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We developed a model describing the structure and contractile mechanism of the actomyosin ring in fission yeast, Schizosaccharomyces pombe. The proposed ring includes actin, myosin, and α-actinin, and is organized into a structure similar to that of muscle sarcomeres. This structure justifies the use of the sliding-filament mechanism developed by Huxley and Hill, but it is probably less organized relative to that of muscle sarcomeres. Ring contraction tension was generated via the same fundamental mechanism used to generate muscle tension, but some physicochemical parameters were adjusted to be consistent with the proposed ring structure. Simulations allowed an estimate of ring constriction tension that reproduced the observed ring constriction velocity using a physiologically possible, self-consistent set of parameters. Proposed molecular-level properties responsible for the thousand-fold slower constriction velocity of the ring relative to that of muscle sarcomeres include fewer myosin molecules involved, a less organized contractile configuration, a low α-actinin concentration, and a high resistance membrane tension. Ring constriction velocity is demonstrated as an exponential function of time despite a near linear appearance. We proposed a hypothesis to explain why excess myosin heads inhibit constriction velocity rather than enhance it. The model revealed how myosin concentration and elastic resistance tension are balanced during cytokinesis in S. pombe.

The functional design of spine muscles in part dictates their role in moving, loading, and stabilizing the lumbar spine. There have been numerous studies that have examined the isolated properties of these individual muscles. Understanding how these muscles interact and work together, necessary for the prediction of muscle function, spine loading, and stability, is lacking. The objective of this study was to measure sarcomere lengths of lumbar muscles in a neutral cadaveric position and predict the sarcomere operating ranges of these muscles throughout full ranges of spine movements. Sarcomere lengths of seven lumbar muscles in each of seven cadaveric donors were measured using laser diffraction. Using published anatomical coordinate data, superior muscle attachment sites were rotated about each intervertebral joint and the total change in muscle length was used to predict sarcomere length operating ranges. The extensor muscles had short sarcomere lengths in a neutral spine posture and there were no statistically significant differences between extensor muscles. The quadratus lumborum was the only muscle with sarcomere lengths that were optimal for force production in a neutral spine position, and the psoas muscles had the longest lengths in this position. During modeled flexion the extensor, quadratus lumborum, and intertransversarii muscles lengthened so that all muscles operated in the approximate same location on the descending limb of the force-length relationship. The intrinsic properties of lumbar muscles are designed to complement each other. The extensor muscles are all designed to produce maximum force in a mid-flexed posture, and all muscles are designed to operate at similar locations of the force-length relationship at full spine flexion., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The sarcomeres of striated muscle are among the most elaborate and dynamic eukaryotic cellular protein machinery, and the mechanisms by which these semicrystalline filament networks are initially patterned and assembled remain contentious. In addition to the acto-myosin filaments that provide motor function, the sarcomere contains titin filaments, comprised of individual molecules of the giant Ig- and fibronectin domain-rich protein titin. Titin is the largest known protein, containing many structurally distinct domains with a variety of proposed functions, including sarcomere stabilization, the prevention of over-stretching, and returning to resting length after contraction. One molecule of titin, which binds to both the Z-disk and the M-line, spans a half-sarcomere, and is proposed to serve as a "molecular ruler" that dictates the spacing of sarcomeres. The semirigid rod-like A-band region of titin has also been proposed to act as a scaffold for thick filament formation during muscle development, but despite decades of research, this hypothesis has not been rigorously tested. Recent studies in zebrafish have brought into question the necessity for the A-band region of titin during the early stages of sarcomere patterning. In this review, we give an overview of the many different roles of titin in the development and function of striated muscle, and address the validity of the "molecular ruler" model of myofibrillogenesis in light of the current literature., (© 2014 Wiley Periodicals, Inc.)

It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, nonmuscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that nonmuscle myosins II A and B are components of the Z-bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells., (© 2014 Wiley Periodicals, Inc.)

The nematode Caenorhabditis elegans has been used as a valuable system to study structure and function of striated muscle. The body wall muscle of C. elegans is obliquely striated muscle with highly organized sarcomeric assembly of actin, myosin, and other accessory proteins. Genetic and molecular biological studies in C. elegans have identified a number of genes encoding structural and regulatory components for the muscle contractile apparatuses, and many of them have counterparts in mammalian cardiac and skeletal muscles or striated muscles in other invertebrates. Applicability of genetics, cell biology, and biochemistry has made C. elegans an excellent system to study mechanisms of muscle contractility and assembly and maintenance of myofibrils. This review focuses on the regulatory mechanisms of structure and function of actin filaments in the C. elegans body wall muscle. Sarcomeric actin filaments in C. elegans muscle are associated with the troponin-tropomyosin system that regulates the actin-myosin interaction. Proteins that bind to the side and ends of actin filaments support ordered assembly of thin filaments. Furthermore, regulators of actin dynamics play important roles in initial assembly, growth, and maintenance of sarcomeres. The knowledge acquired in C. elegans can serve as bases to understand the basic mechanisms of muscle structure and function., (© 2014 Wiley Periodicals, Inc.)

Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT., (Copyright © 2014. Published by Elsevier Ltd.)

The epicardium, which is derived from the proepicardial organ (PE) as the third epithelial layer of the developing heart, is crucial for ventricular morphogenesis. An epicardial deficiency leads to a thin compact layer for the developing ventricle; however, the mechanisms leading to the impaired development of the compact layer are not well understood. Using chick embryonic hearts, we produced epicardium-deficient hearts by surgical ablation or blockade of the migration of PE and examined the mechanisms underlying a thin compact myocardium. Sarcomeric maturation (distance between Z-lines) and cardiomyocyte growth (size) were affected in the thin compact myocardium of epicardium-deficient ventricles, in which the amounts of phospho-smad2 and phospho-ERK as well as expression of transforming growth factor (TGF)β2 and fibroblast growth factor (FGF)2 were reduced. TGFβ and FGF were required for the maturation of sarcomeres and growth of cardiomyocytes in cultured ventricles. In ovo co-transfection of dominant negative (dN)-Alk5 (dN-TGFβ receptor I) and dN-FGF receptor 1 to ventricles caused a thin compact myocardium. Our results suggest that immature sarcomeres and small cardiomyocytes are the causative architectures of an epicardium-deficient thin compact layer and also that epicardium-dependent signaling mediated by TGFβ and FGF plays a role in the development of the ventricular compact layer before the onset of coronary circulation., (© 2014 Japanese Teratology Society.)

