Sarcomere Dynamics Research Articles

CARDIAC CONTRACTILITY MEASURES OF LEFT VENTRICULAR SYSTOLIC FUNCTIONAL ASSESSMENT OF NORMAL AND DISEASED HEARTS

Left ventricular (LV) contraction is the basis of LV systolic function, impairment of which underlies heart failure pathophysiology. Its accurate quantification in the form of LV contractility indices is imperative for diagnostic and follow-up assessment of LV systolic function in heart failure. Herein, we analyze LV contractile performance by focusing on LV contractility indices at different physiological organizational levels: from sarcomere dynamics to LV myocardial properties (such as elastic modulus and elastance), and from LV wall contractile stress development to the generation of intra-LV blood flow velocities and pressure distributions. Further, we present the development analyses of these indices and their medical applications. Using improved development of invasive and noninvasive techniques for measuring ventricular pressure, geometry, and volume, we show how these indices have become more amenable for clinical usage to obtain better patient assessment. The purpose of this paper is to present a comprehensive coverage of LV contraction physiology, indices to qualify LV contraction, formulation, and medical applications of some major intrinsic LV contractility indices, so as to provide the basis of functional assessment of normal versus diseased hearts.

Sarcomere dynamics in skeletal muscle myofibrils during isometric contractions

The main goal of this study was to evaluate the dynamics of sarcomeres during isometric activation of skeletal muscle myofibrils. Rabbit psoas myofibrils ( n=14) were attached between a pair of cantilevers for force measurements at one side and a rigid glass needle at the other side, and their images were used for measurements of individual sarcomere lengths (SL) during contractions. Myofibrils were set at average SL between 2.13 and 3.06 μm, and were activated and held isometric for 20–35 s during which SL and force were continuously measured. SL dispersion increased from the rest state to activation, but it remained mostly constant during the activation period. Even with the length non-uniformity developed during myofibril activation, most sarcomeres stabilized their length changes during the isometric contraction. As a result, sarcomeres contracted at different degrees of filament overlap while producing similar forces. When the myofibrils were separated in two groups that produced force at averaged short (≤2.5 μm) or long (≥2.5 μm) SL, the initial non-uniformity was greater in long lengths, but changes observed in sarcomeres during the activation period were similar, suggesting that sarcomere stability is not length-dependent.

Titin-induced force enhancement and force depression: A ‘sticky-spring’ mechanism in muscle contractions?

The sliding filament and crossbridge theories do not suffice to explain a number of muscle experiments. For example, from the entire muscle to myofibrils, predictions of these theories were shown to underestimate the force output during and after active tissue stretch. The converse applies to active tissue shortening. In addition to the crossbridge cycle, we propose that another molecular mechanism is effective in sarcomere force generation. We suggest that, when due to activation, myosin binding sites are available on actin, the giant protein titin's PEVK region attaches itself to the actin filament at those sites. As a result, the molecular spring length is dramatically reduced. This leads to increased passive force when the sarcomere is stretched and to decreased or even negative passive force when the sarcomere shortens. Moreover, during shortening, the proposed mechanism interferes with active-force production by inhibiting crossbridges. Incorporation of a simple ‘sticky-spring’ mechanism model into a Hill-type model of sarcomere dynamics offers explanations for several force-enhancement and force-depression effects. For example, the increase of the sarcomere force compared to the force predicted solely by the sliding filament and crossbridge theories depends on the stretch amplitude and on the working range. The same applies to the decrease of sarcomere force during and after shortening. Using only literature data for its parameterization, the model predicts forces similar to experimental results.

Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

Muscle contraction results from force-generating interactions between myosin cross-bridges on the thick filament and actin on the thin filament. The force-generating interactions are regulated by Ca(2+) via specialised proteins of the thin filament. It is controversial how the contractile and regulatory systems dynamically interact to determine the time course of muscle contraction and relaxation. Whereas kinetics of Ca(2+)-induced thin-filament regulation is often investigated with isolated proteins, force kinetics is usually studied in muscle fibres. The gap between studies on isolated proteins and structured fibres is now bridged by recent techniques that analyse the chemical and mechanical kinetics of small components of a muscle fibre, subcellular myofibrils isolated from skeletal and cardiac muscle. Formed of serially arranged repeating units called sarcomeres, myofibrils have a complete fully structured ensemble of contractile and Ca(2+) regulatory proteins. The small diameter of myofibrils (few micrometres) facilitates analysis of the kinetics of sarcomere contraction and relaxation induced by rapid changes of [ATP] or [Ca(2+)]. Among the processes studied on myofibrils are: (1) the Ca(2+)-regulated switch on/off of the troponin complex, (2) the chemical steps in the cross-bridge adenosine triphosphatase cycle, (3) the mechanics of force generation and (4) the length dynamics of individual sarcomeres. These studies give new insights into the kinetics of thin-filament regulation and of cross-bridge turnover, how cross-bridges transform chemical energy into mechanical work, and suggest that the cross-bridge ensembles of each half-sarcomere cooperate with each other across the half-sarcomere borders. Additionally, we now have a better understanding of muscle relaxation and its impairment in certain muscle diseases.

Automatic step-detection algorithm for analysis of sarcomere dynamics

Motile systems exhibit a stepwise nature, seen most prominently in muscle contraction. A novel algorithm has been developed that detects steps automatically in sarcomere-length change data and computes their size. The method is based on a nonlinear filter and a step detection protocol that detects local slope values in comparison to a threshold. The algorithm was first evaluated using artificial data with various degrees of Gaussian noise. Steps were faithfully detected even with significant noise. With actual data, the algorithm detected 2.7 nm steps and integer multiples thereof. The results confirm a quantal 2.7 nm step-size reported earlier. As stepwise phenomena become increasingly evident, automatic step-detection algorithms become increasingly useful since the limiting factor is almost always noise. The algorithm presented here offers a versatile and accurate tool that should be useful not only within muscle contraction and motility fields, but in fields which quantal phenomena play a role.

Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans

Sarcomeres are the basic contractile units of striated muscle. Our knowledge about sarcomere dynamics has primarily come from in vitro studies of muscle fibres and analysis of optical diffraction patterns obtained from living muscles. Both approaches involve highly invasive procedures and neither allows examination of individual sarcomeres in live subjects. Here we report direct visualization of individual sarcomeres and their dynamical length variations using minimally invasive optical microendoscopy to observe second-harmonic frequencies of light generated in the muscle fibres of live mice and humans. Using microendoscopes as small as 350 microm in diameter, we imaged individual sarcomeres in both passive and activated muscle. Our measurements permit in vivo characterization of sarcomere length changes that occur with alterations in body posture and visualization of local variations in sarcomere length not apparent in aggregate length determinations. High-speed data acquisition enabled observation of sarcomere contractile dynamics with millisecond-scale resolution. These experiments point the way to in vivo imaging studies demonstrating how sarcomere performance varies with physical conditioning and physiological state, as well as imaging diagnostics revealing how neuromuscular diseases affect contractile dynamics.

Spontaneous waves in muscle fibres

Mechanical oscillations are important for many cellular processes, e.g. the beating of cilia and flagella or the sensation of sound by hair cells. These dynamic states originate from spontaneous oscillations of molecular motors. A particularly clear example of such oscillations has been observed in muscle fibers under non-physiological conditions. In that case, motor oscillations lead to contraction waves along the fiber. By a macroscopic analysis of muscle fiber dynamics we find that the spontaneous waves involve non-hydrodynamic modes. A simple microscopic model of sarcomere dynamics highlights mechanical aspects of the motor dynamics and fits with the experimental observations.

