Range Of Sarcomere Lengths Research Articles

Titin function in the active sarcomere studied in frog muscle fibers upon inhibition of myosin motors by para-nitro-blebbistatin.

Titin spans the whole half-sarcomere, through the I-band connecting the Z-line to the myosin filament tip, and through the A-band, associated to myosin filament, up to M-line. Titin in the I-band transmits the stress also without attached myosin motors and is responsible for the passive force developed when a skeletal muscle fibre is stretched at rest. However, the passive stiffness in the range of physiological sarcomere lengths (SL, <2.7 µm) is too low to account for a putative role of I-band titin as mechanical link able to sustain mechanosensing in myosin filaments and prevent development of sarcomere inhomogeneity during contraction. Here we define titin mechanics during tetanic stimulation of intact frog muscle fibers using 20 μM para-nitro-blebbistatin (which fully inhibits myosin ATPase in vitro) to suppress the myosin-based mechanical responses. Small-angle X-ray fiber diffraction at the European Synchrotron ESRF demonstrates that the resting structure of thick filament is fully preserved upon tetanic stimulation. Under these conditions we use stepwise changes in force (10-50 pN per half-thick filament, htf) to elicit the lengthening transient that defines titin dynamic stiffness in situ at 2.3–3.0 µm SL. We show that, upon tetanic stimulation titin switches from the OFF-state characterised by a large, SL-dependent, extensibility, to the ON-state in which it opposes stretching with an effective stiffness one-two order of magnitude larger (∼3 pN·nm−1·htf−1) and independent of SL. The results demonstrate that, in the active sarcomere working at physiological lengths, I-band titin is an efficient mechanical link between the Z-line and the myosin filament tip, a condition for playing a role in mechanosensing-based myosin filament activation and for opposing to development of SL inhomogeneity during contraction. Supported by ECRF, University of Florence and MUR (Italy), ESRF (France) and EJP-RD.

Corn Snakes Show Consistent Sarcomere Length Ranges Across Muscle Groups and Ontogeny.

The force-generating capacity of muscle depends upon many factors including the actin-myosin filament overlap due to the relative length of the sarcomere. Consequently, the force output of a muscle may vary throughout its range of motion, and the body posture allowing maximum force generation may differ even in otherwise similar species. We hypothesized that corn snakes would show an ontogenetic shift in sarcomere length range from being centered on the plateau of the length-tension curve in small individuals to being on the descending limb in adults. Sarcomere lengths across the plateau would be advantageous for locomotion, while the descending limb would be advantageous for constriction due to the increase in force as the coil tightens around the prey. To test this hypothesis, we collected sarcomere lengths from freshly euthanized corn snakes, preserving segments in straight and maximally curved postures, and quantifying sarcomere length via light microscopy. We dissected 7 muscles (spinalis, semispinalis, multifidus, longissimus dorsi, iliocostalis (dorsal and ventral), and levator costae) in an ontogenetic series of corn snakes (mass = 80–335 g) at multiple regions along the body (anterior, middle, and posterior). Our data shows all of the muscles analyzed are on the descending limb of the length-tension curve at rest across all masses, regions, and muscles analyzed, with muscles shortening onto or past the plateau when flexed. While these results are consistent with being advantageous for constriction at all sizes, there could also be unknown benefits of this sarcomere arrangement for locomotion or striking.

Shortening the thick filament by partial deletion of titin's C-zone alters cardiac function by reducing the operating sarcomere length range

Titin's C-zone is an inextensible segment in titin, comprised of 11 super-repeats and located in the cMyBP-C-containing region of the thick filament. Previously we showed that deletion of titin's super-repeats C1 and C2 (TtnΔC1–2 model) results in shorter thick filaments and contractile dysfunction of the left ventricular (LV) chamber but that unexpectedly LV diastolic stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the LV and intact cardiomyocyte, cellular work loops of intact cardiomyocytes, Ca2+ transients, cross-bridge kinetics, and myofilament Ca2+ sensitivity. Intact cardiomyocytes of TtnΔC1–2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance at both LV and single-cell levels showed that activation kinetics are normal in TtnΔC1–2 mice, but that relaxation is slower. The slowed relaxation is, in part, attributable to an increased myofilament Ca2+ sensitivity and slower early Ca2+ reuptake. Cross-bridge dynamics showed that cross-bridge kinetics are normal but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements revealed that in TtnΔC1–2 mice the operating SL range of the LV is shifted towards shorter lengths. This normalizes the apparent cell and LV diastolic stiffness but further reduces systolic force as systole occurs further down on the ascending limb of the force-SL relation. We propose that the reduced working SLs reflect titin's role in regulating diastolic stiffness by altering the number of sarcomeres in series. Overall, our study reveals that thick filament length regulation by titin's C-zone is critical for normal cardiac function.