Complex 3D beating heart models are now available, but their complexity makes calibration and validation very difficult tasks. We thus propose a systematic approach of deriving simplified reduced-dimensional models, in "0D"-typically, to represent a cardiac cavity, or several coupled cavities-and in "1D"-to model elongated structures such as muscle samples or myocytes. We apply this approach with an earlier-proposed 3D cardiac model designed to capture length-dependence effects in contraction, which we here complement by an additional modeling component devised to represent length-dependent relaxation. We then present experimental data produced with rat papillary muscle samples when varying preload and afterload conditions, and we achieve some detailed validations of the 1D model with these data, including for the length-dependence effects that are accurately captured. Finally, when running simulations of the 0D model pre-calibrated with the 1D model parameters, we obtain pressure-volume indicators of the left ventricle in good agreement with some important features of cardiac physiology, including the so-called Frank-Starling mechanism, the End-Systolic Pressure-Volume Relationship, as well as varying elastance properties. This integrated multi-dimensional modeling approach thus sheds new light on the relations between the phenomena observed at different scales and at the local versus organ levels.

Aim: The study was undertaken to explore the force-velocity relationship under conditions where the myofilament system is subjected to an external force that serves as a negative load and assists the shortening movement., Methods: The experiments were carried out on single muscle fibres isolated from the anterior tibialis muscle of Rana temporaria. The fibres, being operated under load-clamp control, were released to shorten during tetanic stimulation at sarcomere lengths where the fibres carried different degrees of passive tension. The shortening thus occurred while the sarcomeres were subjected to a force that may be characterized as a 'negative load', that is, a force assisting the shortening movement., Results: The force-velocity relationship below zero load was found to be a smooth continuation of the force-velocity curve recorded at positive loads with the shortening velocity increasing steeply at loads below zero. A negative load amounting to merely 10% of the isometric force, thus raised the shortening velocity to a level two to three times higher than V0 , the velocity recorded at zero load., Conclusions: The results provide evidence that, even in the presence of a longitudinal compressive force, the speed of shortening of the muscle fibre is determined by the cycling rate of the interacting cross-bridges. The force-velocity relationship at negative loads may play a relevant part during fast movements of striated muscle as pointed out in the discussion., (© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain (β-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCMsmn). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P < 0.0001) than in HCMsmn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s(-1), respectively). Moreover, compared to HCMsmn, tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 μmol l(-1) s(-1) kN(-1) m(-2), respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCMsmn. Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Many snakes swallow large prey whole, and this process requires large displacements of the unfused tips of the mandibles and passive stretching of the soft tissues connecting them. Under these conditions, the intermandibular muscles are highly stretched but subsequently recover normal function. In the highly stretched condition we observed in snakes, sarcomere length (SL) increased 210% its resting value (SL0), and actin and myosin filaments no longer overlapped. Myofibrils fell out of register and triad alignment was disrupted. Following passive recovery, SLs returned to 82% SL0, creating a region of double-overlapping actin filaments. Recovery required recoil of intracellular titin filaments, elastic cytoskeletal components for realigning myofibrils, and muscle activation. Stretch of whole muscles exceeded that of sarcomeres as a result of extension of folded terminal tendon fibrils, stretching of extracellular elastin and independent slippage of muscle fibers. Snake intermandibular muscles thus provide a unique model of how basic components of vertebrate skeletal muscle can be modified to permit extreme extensibility., (© 2014. Published by The Company of Biologists Ltd.)

Muscle differentiation requires the assembly of high-order structures called myofibrils, composed of sarcomeres. Even though the molecular organization of sarcomeres is well known, the mechanisms underlying myofibrillogenesis are poorly understood. It has been proposed that integrin-dependent adhesion nucleates myofibrils at the periphery of the muscle cell to sustain sarcomere assembly. Here, we report a role for the gene perdido (perd, also known as kon-tiki, a transmembrane chondroitin proteoglycan) in myofibrillogenesis. Expression of perd RNAi in muscles, prior to adult myogenesis, can induce misorientation and detachment of Drosophila adult abdominal muscles. In comparison to controls, perd-depleted muscles contain fewer myofibrils, which are localized at the cell periphery. These myofibrils are detached from each other and display a defective sarcomeric structure. Our results demonstrate that the extracellular matrix receptor Perd has a specific role in the assembly of myofibrils and in sarcomeric organization. We suggest that Perd acts downstream or in parallel to integrins to enable the connection of nascent myofibrils to the Z-bands. Our work identifies the Drosophila adult abdominal muscles as a model to investigate in vivo the mechanisms behind myofibrillogenesis., (© 2014. Published by The Company of Biologists Ltd.)

Cerebral palsy (CP) of the spastic type is a neurological disorder characterized by a velocity-dependent increase in tonic stretch reflexes with exaggerated tendon jerks. Secondary to the spasticity, muscle adaptation is presumed to contribute to limitations in the passive range of joint motion. However, the mechanisms underlying these limitations are unknown. Using biopsies, we compared mechanical as well as histological properties of flexor carpi ulnaris muscle (FCU) from CP patients (n = 29) and healthy controls (n = 10). The sarcomere slack length (mean 2.5 µm, SEM 0.05) and slope of the normalized sarcomere length-tension characteristics of spastic fascicle segments and single myofibre segments were not different from those of control muscle. Fibre type distribution also showed no significant differences. Fibre size was significantly smaller (1933 µm2, SEM 190) in spastic muscle than in controls (2572 µm2, SEM 322). However, our statistical analyses indicate that the latter difference is likely to be explained by age, rather than by the affliction. Quantities of endomysial and perimysial networks within biopsies of control and spastic muscle were unchanged with one exception: a significant thickening of the tertiary perimysium (3-fold), i.e. the connective tissue reinforcement of neurovascular tissues penetrating the muscle. Note that this thickening in tertiary perimysium was shown in the majority of CP patients, however a small number of patients (n = 4 out of 23) did not have this feature. These results are taken as indications that enhanced myofascial loads on FCU is one among several factors contributing in a major way to the aetiology of limitation of movement at the wrist in CP and the characteristic wrist position of such patients.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin-/- mice. An ∼ 2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼ 91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin-/-, mdx4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD.