Abstract 144: Erythropoietin Induces Positive Inotropy And Lusitropy Mediated By Phosphatidylinositol 3-kinase And Novel PKC Isoforms In Murine Cardiomyocytes

Introduction Clinical studies indicate a potentially beneficial effect of erythropoietin (EPO) in patients with anemia and heart failure. Moreover, EPO is known to be cardioprotective in ischemia-reperfusion models, an effect mediated through a cardiac EPO receptor. However it is not known if EPO has a direct effect on cardiac contractility. Methods Isolated cardiomyocytes were obtained from C57BL/6 mice and exposed to either 50 U/mL of EPO or to a buffer solution. Cells were paced at 8 Hz. Sarcomere shortening dynamics were measured using a Fourier video transformation method. Global intracellular Ca 2+ dynamics were determined after loading cells with fura 2-AM. EPO concentrations from 0.1 – 50 U/mL and exposure times from 0 – 90 min were used for dose finding and time course experiments. The phosphatidylinositol 3-kinase (PI3K) blocker Wortmannin, the non-isozyme specific PKC blocker Chelerythrine or the PKC α and β blocker Gö6976 were administered concurrently with EPO to determine the signaling pathways involved. Results In control cells peak sarcomere shortening was 2.3 ± 0.3 %, while in EPO treated cells it was 4.4 ± 0.5 % (P=0.007, N=40 cells in each group from 5 hearts). Baseline sarcomere length was 1.55 ± 0.04 μm in controls compared with 1.72 ± 0.04 μm in EPO cells (P=0.008). Both shortening velocity [2.2 ± 0.3 μm/s versus 4.2 ± 0.5 μm/s (P=0.01)] and relengthening velocity [1.3 ± 0.2 μm/s versus 3.5 ± 0.5 μm/s (P=0.002)] were higher with EPO treatment. Ca 2+ dynamics studied in 25 cells in each group showed no differences between EPO treated and control cells. Positive inotropic and lusitropic effects occurred at EPO concentrations above 1 U/mL and after an incubation period of at least 30 min. The EPO mediated increase in peak sarcomere shortening was abrogated by concurrent PI3K blockade or by non-isozyme specific PKC blockade. However, it was preserved if only the classical PKC isozymes αand β were blocked. Conclusions In addition to its antiapoptotic and cytoprotective effects, EPO has direct positive inotropic and lusitropic effects in single murine cardiomyocytes. These are mediated by PI3K and novel PKC isoforms and are independent of changes in intracellular Ca 2+ handling, suggesting a PKC δor PKC ϵ mediated increase in myofilament contractile function.

Sarcomere dynamics during muscular contraction and their implications to muscle function

This article attempts to identify the key aspects of sarcomere inhomogeneity and the dynamics of sarcomere length changes in muscle contraction experiments and focuses on understanding the mechanics of myofibrils or muscle fibres when viewed as independent units of biological motors (the half-sarcomeres) connected in series. Muscle force generation has been interpreted traditionally on the basis of the kinetics of crossbridge cycling, i.e. binding of myosin heads to actin and consecutive force generating conformational change of the head, under controlled conditions and assuming uniformity of sarcomere or half-sarcomere behaviour. However, several studies have shown that re-distribution of internal strain within myofibrils and muscle fibres may be a key player, particularly, during stretch or relaxation so that force kinetics parameters are strongly affected by sarcomere dynamics. Here, we aim to shed light on how force generation, crossbridge kinetics, and the complex sarcomere movements are to be linked and which mechanical concepts are necessary to develop a comprehensive contraction model of a myofibril.

Reply from I. A. Telley, R. Stehle, K. W. Ranatunga, G. Pfitzer, E. Stüssi and J. Denoth

Reply from I. A. Telley, R. Stehle, K. W. Ranatunga, G. Pfitzer, E. Stüssi and J. Denoth

Sarcomere popping requires stretch over a range where total tension decreases with length

Sarcomere popping requires stretch over a range where total tension decreases with length

Origin of complex force kinetics in striated muscles: cross-bridge kinetics or sarcomere dynamics?

Origin of complex force kinetics in striated muscles: cross-bridge kinetics or sarcomere dynamics?

