) phenotype. Thus, regulation of thick filament length depends on titin and is critical for maintaining muscle health.
Asunto(s)
Conectina/fisiología , Sarcómeros/ultraestructura , Animales , Cardiomiopatía Dilatada/fisiopatología , Conectina/genética , Masculino , Ratones , Contracción Muscular , Eliminación de SecuenciaRESUMEN
In contrast to experimentally observed progressive forces in eccentric contractions, cross-bridge and sliding-filament theories of muscle contraction predict that varying myofilament overlap will lead to increases and decreases in active force during eccentric contractions. Non-cross-bridge contributions potentially explain the progressive total forces. However, it is not clear whether underlying abrupt changes in the slope of the nonlinear force-length relationship are visible in long isokinetic stretches, and in which proportion cross-bridges and non-cross-bridges contribute to muscle force. Here, we show that maximally activated single skinned rat muscle fibres behave (almost across the entire working range) like linear springs. The force slope is about three times the maximum isometric force per optimal length. Cross-bridge and non-cross-bridge contributions to the muscle force were investigated using an actomyosin inhibitor. The experiments revealed a nonlinear progressive contribution of non-cross-bridge forces and suggest a nonlinear cross-bridge contribution similar to the active force-length relationship (though with increased optimal length and maximum isometric force). The linear muscle behaviour might significantly reduce the control effort. Moreover, the observed slight increase in slope with initial length is in accordance with current models attributing the non-cross-bridge force to titin.
Asunto(s)
Contracción Muscular , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Citoesqueleto de Actina , Actomiosina/fisiología , Animales , Conectina/fisiología , Contracción Isométrica , RatasRESUMEN
Striated cardiac and skeletal muscles play very different roles in the body, but they are similar at the molecular level. In particular, contraction, regardless of the type of muscle, is a precise and complex process involving the integral protein myofilaments and their associated regulatory components. The smallest functional unit of muscle contraction is the sarcomere. Within the sarcomere can be found a sophisticated ensemble of proteins associated with the thick filaments (myosin, myosin binding protein-C, titin, and obscurin) and thin myofilaments (actin, troponin, tropomyosin, nebulin, and nebulette). These parallel thick and thin filaments slide across one another, pulling the two ends of the sarcomere together to regulate contraction. More specifically, the regulation of both timing and force of contraction is accomplished through an intricate network of intra- and interfilament interactions belonging to each myofilament. This review introduces the sarcomere proteins involved in striated muscle contraction and places greater emphasis on the more recently identified and less well-characterized myofilaments: cardiac myosin binding protein-C, titin, nebulin, and obscurin. © 2017 American Physiological Society. Compr Physiol 7:675-692, 2017.
Asunto(s)
Miofibrillas/fisiología , Sarcómeros/fisiología , Conectina/fisiología , Humanos , Contracción Muscular/fisiología , Proteínas Musculares/fisiología , Músculo Esquelético/fisiología , Proteínas Serina-Treonina Quinasas , Factores de Intercambio de Guanina Nucleótido Rho/fisiología , Troponina/fisiologíaRESUMEN
KEY POINTS: When a skeletal muscle is stretched while it contracts, the muscle produces a relatively higher force than the force from an isometric contraction at the same length: a phenomenon referred to as residual force enhancement. Residual force enhancement is puzzling because it cannot be directly explained by the classical force-length relationship and the sliding filament theory of contraction, the main paradigms in the muscle field. We used custom-built instruments to measure residual force enhancement in skeletal myofibrils, and, for the first time, in cardiac myofibrils. Our data report that residual force enhancement is present in skeletal muscles, but not cardiac muscles, and is regulated by the different isoforms of the titin protein filaments. ABSTRACT: When a skeletal muscle contracts isometrically, the muscle produces a force that is relative to the final isometric sarcomere length (SL). However, when the same final SL is reached by stretching the muscle while it contracts, the muscle produces a relatively higher force: a phenomenon commonly referred to as residual force enhancement. In this study, we investigated residual force enhancement in rabbit skeletal psoas myofibrils and, for the first time, cardiac papillary myofibrils. A custom-built atomic force microscope was used in experiments that stretched myofibrils before and after inhibiting myosin and actin interactions to determine whether the different cardiac and skeletal titin isoforms regulate residual force enhancement. At SLs ranging from 2.24 to 3.13 µm, the skeletal myofibrils enhanced the force by an average of 9.0%, and by 29.5% after hindering myosin and actin interactions. At SLs ranging from 1.80 to 2.29 µm, the cardiac myofibrils did not enhance the force before or after hindering myosin and actin interactions. We conclude that residual force enhancement is present only in skeletal muscles and is dependent on the titin isoforms.