Background: RV and LV have different embryologic, structural, metabolic, and electrophysiologic characteristics, but whether interventricular differences exist in β-adrenergic (β-AR) responsiveness is unknown. In this study, we examine whether β-AR response and signaling differ in right (RV) versus left (LV) ventricles., Methods and Results: Sarcomere shortening, Ca(2+) transients, ICa,L and IKs currents were recorded in isolated dog LV and RV midmyocytes. Intracellular [cAMP] and PKA activity were measured by live cell imaging using FRET-based sensors. Isoproterenol increased sarcomere shortening ≈10-fold and Ca(2+)-transient amplitude ≈2-fold in LV midmyocytes (LVMs) versus ≈25-fold and ≈3-fold in RVMs. FRET imaging using targeted Epac2camps sensors revealed no change in subsarcolemmal [cAMP], but a 2-fold higher β-AR stimulation of cytoplasmic [cAMP] in RVMs versus LVMs. Accordingly, β-AR regulation of ICa,L and IKs were similar between LVMs and RVMs, whereas cytoplasmic PKA activity was increased in RVMs. Both PDE3 and PDE4 contributed to the β-AR regulation of cytoplasmic [cAMP], and the difference between LVMs and RVMs was abolished by PDE3 inhibition and attenuated by PDE4 inhibition. Finally LV and RV intracavitary pressures were recorded in anesthetized beagle dogs. A bolus injection of isoproterenol increased RV dP/dtmax≈5-fold versus 3-fold in LV., Conclusion: Canine RV and LV differ in their β-AR response due to intrinsic differences in myocyte β-AR downstream signaling. Enhanced β-AR responsiveness of the RV results from higher cAMP elevation in the cytoplasm, due to a decreased degradation by PDE3 and PDE4 in the RV compared to the LV., (© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Mechanosensation and mechanotransduction are fundamental aspects of biology, but the link between physical stimuli and biological responses remains not well understood. The perception of mechanical stimuli, their conversion into biochemical signals, and the transmission of these signals are particularly important for dynamic organs such as the heart. Various concepts have been introduced to explain mechanosensation at the molecular level, including effects on signalosomes, tensegrity, or direct activation (or inactivation) of enzymes. Striated muscles, including cardiac myocytes, differ from other cells in that they contain sarcomeres which are essential for the generation of forces and which play additional roles in mechanosensation. The majority of cardiomyopathy causing candidate genes encode structural proteins among which titin probably is the most important one. Due to its elastic elements, titin is a length sensor and also plays a role as a tension sensor (i.e., stress sensation). The recent discovery of titin mutations being a major cause of dilated cardiomyopathy (DCM) also underpins the importance of mechanosensation and mechanotransduction in the pathogenesis of heart failure. Here, we focus on sarcomere-related mechanisms, discuss recent findings, and provide a link to cardiomyopathy and associated heart failure.

In contrast to isometric and shortening contractions, many observations made on actively lengthening muscles cannot be readily explained with the sliding filament and cross-bridge theory. Specifically, residual force enhancement, the persistent increase in force following active muscle lengthening, beyond what one would expect based on muscle length, has not been explained satisfactorily. Here, we summarize the experimental evidence on residual force enhancement, critically evaluate proposed mechanisms for the residual force enhancement, and propose a mechanism for residual force enhancement that explains all currently agreed upon experimental observations. The proposed mechanism is based on the engagement of the structural protein titin upon muscle activation and an increase in titin's resistance to active compared with passive stretching. This change in resistance from the passive to the active state is suggested to be based on 1) calcium binding by titin upon activation, 2) binding of titin to actin upon activation, and 3) as a consequence of titin-actin binding--a shift toward stiffer titin segments that are used in active compared with passive muscle elongation. Although there is some experimental evidence for the proposed mechanism, it must be stressed that much of the details proposed here remain unclear and should provide ample research opportunities for scientists in the future. Nevertheless, the proposed mechanism for residual force enhancement explains all basic findings in this area of research., (Copyright © 2014 the American Physiological Society.)

In this brief review, recent evidence is presented to indicate a role for specific components of the cardiomyocyte costamere (and its related structure the focal adhesion complex of cultured cardiomyocytes) in initiating and sustaining the aberrant signal transduction that contributes to myocardial remodeling and the progression to heart failure (HF). Special attention is devoted to the focal adhesion kinase family of nonreceptor protein tyrosine kinases in bidirectional signal transduction during cardiac remodeling and HF progression. Finally, some speculations and directions for future study are provided for this rapidly developing field of research.

Recent experiments involving muscle force measurements over a range of muscle lengths show that effects of botulinum toxin (BTX) are complex e.g., force reduction varies as a function of muscle length. We hypothesized that altered conditions of sarcomeres within active parts of partially paralyzed muscle is responsible for this effect. Using finite element modeling, the aim was to test this hypothesis and to study principles of how partial activation as a consequence of BTX affects muscle mechanics. In order to model the paralyzing effect of BTX, only 50% of the fascicles (most proximal, or middle, or most distal) of the modeled muscle were activated. For all muscle lengths, a vast majority of sarcomeres of these BTX-cases were at higher lengths than identical sarcomeres of the BTX-free muscle. Due to such "longer sarcomere effect", activated muscle parts show an enhanced potential of active force exertion (up to 14.5%). Therefore, a muscle force reduction originating exclusively from the paralyzed muscle fiber populations, is compromised by the changes of active sarcomeres leading to a smaller net force reduction. Moreover, such "compromise to force reduction" varies as a function of muscle length and is a key determinant of muscle length dependence of force reduction caused by BTX. Due to longer sarcomere effect, muscle optimum length tends to shift to a lower muscle length. Muscle fiber-extracellular matrix interactions occurring via their mutual connections along full peripheral fiber lengths (i.e., myofascial force transmission) are central to these effects. Our results may help improving our understanding of mechanisms of how the toxin secondarily affects the muscle mechanically., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Frank-Starling's law reflects the ability of the heart to adjust the force of its contraction to changes in ventricular filling, a property based on length-dependent myofilament activation (LDA). The threonine at amino acid 143 of cardiac troponin I (cTnI) is prerequisite for the length-dependent increase in Ca(2+) sensitivity. Thr143 is a known target of protein kinase C (PKC) whose activity is increased in cardiac disease. Thr143 phosphorylation may modulate length-dependent myofilament activation in failing hearts. Therefore, we investigated if pseudo-phosphorylation at Thr143 modulates length dependence of force using troponin exchange experiments in human cardiomyocytes. In addition, we studied effects of protein kinase A (PKA)-mediated cTnI phosphorylation at Ser23/24, which has been reported to modulate LDA. Isometric force was measured at various Ca(2+) concentrations in membrane-permeabilized cardiomyocytes exchanged with recombinant wild-type (WT) troponin or troponin mutated at the PKC site Thr143 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after incubation with exogenous PKA. Pseudo-phosphorylation of Thr143 increased myofilament Ca(2+) sensitivity compared with WT without affecting LDA in failing and donor cardiomyocytes. Subsequent PKA treatment enhanced the length-dependent shift in Ca(2+) sensitivity after WT and 143D exchange. Exchange with Ser23/24 variants demonstrated that pseudo-phosphorylation of both Ser23 and Ser24 is needed to enhance the length-dependent increase in Ca(2+) sensitivity. cTnI pseudo-phosphorylation did not alter length-dependent changes in maximal force. Thus phosphorylation at Thr143 enhances myofilament Ca(2+) sensitivity without affecting LDA, while Ser23/24 bisphosphorylation is needed to enhance the length-dependent increase in myofilament Ca(2+) sensitivity.