Cardiomyocyte function associated with hyperactivity and/or hypertension in genetic models of LV hypertrophy

We examined cardiomyocyte intracellular calcium ([Ca2+]i) dynamics and sarcomere shortening dynamics in genetic rat models of left ventricular (LV) hypertrophy associated with or without hypertension (HT) and with or without hyperactive (HA) behavior. Previous selective breeding of the spontaneously hypertensive rat (SHR) strain, which is HA and HT, with the Wistar-Kyoto (WKY) rat strain, which is not hyperactive (NA) and not hypertensive (NT), has led to two unique strains: the WKHA strain, selected for HA and NT, and the WKHT strain, selected for NA and HT. Cardiomyocytes were isolated from young adult males and females of each strain, paced at 2, 3, and 4 Hz in 1.2 mM external Ca2+ concentration at 37 degrees C, and cardiomyocyte [Ca2+]i and sarcomere dynamics were recorded simultaneously. Under these conditions, LV cardiomyocyte systolic and diastolic [Ca2+]i dynamics and diastolic sarcomere dynamics in the WKHT were significantly enhanced compared with WKY controls, suggesting an underlying LV hypertrophic response that successfully compensated for HT in the absence of HA. LV cardiomyocyte [Ca2+]i dynamics in the WKHA and SHR were strikingly similar to each other and only slightly reduced compared with WKY. LV cardiomyocyte systolic and diastolic sarcomere dynamics, on the other hand, were significantly reduced in the SHR compare with WKHA and more so in male than in female SHR. We conclude from these data that HT alone is an insufficient descriptor of the cause of LV hypertrophy and diminished LV cardiomyocyte function in the SHR rat. These data further suggest that HA (augmented by male sex) in the SHR may interact with the HT state to initiate impaired cardiomyocyte function and thereby inhibit or undermine an otherwise compensatory response that may occur with HT in the absence of HA.

Caveolin-3 at the T-tubule Colocalizes with .ALPHA.-actinin in the Adult Murine Cardiac Muscle

Caveolin-3 is thought to serve as a structural and a scaffolding protein in the caveolae of striated muscle. The present study aims to clarify the precise localization of caveolin-3 and its interactive protein partners in adult murine cardiac muscle by immunohistochemistry, immunoblot and immunoprecipitation. Double labeling confocal images showed that caveolin-3 is colocalized with dihydropyridine receptor α1, ryanodine receptor, and α-actinin, but not sarcoplasmic or endoplasmic reticulum calcium ATPase 2. Immunoelectron microscopy demonstrated that caveolin-3 is localized at the T-tubules. Subsequently, we investigated the protein interaction between caveolin-3 and α-actinin, a major component of the Z-band, using immunoprecipitation and immunoblot. It was of interest to note that caveolin-3 was coprecipitated with α-actinin in cardiac muscle extraction but not in skeletal muscle. Taken together, these data indicate that caveolin-3 serves as a mediator between sarcomeric dynamics via the Z-bands and T-tubule function in cardiac muscle.

Dynamics of individual sarcomeres during and after stretch in activated single myofibrils.

It is generally assumed that sarcomere lengths (SLs) change in isometric fibres following activation and following stretch on the descending limb of the force-length relationship, because of an inherent instability. Although this assumption has never been tested directly, instability and SL non-uniformity have been associated with several mechanical properties, such as 'creep' and force enhancement. The aim of this study was to test directly the hypothesis that sarcomeres are unstable on the descending limb of the force-length relationship. We used single myofibrils, isolated from rabbit psoas, that were attached to glass needles that allowed for controlled stretching of myofibrils. Images of the sarcomere striation pattern were projected onto a linear photodiode array, which was scanned at 20 Hz to produce dark-light patterns corresponding to the A- and I-bands, respectively. Starting from a mean SL of 2.55 +/- 0.07 microm, stretches of 11.2 +/- 1.6% of SL at a speed of 118.9 +/- 5.9 nm s(-1) were applied to the activated myofibrils (pCa(2+) = 4.75). SLs along the myofibril were non-uniform before, during and after the stretch, but with few exceptions, they remained constant during the isometric period before stretch, and during the extended isometric period after stretch. Sarcomeres never lengthened to a point beyond thick and thin filament overlap. We conclude that sarcomeres are non-uniform but generally stable on the descending limb of the force-length relationship.