Asunto(s)
Conectina/fisiología , Miofibrillas/fisiología , Músculos Psoas/fisiología , Animales , Femenino , Músculos Papilares/fisiología , Isoformas de Proteínas/fisiología , ConejosRESUMEN
This review considers data on structural and functional features of titin, on the role of this protein in determination of mechanical properties of sarcomeres, and on specific features of regulation of the stiffness and elasticity of its molecules, amyloid aggregation of this protein in vitro, and possibilities of formation of intramolecular amyloid structure in vivo. Molecular mechanisms are described of protection of titin against aggregation in muscle cells. Based on the data analysis, it is supposed that titin and the formed by it elastic filaments have features of amyloid.
Asunto(s)
Proteínas Amiloidogénicas/química , Conectina/química , Conectina/fisiología , Animales , Elasticidad , Humanos , SarcómerosRESUMEN
BACKGROUND: Left ventricular (LV) stiffening contributes to heart failure with preserved ejection fraction (HFpEF), a syndrome with no effective treatment options. Increasing the compliance of titin in the heart has become possible recently through inhibition of the splicing factor RNA binding motif-20. Here, we investigated the effects of increasing the compliance of titin in mice with diastolic dysfunction. METHODS: Mice in which the RNA recognition motif (RRM) of one of the RNA binding motif-20 alleles was floxed and that expressed the MerCreMer transgene under control of the αMHC promoter (referred to as cRbm20ΔRRM mice) were used. Mice underwent transverse aortic constriction (TAC) surgery and deoxycorticosterone acetate (DOCA) pellet implantation. RRM deletion in adult mice was triggered by injecting raloxifene (cRbm20ΔRRM-raloxifene), with dimethyl sulfoxide (DMSO)-injected mice (cRbm20ΔRRM-DMSO) as the control. Diastolic function was investigated with echocardiography and pressure-volume analysis; passive stiffness was studied in LV muscle strips and isolated cardiac myocytes before and after elimination of titin-based stiffness. Treadmill exercise performance was also studied. Titin isoform expression was evaluated with agarose gels. RESULTS: cRbm20ΔRRM-raloxifene mice expressed large titins in the hearts, called supercompliant titin (N2BAsc), which, within 3 weeks after raloxifene injection, made up ≈45% of total titin. TAC/DOCA cRbm20ΔRRM-DMSO mice developed LV hypertrophy and a marked increase in LV chamber stiffness as shown by both pressure-volume analysis and echocardiography. LV chamber stiffness was normalized in TAC/DOCA cRbm20ΔRRM-raloxifene mice that expressed N2BAsc. Passive stiffness measurements on muscle strips isolated from the LV free wall revealed that extracellular matrix stiffness was equally increased in both groups of TAC/DOCA mice (cRbm20ΔRRM-DMSO and cRbm20ΔRRM-raloxifene). However, titin-based muscle stiffness was reduced in the mice that expressed N2BAsc (TAC/DOCAcRbm20ΔRRM-raloxifene). Exercise testing demonstrated significant improvement in exercise tolerance in TAC/DOCA mice that expressed N2BAsc. CONCLUSIONS: Inhibition of the RNA binding motif-20-based titin splicing system upregulates compliant titins, which improves diastolic function and exercise tolerance in the TAC/DOCA model. Titin holds promise as a therapeutic target for heart failure with preserved ejection fraction.