The etiology of hypertrophic cardiomyopathy (HCM) has been ascribed to mutations in genes encoding sarcomere proteins. In particular, mutations in MYBPC3, a gene which encodes cardiac myosin binding protein-C (cMyBP-C), have been implicated in over one third of HCM cases. Of these mutations, 70% are predicted to result in C'-truncated protein products, which are undetectable in tissue samples. Heterozygous carriers of these truncation mutations exhibit varying penetrance of HCM, with symptoms often occurring later in life. We hypothesize that heterozygous carriers of MYBPC3 mutations, while seemingly asymptomatic, have subtle functional impairments that precede the development of overt HCM. This study compared heterozygous (+/t) knock-in MYBPC3 truncation mutation mice with wild-type (+/+) littermates to determine whether functional alterations occur at the whole-heart or single-cell level before the onset of hypertrophy. The +/t mice show ∼40% reduction in MYBPC3 transcription, but no changes in cMyBP-C level, phosphorylation status, or cardiac morphology. Nonetheless, +/t mice show significantly decreased maximal force development at sarcomere lengths of 1.9 μm (+/t 68.5 ± 4.1 mN/mm(2) vs. +/+ 82.2 ± 3.2) and 2.3 μm (+/t 79.2 ± 3.1 mN/mm(2) vs. +/+ 95.5 ± 2.4). In addition, heterozygous mice show significant reductions in vivo in the early/after (E/A) (+/t 1.74 ± 0.12 vs. +/+ 2.58 ± 0.43) and E'/A' (+/t 1.18 ± 0.05 vs. +/+ 1.52 ± 0.15) ratios, indicating diastolic dysfunction. These results suggest that seemingly asymptomatic heterozygous MYBPC3 carriers do suffer impairments that may presage the onset of HCM.

Force in striated muscle is due to attachment of the heads of the myosin, the molecular motors extending from the myosin filament, to the actin filament in each half-sarcomere, the functional unit where myosin motors act in parallel. Mechanical and X-ray structural evidence indicates that at the plateau of isometric contraction (force T0), less than half of the elastic strain of the half-sarcomere is due to the strain in the array of myosin motors (s), with the remainder being accounted for by the compliance of filaments acting as linear elastic elements in series with the motor array. Early during the development of isometric force, however, the half-sarcomere compliance has been found to be less than that expected from the linear elastic model assumed above, and this non-linearity may affect the estimate of s. This question is investigated here by applying nanometre-microsecond-resolution mechanics to single intact fibres from frog skeletal muscle at 4 °C, to record the mechanical properties of the half-sarcomere throughout the development of force in isometric contraction. The results are interpreted with mechanical models to estimate the compliance of the myosin motors. Our conclusions are as follows: (i) early during the development of an isometric tetanus, an elastic element is present in parallel with the myosin motors, with a compliance of ∼200 nm MPa(-1) (∼20 times larger than the compliance of the motor array at T0); and (ii) during isometric contraction, s is 1.66 ± 0.05 nm, which is not significantly different from the value estimated with the linear elastic model.

Cardiac myosin-binding protein C is a key regulator of cardiac contractility and is capable of both activating the thin filament to initiate actomyosin motion generation and governing maximal sliding velocities. While MyBP-C's C terminus localizes the molecule within the sarcomere, the N terminus appears to confer regulatory function by binding to the myosin motor domain and/or actin. Literature pertaining to how MyBP-C binding to the myosin motor domain and or actin leads to MyBP-C's dual modulatory roles that can impact actomyosin interactions are discussed.

X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick filament is maintained by an interaction between MyBP-C and the thin filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the filaments decreased in proportion to the overlap between thick and thin filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the thick filaments that do not overlap with thin filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin filaments.

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin.

Cardiac myosin binding protein-C (cMyBP-C) is a cardiac-specific thick filament assembly, accessory, and regulatory protein. Physiologically, it is a key regulator of cardiac contractility. With more than 200 mutations in the cMyBP-C gene directly linked to the development of cardiomyopathy and heart failure, cMyBP-C clearly plays a critical role in heart function. At baseline, cMyBP-C is highly phosphorylated, a condition required for normal cardiac function. However, the level of cMyBP-C phosphorylation is significantly decreased during heart failure, indicating that the level of cMyBP-C phosphorylation is directly linked to signaling and cardiac function. Early studies indicated that cMyBP-C interacts with myosin and titin, whereas studies now show that it also interacts with thin filament proteins. However, the exact role(s) of cMyBP-C in the heart remain(s) to be elucidated. As such, we invited experts in the field of cardiac muscle to identify and address key issues related to cMyBP-C by contributing a mini review on such topics as structure, function, regulation, cardiomyopathy, proteolysis, and gene therapy. Starting from this issue, Pflügers Archiv European Journal of Physiology will publish two invited mini review articles each month to discuss the most recent advances in the study of cMyBP-C.

The response of skeletal muscle to unaccustomed eccentric exercise has been studied widely, yet it is incompletely understood. This review is intended to provide an up-to-date overview of our understanding of how skeletal muscle responds to eccentric actions, with particular emphasis on the underlying molecular and cellular mechanisms of damage and recovery. This review begins by addressing the question of whether eccentric actions result in physical damage to muscle fibers and/or connective tissue. We next review the symptomatic manifestations of eccentric exercise (i.e., indirect damage markers, such as delayed onset muscle soreness), with emphasis on their relatively poorly understood molecular underpinnings. We then highlight factors that potentially modify the muscle damage response following eccentric exercise. Finally, we explore the utility of using eccentric training to improve muscle function in populations of healthy and aging individuals, as well as those living with neuromuscular disorders., (Copyright © 2013 Wiley Periodicals, Inc.)

Skeletal muscles power body movement by converting free energy of ATP hydrolysis into mechanical work. During the landing phase of running or jumping some activated skeletal muscles are subjected to stretch. Upon stretch they absorb body energy quickly and effectively thus protecting joints and bones from impact damage. This is achieved because during lengthening, skeletal muscle bears higher force and has higher instantaneous stiffness than during isometric contraction, and yet consumes very little ATP. We wish to understand how the actomyosin molecules change their structure and interaction to implement these physiologically useful mechanical and thermodynamical properties. We monitored changes in the low angle x-ray diffraction pattern of rabbit skeletal muscle fibers during ramp stretch compared to those during isometric contraction at physiological temperature using synchrotron radiation. The intensities of the off-meridional layer lines and fine interference structure of the meridional M3 myosin x-ray reflection were resolved. Mechanical and structural data show that upon stretch the fraction of actin-bound myosin heads is higher than during isometric contraction. On the other hand, the intensities of the actin layer lines are lower than during isometric contraction. Taken together, these results suggest that during stretch, a significant fraction of actin-bound heads is bound non-stereo-specifically, i.e. they are disordered azimuthally although stiff axially. As the strong or stereo-specific myosin binding to actin is necessary for actin activation of the myosin ATPase, this finding explains the low metabolic cost of energy absorption by muscle during the landing phase of locomotion.