Abstract P315: Shortening The Thick Filament By Partial Deletion Of Titin’S C-zone Alters Cardiac Function By Reducing The Operating Sarcomere Length Range Of The Heart.

Titin’s C-zone is the inextensible part of titin that binds along the thick filament at its cMyBP-C -containing region. Previously it was shown that deletion of titin’s super-repeats C1 and C2 ( Ttn ΔC1-2 mouse model) results in shorter thick filaments and contractile dysfunction, but LV chamber stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the left ventricle (LV) and from cellular work loops of intact loaded cardiac myocytes. Ca 2+ transients were also measured as well as crossbridge cycling kinetics and Ca 2+ sensitivity of force. It was found that intact cardiomyocytes of Ttn ΔC1-2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance of the LV chamber showed that the kinetics of LV activation are normal but that relaxation is slower in Ttn ΔC1-2 mice. The slowed relaxation was, in part, attributable to an increased myofilament Ca 2+ sensitivity and slower early Ca 2+ reuptake. Dynamic stiffness at the myofilament level showed that cross-bridge kinetics are normal, but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements in the mid-wall region of the LV revealed that the operating SL range is shifted in Ttn ΔC1-2 mice towards shorter lengths. This normalizes the apparent cell and LV chamber stiffness but reduces the number of force generating cross-bridges due to suboptimal thin and thick filament overlap. Thus the contractile dysfunction in Ttn ΔC1-2 mice is not only due to shorter thick filaments but also to a reduced operating sarcomere length range. Overall these results reveal that for normal cardiac function, thick filament length regulation by titin’s C-zone is critical.

The effects of inorganic phosphate on contractile function of slow skeletal muscle fibres are length-dependent

Muscle operates across a wide range of sarcomere lengths. Inorganic phosphate (Pi) diminishes force output of striated muscle, with greater influence at short relative to long sarcomere lengths in fast skeletal and cardiac muscle fibres. The purpose of this study was to fill a gap in the literature regarding the length-dependent effects of Pi on contractile function of slow skeletal muscle fibres. Permeabilized slow skeletal muscle fibres from rabbit soleus were assessed at average sarcomere lengths of 2.0, 2.4, or 2.8 μm, with and without 20 mM Pi added to activating solutions (22±1 °C). The magnitude of Pi-induced reductions in peak force (43 ± 7% at 2.0 μm, 38 ± 7% at 2.4 μm, and 31 ± 8% at 2.8 μm) and peak stiffness (41 ± 9% at 2.0 μm, 36 ± 8% at 2.4 μm, and 26 ± 9% at 2.8 μm) were length dependent. Peak stiffness was less affected by Pi than peak force. Pi diminished the Ca2+-sensitivity of the force-pCa and stiffness-pCa relationships to a greater extent at 2.8 μm than 2.0 μm. Comparable results were obtained from a cooperative model of Ca2+ and myosin binding to regulated actin. In conclusion, Pi is more detrimental to the peak force output of slow skeletal muscle fibres held at short relative to long sarcomere lengths, whereas Pi has a greater effect on the Ca2+-sensitivity of force production at long relative to short sarcomere lengths. Stiffness data suggest that Pi-induced reductions in force are primarily due to fewer bound cross-bridges, with a lesser contribution attributable to lower average force per cross-bridge.

Alterations of Extracellular Matrix Mechanical Properties Contribute to Age-Related Functional Impairment of Human Skeletal Muscles.

Aging of human skeletal muscles is associated with increased passive stiffness, but it is still debated whether muscle fibers or extracellular matrix (ECM) are the determinants of such change. To answer this question, we compared the passive stress generated by elongation of fibers alone and arranged in small bundles in young healthy (Y: 21 years) and elderly (E: 67 years) subjects. The physiological range of sarcomere length (SL) 2.5–3.3 μm was explored. The area of ECM between muscle fibers was determined on transversal sections with picrosirius red, a staining specific for collagen fibers. The passive tension of fiber bundles was significantly higher in E compared to Y at all SL. However, the resistance to elongation of fibers alone was not different between the two groups, while the ECM contribution was significantly increased in E compared to Y. The proportion of muscle area occupied by ECM increased from 3.3% in Y to 8.2% in E. When the contribution of ECM to bundle tension was normalized to the fraction of area occupied by ECM, the difference disappeared. We conclude that, in human skeletal muscles, the age-related reduced compliance is due to an increased stiffness of ECM, mainly caused by collagen accumulation.