Asunto(s)
Diástole/genética , Tolerancia al Ejercicio/genética , Insuficiencia Cardíaca/genética , Proteínas de Unión al ARN/genética , Función Ventricular Izquierda/genética , Animales , Adaptabilidad , Conectina/fisiología , Diástole/fisiología , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Hipertrofia Ventricular Izquierda/metabolismo , Ratones , Ratones Transgénicos , Motivos de Unión al ARN/genética , Volumen Sistólico/fisiología , Función Ventricular Izquierda/fisiologíaRESUMEN
Eccentric muscle properties are not well characterized by the current paradigm of the molecular mechanism of contraction: the cross-bridge theory. Findings of force contributions by passive structural elements a decade ago paved the way for a new theory. Here, we present experimental evidence and theoretical support for the idea that the structural protein titin contributes to active force production, thereby explaining many of the unresolved properties of eccentric muscle contraction.
Asunto(s)
Conectina/fisiología , Contracción Muscular , Músculo Esquelético/fisiología , Animales , Conectina/metabolismo , Humanos , Modelos Biológicos , Músculo Esquelético/metabolismo , Sarcómeros/fisiologíaRESUMEN
Passive forces in sarcomeres are mainly related to the giant protein titin. Titin's extensible region consists of spring-like elements acting in series. In skeletal muscles these elements are the PEVK segment, two distinct immunoglobulin (Ig) domain regions (proximal and distal), and a N2A portion. While distal Ig domains are thought to form inextensible end filaments in intact sarcomeres, proximal Ig domains unfold in a force- and time-dependent manner. In length-ramp experiments of single titin strands, sequential unfolding of Ig domains leads to a typical saw-tooth pattern in force-elongation curves which can be simulated by Monte Carlo simulations. In sarcomeres, where more than a thousand titin strands are arranged in parallel, numerous Monte Carlo simulations are required to estimate the resultant force of all titin filaments based on the non-uniform titin elongations. To simplify calculations, the stochastic model of passive forces is often replaced by linear or non-linear deterministic and phenomenological functions. However, new theories of muscle contraction are based on the hypothesized binding of titin to the actin filament upon activation, and thereby on a prominent role of the structural properties of titin. Therefore, these theories necessitate a detailed analysis of titin forces in length-ramp experiments. In our study we present a simple and efficient alternative to Monte Carlo simulations. Based on a structural titin model, we calculate the exact probability distributions of unfolded Ig domains under length-ramp conditions needed for rigorous analysis of expected forces, distribution of unfolding forces, etc. Due to the generality of our model, the approach is applicable to a wide range of stochastic protein unfolding problems.
Asunto(s)
Conectina/química , Conectina/fisiología , Modelos Biológicos , Modelos Químicos , Sarcómeros/química , Sarcómeros/fisiología , Simulación por Computador , Conectina/ultraestructura , Modelos Moleculares , Sarcómeros/ultraestructura , Estrés Mecánico , Relación Estructura-Actividad , Resistencia a la Tracción/fisiologíaRESUMEN
The force maintained following stretching of an active muscle exceeds the isometric force at the same muscle length. This residual force enhancement (RFE) is different for various muscles. It is currently unknown whether training induces changes in RFE. Weightlifters perform a large number of eccentric contractions during training, and RFE might be functionally relevant. The aim of this study was to examine whether there is increased RFE in weightlifters versus a reference group. Therefore, we measured external reaction forces during a multi-joint leg extension in weightlifters (n=10) and a reference group (n=11) using a motor driven leg press dynamometer (ISOMED 2000). Steady state isometric forces after stretching were compared to the corresponding forces obtained during isometric reference contractions. Statistical analyses yielded a significant RFE for both groups (p<0.001), but there were no RFE differences between the groups (p=0.320). However, RFE tends to decrease slower in the weightlifting group versus the reference group. We conclude that long-term weightlifting has only a minor influence on RFE. We speculate that the specific training including a combination of eccentric and concentric exercises induced almost no changes in titin-isoform expression which may be responsible for generation of RFE after active muscle stretching.