Background: A chronic regimen of flexibility training can increase range of motion, with the increase mechanisms believed to be a change in the muscle material properties or in the neural components associated with this type of training., Methods: This study followed chronic structural adaptations of lateral gastrocnemius muscle of rats submitted to stretching training (3 times a week during 8weeks), based on muscle architecture measurements including pennation angle, muscle thickness and tendon length obtained from ultrasound biomicroscopic images, in vivo. Fiber length and sarcomere number per 100μm were determined in 3 fibers of each muscle (ex vivo and in vitro, respectively), using conventional optical microscopy., Findings: Stretching training resulted in a significant pennation angle reduction of the stretched leg after 12 sessions (25%, P=0.002 to 0.024). Muscle thickness and tendon length presented no significant changes. Fiber length presented a significant increase for the stretched leg (8.5%, P=0.00006), with the simultaneous increase in sarcomere length (5%, P=0.041) since the stretched muscles presented less sarcomeres per 100μm., Interpretation: A stretching protocol with characteristics similar to those applied in humans was sufficient to modify muscle architecture of rats with absence of a sarcomerogenesis process. The results indicate that structural adaptations take place in skeletal muscle tissue submitted to moderate-intensity stretching training., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Skeletal muscle exhibiting complex and excellent precision has evolved for millions of years. Skeletal muscle has better performance and simpler structure compared with existing driving modes. Artificial muscle may be designed by analyzing and imitating properties and structure of skeletal muscle based on bionics, which has been focused on by bionic researchers, and a structure mode of linear electromagnetic array artificial muscle has been designed in this paper. Half sarcomere is the minimum unit of artificial muscle and electromagnetic model has been built. The structural parameters of artificial half sarcomere actuator were optimized to achieve better movement performance. Experimental results show that artificial half sarcomere actuator possesses great motion performance such as high response speed, great acceleration, small weight and size, robustness, etc., which presents a promising application prospect of artificial half sarcomere actuator.

In development and differentiation, morphological changes often accompany mechanical changes [1], but it is unclear whether or when cells in embryos sense tissue elasticity. The earliest embryo is uniformly pliable, while adult tissues vary widely in mechanics from soft brain and stiff heart to rigid bone [2]. However, cell sensitivity to microenvironment elasticity is debated based in part on results from complex three-dimensional culture models [3]. Regenerative cardiology provides strong motivation to clarify any cell-level sensitivities to tissue elasticity because rigid postinfarct regions limit pumping by the adult heart [4]. Here, we focus on the spontaneously beating embryonic heart and sparsely cultured cardiomyocytes, including cells derived from pluripotent stem cells. Tissue elasticity, Et, increases daily for heart to 1-2 kPa by embryonic day 4 (E4), and although this is ~10-fold softer than adult heart, the beating contractions of E4 cardiomyocytes prove optimal at ~Et,E4 both in vivo and in vitro. Proteomics reveals daily increases in a small subset of proteins, namely collagen plus cardiac-specific excitation-contraction proteins. Rapid softening of the heart's matrix with collagenase or stiffening it with enzymatic crosslinking suppresses beating. Sparsely cultured E4 cardiomyocytes on collagen-coated gels likewise show maximal contraction on matrices with native E4 stiffness, highlighting cell-intrinsic mechanosensitivity. While an optimal elasticity for striation proves consistent with the mathematics of force-driven sarcomere registration, contraction wave speed is linear in Et as theorized for excitation-contraction coupled to matrix elasticity. Pluripotent stem cell-derived cardiomyocytes also prove to be mechanosensitive to matrix and thus generalize the main observation that myosin II organization and contractile function are optimally matched to the load contributed by matrix elasticity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The 3-O-sulfotransferase (3-OST) family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4) expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function., Competing Interests: The authors have declared that no competing interests exist.

Lipoteichoic acid (LTA) is the key pathogenic factor of gram-positive bacteria and contributes significantly to organ dysfunction in sepsis, a frequent complication in critical care patients. We hypothesized that LTA directly affects cardiomyocyte function, thus contributing to cardiac failure in sepsis. This study was designed to evaluate the effects of LTA on contractile properties and calcium-transients of isolated adult rat cardiomyocytes. When myocytes were exposed to LTA for 1h prior to analysis, the amplitudes of calcium-transients as well as sarcomere shortening increased to 130% and 142% at 1 Hz stimulation frequency. Relengthening of sarcomeres as well as decay of calcium-transients was accelerated after LTA incubation. Exposure to LTA for 24 h resulted in significant depression of calcium-transients as well as of sarcomere shortening compared to controls. One of the major findings of our experiments is that LTA most likely affects calcium-handling of the cardiomyocytes. The effect is exacerbated by reduced extracellular calcium, which resembles the clinical situation in septic patients. Functionally, an early stimulating effect of LTA with increased contractility of the cardiomyocytes may be an in vitro reflection of early hyperdynamic phases in clinical sepsis. Septic disorders have been shown to induce late hypodynamic states of the contractile myocardium, which is also supported at the single-cell level in vitro by results of our 24h-exposure to LTA., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT), and related drug raloxifene (Ral) on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca²⁺ transient amplitude. Effects were primarily observed at 5 and 10 μM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca²⁺ transient amplitudes. At 10 μM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca²⁺ handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy.

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg's release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin's increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.

Skeletal muscle shortens faster against a lower load. This force-velocity relationship is the fundamental determinant of muscle performance in vivo and is due to ATP-driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half-sarcomere. Crystallographic studies suggest that the working stroke is associated with the release of phosphate (Pi) and consists of 70 deg tilting of a light-chain domain that connects the catalytic domain of the myosin motor to the myosin tail and filament. However, the coupling of the working stroke with Pi release is still an unsolved question. Using nanometre-microsecond mechanics on skinned muscle fibres, we impose stepwise drops in force on an otherwise isometric contraction and record the isotonic velocity transient, to measure the mechanical manifestation of the working stroke of myosin motors and the rate of its regeneration in relation to the half-sarcomere load and [Pi]. We show that the rate constant of the working stroke is unaffected by [Pi], while the subsequent transition to steady velocity shortening is accelerated. We propose a new chemo-mechanical model that reproduces the transient and steady state responses by assuming that: (i) the release of Pi from the catalytic site of a myosin motor can occur at any stage of the working stroke, and (ii) a myosin motor, in an intermediate state of the working stroke, can slip to the next actin monomer during filament sliding. This model explains the efficient action of muscle molecular motors working as an ensemble in the half-sarcomere.