Age-related changes in human single muscle fibre passive elastic properties are sarcomere length dependent.

Studies have revealed an age-related decrease in the ability to produce force as well as an increase in the contractile stiffness and passive stress of single muscle fibres. However, further insight into age-related changes to the passive properties of human skeletal muscles is needed. The aim of this study was to characterize single muscle fibre passive properties from young and old males across a physiologic range of sarcomere lengths (SLs). Ten young ([YM]: mean age: 25.4years) and ten old ([OM]: mean age: 68.9years) males participated. Vastus lateralis muscles were biopsied and 182 fibres were tested and analyzed (90 YM and 92 OM) using a cumulative stretch-relaxation protocol. Passive mechanical characteristics of each fibre were obtained by fitting the integral of the logistic function to experimental stress-SL data. Muscle fibres from older individuals had significantly greater passive elastic moduli at short SL (1.9-2.65μm) compared with young (p<0.05). This resulted in a significantly larger passive stress for SLs between 2.1μm and 3.55μm for older individuals when compared with young (p<0.05). These results provide novel insight into the length dependency of changes in single muscle fibre passive mechanical properties with age.

Contracting striated muscle has a dynamic I-band spring with an undamped stiffness 100 times larger than the passive stiffness.

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 2.7-3.1µm, 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. 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 4kHz 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>2.7µm attains a maximum constant value of ∼6pNnm-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.

Stretch-Induced Activation of the Myosin Motors on the Thick Filament in Rat Cardiac Trabeculae

The contractility of cardiac muscle is regulated by thick-filament based mechanisms in addition to the classical calcium/thin-filament mediated mechanisms. Here we studied the role of structural changes of the thick filament in length-dependent activation in cardiac muscle. We used fluorescence polarization from bifunctional sulphorhodamine probes on the N- and C-lobes of the myosin regulatory light chain (RLC) to monitor changes in the orientation of the myosin motors induced by increasing sarcomere length in relaxed and partially calcium-activated demembranated trabeculae. Under relaxing conditions at near physiological temperature and lattice spacing the myosin motors are roughly parallel to the filament axis, consistent with the OFF structure of the thick filament. Cooling of the relaxed trabecula induced a more perpendicular orientation of the myosin motors indicating disruption of the OFF-state. Application of a staircase stretch protocol in the sarcomere length range 2.0-2.3 μm (25°C, 3% dextran T500) at pCa 9.0 also induced a more perpendicular orientation of the myosin motors, but despite an increase in passive tension to ∼30% of the maximum active force (T0), the orientation change was only ∼4% of that associated with full calcium activation. Larger stretch-induced orientation changes were observed at pCa 7 (∼7%) and at pCa 6.6 (∼20%), the latter accompanied by an increase in active force of ∼0.05 T0. RLC phosphorylation further increased the stretch-induced orientation changes to ∼14% and ∼33% respectively, and the active force response at pCa 6.6 increased to ∼0.2 T0. These results indicate that increasing sarcomere length in the physiological range at diastolic calcium concentration induces activation of the myosin motors and that this effect is enhanced by RLC phosphorylation, suggesting that changes in thick filament structure may mediate length-dependent activation. Supported by Wellcome Trust and BHF, UK.

Mitochondrial Deformation During the Cardiac Mechanical Cycle.

ABSTRACTCardiomyocytes both cause and experience continual cyclic deformation. The exact effects of this deformation on the properties of intracellular organelles are not well characterized, although they are likely to be relevant for cardiomyocyte responses to active and passive changes in their mechanical environment. In the present study we provide three‐dimensional ultrastructural evidence for mechanically induced mitochondrial deformation in rabbit ventricular cardiomyocytes over a range of sarcomere lengths representing myocardial tissue stretch, an unloaded “slack” state, and contracture. We also show structural indications for interaction of mitochondria with one another, as well as with other intracellular elements such as microtubules, sarcoplasmic reticulum and T‐tubules. The data presented here help to contextualize recent reports on the mechanosensitivity and cell‐wide connectivity of the mitochondrial network and provide a structural framework that may aide interpretation of mechanically‐regulated molecular signaling in cardiac cells. Anat Rec, 302:146–152, 2019. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

High tension in sarcomeres hinders myocardial relaxation: A computational study.