Instabilities in the Transient Response of Muscle

We investigate the isometric transient response of muscle using a quantitative stochastic model of the actomyosin cycle based on the swinging lever-arm hypothesis. We first consider a single pair of filaments, and show that when values of parameters such as the lever-arm displacement and the cross-bridge elasticity are chosen to provide effective energy transduction, the T 2 curve (the tension recovered immediately after a step displacement) displays a region of negative slope. If filament compliance and the discrete nature of the binding sites are taken into account, the negative slope is diminished, but not eliminated. This implies that there is an instability in the dynamics of individual half sarcomeres. However, when the symmetric nature of whole sarcomeres is taken into account, filament rearrangement becomes important during the transient: as tension is recovered, some half sarcomeres lengthen whereas others shorten. This leads to a flat T 2 curve, as observed experimentally. In addition, we investigate the isotonic transient response and show that for a range of parameter values the model displays damped oscillations, as recently observed in experiments on single muscle fibers. We conclude that it is essential to consider the collective dynamics of many sarcomeres, rather than the dynamics of a single pair of filaments, when interpreting the transient response of muscle.

In vivo and in vitro heterogeneity of segment length changes in the semimembranosus muscle of the toad.

Many studies examine sarcomere dynamics in single fibres or length-tension dynamics in whole muscles in vivo or in vitro, but few studies link the various levels of organisation. To relate data addressing in vitro muscle segment behaviour with in vivo whole muscle behaviour during locomotion, we measured in vivo strain patterns of muscle segments using three sonomicrometry crystals implanted along a fascicle of the semimembranosus muscle in the American toad (Bufo americanus; n = 6) during hopping. The centre crystal emitted an ultrasonic signal, while the outer crystals received the signal allowing the instantaneous measurement of lengths from two adjacent muscle segments. On the first day, we recorded from the central and distal segments. On the second day of recordings, the most distal crystal was moved to a proximal position to record from a proximal segment and the same central segment. When the toads hopped a distance of two body lengths, the proximal and central segments strained -15.1 +/- 6.1 and -14.0 +/- 4.9 % (i.e. shortening), respectively. Strain of the distal segment, however, was significantly lower and more variable in pattern, often lengthening before shortening during a hop. From rest length, the distal segment initially lengthened by 2.6 +/- 2.0 % before shortening by 6.5 +/- 3.2 % at the same hop distance. Under in vitro conditions, the central segment always shortened more than the distal segment, except when passively cycled, during which the segments strained similarly. When the whole muscle was cycled sinusoidally and stimulated phasically in vitro, the two adjacent segments strained in opposite directions over much (up to 34 %) of the cycle. These differences in strain amplitude and direction imply that two adjacent segments can not only produce and/or absorb varying amounts of mechanical energy, but can also operate on different regions of their force-length and force-velocity relationships when activated by the same neural signal. Understanding regional differences in contractile dynamics within muscles is therefore important to linking our understanding of sarcomere behaviour with whole muscle behaviour during locomotion.

Force Kinetics and Individual Sarcomere Dynamics in Cardiac Myofibrils after Rapid Ca 2+ Changes

Kinetics of force development and relaxation after rapid application and removal of Ca 2+ were measured by atomic force cantilevers on subcellular bundles of myofibrils prepared from guinea pig left ventricles. Changes in the structure of individual sarcomeres were simultaneously recorded by video microscopy. Upon Ca 2+ application, force developed with an exponential rate constant k ACT almost identical to k TR, the rate constant of force redevelopment measured during steady-state Ca 2+ activation; this indicates that k ACT reflects isometric cross-bridge turnover kinetics. The kinetics of force relaxation after sudden Ca 2+ removal were markedly biphasic. An initial slow linear decline (rate constant k LIN) lasting for a time t LIN was abruptly followed by an ∼20 times faster exponential decay (rate constant k REL). k LIN is similar to k TR measured at low activating [Ca 2+], indicating that k LIN reflects isometric cross-bridge turnover kinetics under relaxed-like conditions (see also Tesi et al., 2002. Biophys. J. 83:2142–2151). Video microscopy revealed the following: invariably at t LIN a single sarcomere suddenly lengthened and returned to a relaxed-type structure. Originating from this sarcomere, structural relaxation propagated from one sarcomere to the next. Propagated sarcomeric relaxation, along with effects of stretch and P i on relaxation kinetics, supports an intersarcomeric chemomechanical coupling mechanism for rapid striated muscle relaxation in which cross-bridges conserve chemical energy by strain-induced rebinding of P i.