Divergent effects of α- and β-myosin heavy chain (MHC) isoforms on contractile behavior arise mainly because of their impact on thin filament cooperativity. The N terminus of cardiac troponin T (cTnT) also modulates thin filament cooperativity. Our hypothesis is that the impact of the N terminus of cTnT on thin filament activation is modulated by a shift from α- to β-MHC isoform. We engineered two recombinant proteins by deleting residues 1-43 and 44-73 in rat cTnT (RcTnT): RcTnT(1-43Δ) and RcTnT(44-73Δ), respectively. Dynamic and steady-state contractile parameters were measured at sarcomere length of 2.3 µm after reconstituting proteins into detergent-skinned muscle fibers from normal (α-MHC) and propylthiouracil-treated (β-MHC) rat hearts. α-MHC attenuated Ca(2+)-activated maximal tension (∼46%) in RcTnT(1-43Δ) fibers. In contrast, β-MHC decreased tension only by 19% in RcTnT(1-43Δ) fibers. Both α- and β-MHC did not affect tension in RcTnT(44-73Δ) fibers. The instantaneous muscle fiber stiffness measurements corroborated the divergent impact of α- and β-MHC on tension in RcTnT(1-43Δ) fibers. pCa50 (-log of [Ca(2+)]free required for half-maximal activation) decreased significantly by 0.13 pCa units in α-MHC + RcTnT(1-43Δ) fibers but remained unaltered in β-MHC + RcTnT(1-43Δ) fibers, demonstrating that β-MHC counteracted the attenuating effect of RcTnT(1-43Δ) on myofilament Ca(2+) sensitivity. β-MHC did not alter the sudden stretch-mediated recruitment of new cross-bridges (ER) in RcTnT(1-43Δ) fibers, but α-MHC attenuated ER by 36% in RcTnT(1-43Δ) fibers. The divergent impact of α- and β-MHC on how the N terminus of cTnT modulates contractile dynamics has implications for heart disease; alterations in cTnT and MHC are known to occur via changes in isoform expression or mutations.

The in situ structural coupling between the cardiac troponin (cTn) Ca(2+)-sensitive regulatory switch (CRS) and strong myosin cross-bridges was investigated using Förster resonance energy transfer (FRET). The double cysteine mutant cTnC(T13C/N51C) was fluorescently labeled with the FRET pair 5-(iodoacetamidoethyl)aminonaphthelene-1-sulfonic acid (IAEDENS) and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) and then incorporated into detergent skinned left ventricular papillary fiber bundles. Ca(2+) titrations of cTnC(T13C/N51C)AEDENS/DDPM-reconstituted fibers showed that the Ca(2+)-dependence of the opening of the N-domain of cTnC (N-cTnC) statistically matched the force-Ca(2+) relationship. N-cTnC opening still occurred steeply during Ca(2+) titrations in the presence of 1mM vanadate, but the maximal extent of ensemble-averaged N-cTnC opening and the Ca(2+)-sensitivity of the CRS were significantly reduced. At nanomolar, resting Ca(2+) levels, treatment with ADP·Mg in the absence of ATP caused a partial opening of N-cTnC. During subsequent Ca(2+) titrations in the presence of ADP·Mg and absence of ATP, further N-cTnC opening was stimulated as the CRS responded to Ca(2+) with increased Ca(2+)-sensitivity and reduced steepness. These findings supported our hypothesis here that strong cross-bridge interactions with the cardiac thin filament exert a Ca(2+)-sensitizing effect on the CRS by stabilizing the interaction between the exposed hydrophobic patch of N-cTnC and the switch region of cTnI., (Copyright © 2013 Elsevier Inc. All rights reserved.)

At an intermediate activation level, striated muscle exhibits autonomous oscillations called SPOC, in which the basic contractile units, sarcomeres, oscillate in length, and various oscillatory patterns such as traveling waves and their disrupted forms appear in a myofibril. Here we show that these patterns are reproduced by mechanically connecting in series the unit model that explains characteristics of SPOC at the single-sarcomere level. We further reduce the connected model to phase equations, revealing that the combination of local and global couplings is crucial to the emergence of these patterns.

Thirty beef carcasses were harvested and the foreshank of each side was independently positioned (cranial, natural, parallel, or caudal) 1h post-mortem to determine the effect of foreshank angle at rigor mortis on the sarcomere length and tenderness of six beef shoulder muscles. The infraspinatus (IS), pectoralis profundus (PP), serratus ventralis (SV), supraspinatus (SS), teres major (TM) and triceps brachii (TB) were excised 48 h post-mortem for Warner-Bratzler shear force (WBSF) and sarcomere length evaluations. All muscles except the SS had altered (P<0.05) sarcomere lengths between positions; the cranial position resulted in the longest sarcomeres for the SV and TB muscles whilst the natural position had longer sarcomeres for the PP and TM muscles. The SV from the cranial position had lower (P<0.05) shear than the caudal position and TB from the natural position had lower (P<0.05) shear than the parallel or caudal positions. Sarcomere length was moderately correlated (r=-0.63; P<0.01) to shear force., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Mutations in cardiac myosin binding protein C (cMyBP-C) are prevalent causes of hypertrophic cardiomyopathy (HCM). Although HCM-causing truncation mutations in cMyBP-C are well studied, the growing number of disease-related cMyBP-C missense mutations remain poorly understood. Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT). Wild-type and human E258K cMyBP-C were expressed in mECT lacking endogenous mouse cMyBP-C through adenoviral-mediated gene transfer. Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere. Functionally, expression of E258K cMyBP-C caused accelerated contractile kinetics and severely compromised twitch force amplitude in mECT. Yeast two-hybrid analysis revealed that E258K cMyBP-C abolished interaction between the N terminal of cMyBP-C and myosin heavy chain sub-fragment 2 (S2). Furthermore, this mutation increased the affinity between the N terminal of cMyBP-C and actin. Assessment of phosphorylation of three serine residues in cMyBP-C showed that aberrant phosphorylation of cMyBP-C is unlikely to be responsible for altering these interactions. We show that the E258K mutation in cMyBP-C abolishes interaction between N-terminal cMyBP-C and myosin S2 by directly disrupting the cMyBP-C-S2 interface, independent of cMyBP-C phosphorylation. Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics. Additionally, the E258K mutation impaired force production of mECT, which suggests that in addition to the loss of physiological function, this mutation disrupts contractility possibly by tethering the thick and thin filament or acting as an internal load.