Experiments have shown that the relaxation phase of cardiac sarcomeres during an isometric twitch is prolonged in muscles that reached a higher peak tension. However, the mechanism is not completely understood. We hypothesize that the binding of calcium to troponin is enhanced by the tension in the thin filament, thus contributing to the prolongation of contraction upon higher peak tension generation. To test this hypothesis, we developed a computational model of sarcomere mechanics that incorporates tension-dependence of calcium binding. The model was used to simulate isometric twitch experiments with time dependency in the form of a two-state cross-bridge cycle model and a transient intracellular calcium concentration. In the simulations, peak isometric twitch tension appeared to increase linearly by 51.1 KPa with sarcomere length from 1.9 μm to 2.2 μm. Experiments showed an increase of 47.3 KPa over the same range of sarcomere lengths. The duration of the twitch also increased with both sarcomere length and peak intracellular calcium concentration, likely to be induced by the inherently coupled increase of the peak tension in the thin filament. In the model simulations, the time to 50% relaxation (tR50) increased over the range of sarcomere lengths from 1.9 μm to 2.2 μm by 0.11s, comparable to the increased duration of 0.12s shown in experiments. Model simulated tR50 increased by 0.12s over the range of peak intracellular calcium concentrations from 0.87 μM to 1.45 μM. Our simulation results suggest that the prolongation of contraction at higher tension is a result of the tighter binding of Ca2+ to troponin in areas under higher tension, thus delaying the deactivation of the troponin.

A Spatially Explicit Model Shows How Titin Stiffness Modulates Muscle Mechanics and Energetics.

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.

Cardiomyocyte Hypocontractility and Reduced Myofibril Density in End-Stage Pediatric Cardiomyopathy.

Dilated cardiomyopathy amongst children (pediatric cardiomyopathy, pediatric CM) is associated with a high morbidity and mortality. Because little is known about the pathophysiology of pediatric CM, treatment is largely based on adult heart failure therapy. The reason for high morbidity and mortality is largely unknown as well as data on cellular pathomechanisms is limited. Here, we assessed cardiomyocyte contractility and protein expression to define cellular pathomechanisms in pediatric CM. Explanted heart tissue of 11 pediatric CM patients and 18 controls was studied. Contractility was measured in single membrane-permeabilized cardiomyocytes and protein expression was assessed with gel electrophoresis and western blot analysis. We observed increased Ca2+-sensitivity of myofilaments which was due to hypophosphorylation of cardiac troponin I, a feature commonly observed in adult DCM. We also found a significantly reduced maximal force generating capacity of pediatric CM cardiomyocytes, as well as a reduced passive force development over a range of sarcomere lengths. Myofibril density was reduced in pediatric CM compared to controls. Correction of maximal force and passive force for myofibril density normalized forces in pediatric CM cardiomyocytes to control values. This implies that the hypocontractility was caused by the reduction in myofibril density. Unlike in adult DCM we did not find an increase in compliant titin isoform expression in end-stage pediatric CM. The limited ability of pediatric CM patients to maintain myofibril density might have contributed to their early disease onset and severity.

Increased Non-Uniformity in In Vivo Sarcomere Length during a Tetanic Contraction

IntroductionThe maximal, steady-state, isometric force produced by a muscle depends on its sarcomere length (SL). In previous studies in single myofibrils, it was found that sarcomere length non-uniformities increased during activation and force production. However, single myofibrils lack much of the structural proteins that provide stability to entire muscles, thus the observed SL non-uniformities in myofibrils might not occur in whole muscles. This study was aimed at investigating the change in SL distribution during tetanic contractions in an intact muscles of live mice.MethodsMice were anaesthetized using isoflurane. The proximal femur and foot of the left lower limb were clamped. The skin over the left tibialis anterior (TA) muscle was opened and stretched to form a bath for a saline solution that allowed for imaging using a water-immersion objective. The TA was supra-maximally stimulated using a nerve cuff electrode on the sciatic nerve using 0.1ms square wave pulses at 60Hz for 1s. SL were measured using second harmonic generation microscopy for the fully stretched TA. Measurements were made for the passive and activated TA over an area of 160x3 µm2 in the mid-belly of the muscle.ResultsSLs for the passive muscle were 2.53±0.06µm (mean±sd). During contraction, sarcomeres shortened by ∼12% to 2.24±0.12µm. The coefficient of variation of the SLs doubled from 2.4% at rest to 5.2% during tetanic contractions. The range of SLs increased from the passive (2.36-2.71µm) to the active state (1.85-2.62µm).ConclusionSL non-uniformity doubled during muscle activation and differed by more than 0.7 µm. The functional implications of these massive SL non-uniformities need to be explored, and the common practice of representing muscles with a single SL value needs to be reconsidered.

Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (ktr) in a length‐dependent fashion

Key points Regulatory light chain (RLC) phosphorylation has been shown to alter the ability of muscle to produce force and power during shortening and to alter the rate of force redevelopment (k tr) at submaximal [Ca2+].Increasing RLC phosphorylation ∼50% from the in vivo level in maximally [Ca2+]‐activated cardiac trabecula accelerates k tr.Decreasing RLC phosphorylation to ∼70% of the in vivo control level slows k tr and reduces force generation. k tr is dependent on sarcomere length in the physiological range 1.85–1.94 μm and RLC phosphorylation modulates this response.We demonstrate that Frank–Starling is evident at maximal [Ca2+] activation and therefore does not necessarily require length‐dependent change in [Ca2+]‐sensitivity of thin filament activation.The stretch response is modulated by changes in RLC phosphorylation, pinpointing RLC phosphorylation as a modulator of the Frank–Starling law in the heart.These data provide an explanation for slowed systolic function in the intact heart in response to RLC phosphorylation reduction. Force and power in cardiac muscle have a known dependence on phosphorylation of the myosin‐associated regulatory light chain (RLC). We explore the effect of RLC phosphorylation on the ability of cardiac preparations to redevelop force (k tr) in maximally activating [Ca2+]. Activation was achieved by rapidly increasing the temperature (temperature‐jump of 0.5–20ºC) of permeabilized trabeculae over a physiological range of sarcomere lengths (1.85–1.94 μm). The trabeculae were subjected to shortening ramps over a range of velocities and the extent of RLC phosphorylation was varied. The latter was achieved using an RLC‐exchange technique, which avoids changes in the phosphorylation level of other proteins. The results show that increasing RLC phosphorylation by 50% accelerates k tr by ∼50%, irrespective of the sarcomere length, whereas decreasing phosphorylation by 30% slows k tr by ∼50%, relative to the k tr obtained for in vivo phosphorylation. Clearly, phosphorylation affects the magnitude of k tr following step shortening or ramp shortening. Using a two‐state model, we explore the effect of RLC phosphorylation on the kinetics of force development, which proposes that phosphorylation affects the kinetics of both attachment and detachment of cross‐bridges. In summary, RLC phosphorylation affects the rate and extent of force redevelopment. These findings were obtained in maximally activated muscle at saturating [Ca2+] and are not explained by changes in the Ca2+‐sensitivity of acto‐myosin interactions. The length‐dependence of the rate of force redevelopment, together with the modulation by the state of RLC phosphorylation, suggests that these effects play a role in the Frank–Starling law of the heart.

The Load Dependence of the Size and the Speed of the Working Stroke of Cardiac Myosin in Situ

Cardiac performance depends on power developed by the myocardium and mutations of cardiac myosin, which affect power output, have been proposed to be responsible for various forms of cardiomyopathy. Precise measurements of force and filament sliding generated by cardiac myosin are lacking. Here sarcomere-level mechanics are applied for the first time to intact trabeculae from the right ventricle of the rat to determine the size and the speed of the working stroke of cardiac myosin in situ and their dependency on the load. The isotonic shortening transients elicited by stepwise drops in force to 0.2-0.8 of peak force (Tp) are recorded at sarcomere length of 1.9 and 2.2 μm and external Ca2+-concentrations ([Ca2+]o) 1 and 2.5 mM (temperature 27 oC). The size and the speed of the early rapid phase of shortening (the mechanical manifestation of the myosin working stroke) increase with the reduction of the load from approximately 3 nm per half-sarcomere and 1000 s−1 at high load to 6 nm per half-sarcomere and 7000 s−1 at low load, but are not affected by the increase of sarcomere length or [Ca2+]o. Thus the two-fold increase of force and power in the range of sarcomere length and [Ca2+]o explored are solely due to the increase in number of myosin motors. These results demonstrate the possibility for in situ studies of the molecular basis of cardiomyopathies. Supported by MIUR-PRIN and Telethon (Italy).