Aims: Familial hypertrophic cardiomyopathy (HCM), frequently caused by sarcomeric gene mutations, is characterized by cellular dysfunction and asymmetric left-ventricular (LV) hypertrophy. We studied whether cellular dysfunction is due to an intrinsic sarcomere defect or cardiomyocyte remodelling., Methods and Results: Cardiac samples from 43 sarcomere mutation-positive patients (HCMmut: mutations in thick (MYBPC3, MYH7) and thin (TPM1, TNNI3, TNNT2) myofilament genes) were compared with 14 sarcomere mutation-negative patients (HCMsmn), eight patients with secondary LV hypertrophy due to aortic stenosis (LVHao) and 13 donors. Force measurements in single membrane-permeabilized cardiomyocytes revealed significantly lower maximal force generating capacity (Fmax) in HCMmut (21 ± 1 kN/m²) and HCMsmn (26 ± 3 kN/m²) compared with donor (36 ± 2 kN/m²). Cardiomyocyte remodelling was more severe in HCMmut compared with HCMsmn based on significantly lower myofibril density (49 ± 2 vs. 63 ± 5%) and significantly higher cardiomyocyte area (915 ± 15 vs. 612 ± 11 μm²). Low Fmax in MYBPC3mut, TNNI3mut, HCMsmn, and LVHao was normalized to donor values after correction for myofibril density. However, Fmax was significantly lower in MYH7mut, TPM1mut, and TNNT2mut even after correction for myofibril density. In accordance, measurements in single myofibrils showed very low Fmax in MYH7mut, TPM1mut, and TNNT2mut compared with donor (respectively, 73 ± 3, 70 ± 7, 83 ± 6, and 113 ± 5 kN/m²). In addition, force was lower in MYH7mut cardiomyocytes compared with MYBPC3mut, HCMsmn, and donor at submaximal [Ca²⁺]., Conclusion: Low cardiomyocyte Fmax in HCM patients is largely explained by hypertrophy and reduced myofibril density. MYH7 mutations reduce force generating capacity of sarcomeres at maximal and submaximal [Ca²⁺]. These hypocontractile sarcomeres may represent the primary abnormality in patients with MYH7 mutations.

The latissimus dorsi is primarily considered a muscle with actions at the shoulder, despite its widespread attachments at the spine. There is some dispute regarding the potential contribution of this muscle to lumbar spine function. The architectural design of a muscle is one of the most accurate predictors of muscle function; however, detailed architectural data on the latissimus dorsi muscle are limited. Therefore, the aim of this study was to quantify the architectural properties of the latissimus dorsi muscle and model mechanical function in light of these new data. One latissimus dorsi muscle was removed from each of 12 human cadavers, separated into regions, and micro-dissected for quantification of fascicle length, sarcomere length, and physiological cross-sectional area. From these data, sarcomere length operating ranges were modelled to determine the force-length characteristics of latissimus dorsi across the spine and shoulder ranges of motion. The physiological cross-sectional area of latissimus dorsi was 5.6±0.5 cm2 and normalized fascicle length was 26.4±1.0 cm, indicating that this muscle is designed to produce a moderate amount of force over a large range of lengths. Measured sarcomere length in the post-mortem neutral spine posture was nearly optimal at 2.69±0.06 μm. Across spine range of motion, biomechanical modelling predicted latissimus dorsi acts across both the ascending and descending limbs of the force-length curve during lateral bend, and primarily at or near the plateau region (where maximum force generation is possible) during flexion/extension and axial twist. Across shoulder range of motion, latissimus dorsi acts primarily on the plateau region and descending limbs of the force length curve during both flexion/extension and abduction/adduction. These data provide novel insights into the ability of the latissimus dorsi muscle to generate force and change length throughout the spine and shoulder ranges of motion. In addition, these findings provide an improved understanding of the spine and shoulder positions at which the force-generating capacity of this muscle can become jeopardized, and consequently how this may affect its spine-stabilizing ability., (© 2013 Anatomical Society.)

Myosin binding protein-C (MyBP-C) exists in three major isoforms: slow skeletal, fast skeletal, and cardiac. While cardiac MyBP-C (cMyBP-C) expression is restricted to the heart in the adult, it is transiently expressed in neonatal stages of some skeletal muscles. However, it is unclear whether this expression is necessary for the proper development and function of skeletal muscle. Our aim was to determine whether the absence of cMyBP-C alters the structure, function, or MyBP-C isoform expression in adult skeletal muscle using a cMyBP-C null mouse model (cMyBP-C((t/t))). Slow MyBP-C was expressed in both slow and fast skeletal muscles, whereas fast MyBP-C was mostly restricted to fast skeletal muscles. Expression of these isoforms was unaffected in skeletal muscle from cMyBP-C((t/t)) mice. Slow and fast skeletal muscles in cMyBP-C((t/t)) mice showed no histological or ultrastructural changes in comparison to the wild-type control. In addition, slow muscle twitch, tetanus tension, and susceptibility to injury were all similar to the wild-type controls. Interestingly, fMyBP-C expression was significantly increased in the cMyBP-C((t/t)) hearts undergoing severe dilated cardiomyopathy, though this does not seem to prevent dysfunction. Additionally, expression of both slow and fast isoforms was increased in myopathic skeletal muscles. Our data demonstrate that i) MyBP-C isoforms are differentially regulated in both cardiac and skeletal muscles, ii) cMyBP-C is dispensable for the development of skeletal muscle with no functional or structural consequences in the adult myocyte, and iii) skeletal isoforms can transcomplement in the heart in the absence of cMyBP-C.

The goal of this study was to compare the effects of Ca(2+) and MgADP activation on force development in skeletal muscles during and after imposed length changes. Single fibres dissected from the rabbit psoas were (i) activated in pCa(2+)4.5 and pCa(2+)6.0, or (ii) activated in pCa(2+)4.5 before and after administration of 10 mM MgADP. Fibres were activated in sarcomere lengths (SL) of 2.65 µm and 2.95 µm, and subsequently stretched or shortened (5%SL at 1.0 SL.s(-1)) to reach a final SL of 2.80 µm. The kinetics of force during stretch were not altered by pCa(2+) or MgADP, but the fast change in the slope of force development (P1) observed during shortening and the corresponding SL extension required to reach the change (L1) were higher in pCa(2+)6.0 (P1 = 0.22 ± 0.02 Po; L1 = 5.26 ± 0.24 nm.HS(.1)) than in pCa(2+)4.5 (P1 = 0.15 ± 0.01 Po; L1 = 4.48 ± 0.25 nm.HS(.1)). L1 was also increased by MgADP activation during shortening. Force enhancement after stretch was lower in pCa(2+)4.5 (14.9 ± 5.4%) than in pCa(2+)6.0 (38.8 ± 7.5%), while force depression after shortening was similar in both Ca(2+) concentrations. The stiffness accompanied the force behavior after length changes in all situations. MgADP did not affect the force behavior after length changes, and stiffness did not accompany the changes in force development after stretch. Altogether, these results suggest that the mechanisms of force generation during and after stretch are different from those obtained during and after shortening.