Nano-imaging of the beating mouse heart in vivo: Importance of sarcomere dynamics, as opposed to sarcomere length per se, in the regulation of cardiac function.

Sarcomeric contraction in cardiomyocytes serves as the basis for the heart's pump functions in mammals. Although it plays a critical role in the circulatory system, myocardial sarcomere length (SL) change has not been directly measured in vivo under physiological conditions because of technical difficulties. In this study, we developed a high speed (100-frames per second), high resolution (20-nm) imaging system for myocardial sarcomeres in living mice. Using this system, we conducted three-dimensional analysis of sarcomere dynamics in left ventricular myocytes during the cardiac cycle, simultaneously with electrocardiogram and left ventricular pressure measurements. We found that (a) the working range of SL was on the shorter end of the resting distribution, and (b) the left ventricular-developed pressure was positively correlated with the SL change between diastole and systole. The present findings provide the first direct evidence for the tight coupling of sarcomere dynamics and ventricular pump functions in the physiology of the heart.

Sarcomere‐length dependence of myosin filament structure in skeletal muscle fibres of the frog

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 3.5μm 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 2.6-3.0μm 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.

Muscle and fascicle excursion in children with cerebral palsy

BackgroundFascicle length and fascicle excursion measurements in children with cerebral palsy have yielded inconsistent results. The purpose of this study was to measure in vivo passive fascicle lengths and fascicle excursions in the Medial Gastrocnemius muscle of children with cerebral palsy and typically developing controls. MethodsWe measured 11 children with spastic cerebral palsy and 14 controls between the ages of 9 and 16years. Ultrasound imaging was used to measure fascicle lengths while a dynamometer moved the ankle joint through the range of motion. A common range of motion for all subjects was used for analysis of fascicle excursion. FindingsFascicle lengths in children with cerebral palsy were 43% smaller than those for control subjects throughout the range of motion. The relative fascicle excursion was 92% greater on average for the cerebral palsy compared to the control group children. The muscle excursion for the control group children was greater than for the cerebral palsy group children. InterpretationSince the fascicles in children with spastic cerebral palsy are shorter, but they go through the same excursion as fascicles in typically developing children, sarcomeres within the medial gastrocnemius muscle must be working over a larger range of sarcomere lengths. Combined with findings of overstretched sarcomeres in spastic muscles reported in the literature, our results suggest that the increased passive forces and the weakness found in spastic muscles may be caused by a decrease in contractile filament overlap as sarcomeres are pulled to extreme lengths in children with cerebral palsy.

Gender Differences in Passive Tension in Hypertrophic Cardiomyopathy Patients

BackgroundHypertrophic cardiomyopathy (HCM) is an inherited cardiac disorder with a prevalence of 1:500. In ∼65% of all HCM patients the causative mutation is identified. HCM patients that are sarcomere-mutation negative tend to have a less severe phenotype. Mutations in the MYBPC3 and MYH7 genes encoding cardiac myosin-binding protein C (cMyMP-C) and β-myosin heavy chain (MyHC) represent >80% of all genotyped HCM cases. HCM is characterized by asymmetric hypertrophy of the left ventricle and diastolic dysfunction. In the present study we investigated if passive stiffness of the sarcomeres may underlie diastolic dysfunction.MethodsIn-vitro passive tension measurements were done at sarcomere lengths of 1.8 to 2.2μm in cardiomyocytes from 10 sarcomere mutation-negative patients(SMN: 5 male, 5 female), 17 patients carrying a MYBPC3 mutation(MYBPC3: 10 male, 7 female), and 10 patients carrying a MYH7 mutation (MYH7: 5 male, 5 female). Tissue was obtained during myectomy surgery from the interventricular septum. Cardiomyocytes were mechanically isolated and Triton-permeabilized.ResultsPassive tension over the entire range of sarcomere lengths did not differ between sarcomere-mutation positive and mutation-negative male HCM patients. Passive tension in myocytes from sarcomere mutation-positive women was significantly higher compared to female mutation-negative HCM patients. Female MYH7 cardiomyocytes showed a higher sarcomere stiffness compared to male MYH7.ConclusionOur measurements suggest that high sarcomere passive stiffness may contribute to diastolic dysfunction in female HCM patients harboring a mutation in genes encoding thick filament proteins.