Background: Diastolic dysfunction is a poorly understood but clinically pervasive syndrome that is characterized by increased diastolic stiffness. Titin is the main determinant of cellular passive stiffness. However, the physiological role that the tandem immunoglobulin (Ig) segment of titin plays in stiffness generation and whether shortening this segment is sufficient to cause diastolic dysfunction need to be established., Methods and Results: We generated a mouse model in which 9 Ig-like domains (Ig3-Ig11) were deleted from the proximal tandem Ig segment of the spring region of titin (IG KO). Exon microarray analysis revealed no adaptations in titin splicing, whereas novel phospho-specific antibodies did not detect changes in titin phosphorylation. Passive myocyte stiffness was increased in the IG KO, and immunoelectron microscopy revealed increased extension of the remaining titin spring segments as the sole likely underlying mechanism. Diastolic stiffness was increased at the tissue and organ levels, with no consistent changes in extracellular matrix composition or extracellular matrix-based passive stiffness, supporting a titin-based mechanism for in vivo diastolic dysfunction. Additionally, IG KO mice have a reduced exercise tolerance, a phenotype often associated with diastolic dysfunction., Conclusions: Increased titin-based passive stiffness is sufficient to cause diastolic dysfunction with exercise intolerance.

The cellular basis of the Frank-Starling "Law of the Heart" is the length-dependence of activation, but the mechanisms by which the sarcomere detects length changes and converts this information to altered calcium sensitivity has remained elusive. Here the effect of titin-based passive tension on the length-dependence of activation (LDA) was studied by measuring the tension-pCa relation in skinned mouse LV muscle at two sarcomere lengths (SLs). N2B KO myocardium, where the N2B spring element in titin is deleted and passive tension is elevated, was compared to WT myocardium. Myofilament lattice structure was studied with low-angle X-ray diffraction; the myofilament lattice spacing (d1,0) was measured as well as the ratio of the intensities of the 1,1 and 1,0 diffraction peaks (I1,1/I1,0) as an estimate of the degree of association of myosin heads with the thin filaments. Experiments were carried out in skinned muscle in which the lattice spacing was reduced with Dextran-T500. Experiments with and without lattice compression were also carried out following PKA phosphorylation of the skinned muscle. Under all conditions that were tested, LDA was significantly larger in N2B KO myocardium compared to WT myocardium, with the largest differences following PKA phosphorylation. A positive correlation between passive tension and LDA was found that persisted when the myofilament lattice was compressed with Dextran and that was enhanced following PKA phosphorylation. Low-angle X-ray diffraction revealed a shift in mass from thin filaments to thick filaments as sarcomere length was increased. Furthermore, a positive correlation was obtained between myofilament lattice spacing and passive tension and the change in I1,1/I1,0 and passive tension and these provide possible explanations for how titin-based passive tension might regulate calcium sensitivity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mutations in cardiac myosin binding protein C (MyBP-C) are a common cause of familial hypertrophic cardiomyopathy (FHC). The majority of MyBP-C mutations are expected to reduce MyBP-C expression; however, the consequences of MyBP-C deficiency on the regulation of myofilament function, Ca²⁺ homeostasis, and in vivo cardiac function are unknown. To elucidate the effects of decreased MyBP-C expression on cardiac function, we employed MyBP-C heterozygous null (MyBP-C+/-) mice presenting decreases in MyBP-C expression (32%) similar to those of FHC patients carrying MyBP-C mutations. The levels of MyBP-C phosphorylation were reduced 53% in MyBP-C+/- hearts compared with wild-type hearts. Skinned myocardium isolated from MyBP-C+/- hearts displayed decreased cross-bridge stiffness at half-maximal Ca²⁺ activations, increased steady-state force generation, and accelerated rates of cross-bridge recruitment at low Ca²⁺ activations (<15% and <25% of maximum, respectively). Protein kinase A treatment abolished basal differences in rates of cross-bridge recruitment between MyBP-C+/- and wild-type myocardium. Intact ventricular myocytes from MyBP-C+/- hearts displayed abnormal sarcomere shortening but unchanged Ca²⁺ transient kinetics. Despite a lack of left ventricular hypertrophy, MyBP-C+/- hearts exhibited elevated end-diastolic pressure and decreased peak rate of LV pressure rise, which was normalized following dobutamine infusion. Furthermore, electrocardiogram recordings in conscious MyBP-C+/- mice revealed prolonged QRS and QT intervals, which are known risk factors for cardiac arrhythmia. Collectively, our data show that reduced MyBP-C expression and phosphorylation in the sarcomere result in myofilament dysfunction, contributing to contractile dysfunction that precedes compensatory adaptations in Ca²⁺ handling, and chamber remodeling. Perturbations in mechanical and electrical activity in MyBP-C+/- mice could increase their susceptibility to cardiac dysfunction and arrhythmia.

Little is known about the contraction and relaxation properties of fetal skeletal muscle, and measurements thus far have been made with non-human mammalian muscle. Data on human fetal skeletal muscle contraction are lacking, and there are no published reports on the kinetics of either fetal or adult human skeletal muscle myofibrils. Understanding the contractile properties of human fetal muscle would be valuable in understanding muscle development and a variety of muscle diseases that are associated with mutations in fetal muscle sarcomere proteins. Therefore, we characterised the contractile properties of developing human fetal skeletal muscle and compared them to adult human skeletal muscle and rabbit psoas muscle. Electron micrographs showed human fetal muscle sarcomeres are not fully formed but myofibril formation is visible. Isolated myofibril mechanical measurements revealed much lower specific force, and slower rates of isometric force development, slow phase relaxation, and fast phase relaxation in human fetal when compared to human adult skeletal muscle. The duration of slow phase relaxation was also significantly longer compared to both adult groups, but was similarly affected by elevated ADP. F-actin sliding on human fetal skeletal myosin coated surfaces in in vitro motility (IVM) assays was much slower compared with adult rabbit skeletal myosin, though the Km(app) (apparent (fitted) Michaelis-Menten constant) of F-actin speed with ATP titration suggests a greater affinity of human fetal myosin for nucleotide binding. Replacing ATP with 2 deoxy-ATP (dATP) increased F-actin speed for both groups by a similar amount. Titrations of ADP into IVM assays produced a similar inhibitory affect for both groups, suggesting ADP binding may be similar, at least under low load. Together, our results suggest slower but similar mechanisms of myosin chemomechanical transduction for human fetal muscle that may also be limited by immature myofilament structure.