Concentration and Elongation of Attached Cross-bridges as Pressure Determinants in a Ventricular Model

A ventricular model based on a muscle model relating sarcomere dynamics to Ca2+kinetics was used to establish the relative contribution to pressure development of the two components of cross-bridge dynamics: attached cross-bridge concentration and elongation of its elastic structure. The model was tested by reproduction of experiments reflecting myofibrillar behavior at the ventricular level as well as chamber mechanical properties. It was then used to study cross-bridge behavior independently of Ca2+activation, by simulation of flow-clamp experiments at constant Ca2+concentration. During the volume ramp, both reduced cross-bridge elongation and decreased concentration by cross-bridge detachment caused a fall of pressure; at end-ejection there was a fast partial increase of pressure by recovery of cross-bridge elongation, and during post-ejection there was a slower pressure change towards the value corresponding to end-ejection volume by cross-bridge reattachment according to the rate of constants of Ca2+kinetics. Likewise, during a physiological normal ejection, results showed that a maximal decrease in cross-bridge elongation (Δh) produced a maximal reduction of ejecting pressure with respect to that at constant cross-bridge elongation (ΔP), both in simulated beats (ΔP=20%, Δh=17%), and in experimentally fitted pressure–volume data from open-chest dogs (ΔP=43.7±3.8%, Δh=30.7±8.3%), Δh being dependent of peak flow (Δh=0.1471 peak flow+6.0788,r=0.72). We conclude that normal ejecting pressure depends not only on cross-bridge concentration, but also on the elongation of its elastic structure, which reduces pressure according to flow.

Sarcomere dynamics and contraction-induced injury to maximally activated single muscle fibres from soleus muscles of rats.

1. The focal nature of contraction-induced injury to skeletal muscle fibres may arise from heterogeneities in sarcomere length that develop during contractions. We tested the hypothesis that when a maximally activated single permeabilized fibre segment is stretched and a deficit in maximum isometric force (force deficit) is produced, the regions of sarcomeres with the longest lengths of prior to the stretch contain the majority of the damaged sarcomeres when the fibre is returned to optimum length (Lo) after the stretch. 2. Single fibre segments (n = 16) were obtained from soleus muscles of rats. Average sarcomere length at five discrete positions along the length of each fibre was determined by lateral deflection of a diode laser spot. Diffraction patterns were obtained while fibres were relaxed and immediately before, during and after a single stretch of 40% strain relative to Lo. Following the stretch, the regions of each fibre that potentially contained damaged sarcomeres were identified by an increased scatter of the first-order diffraction patterns. The damage was confirmed by light and electron microscopy. 3. While single fibre segments were in relaxing solution, the mean value for all of the average sarcomere lengths sampled (n = 80) was 2.53 +/- 0.01 microns (range, 2.40-2.68 microns). During the maximum isometric contraction before each stretch, the mean sarcomere length decreased to 2.42 +/- 0.02 microns and the range increased to 2.12-3.01 microns. 4. During the stretch of 40% strain, all regions of sarcomeres were stretched onto the descending limb of the length-force curve, but sarcomere lengthening was non-uniform. After the stretch, when the maximally activated fibres were returned to Lo, the force deficit was 10 +/- 1%. Microscopic evaluation confirmed that the regions with the longest sarcomere lengths before the stretch contained the majority of the damaged sarcomeres after the stretch. We conclude that when heterogeneities in sarcomere length develop in single permeabilized fibre segments during a maximum isometric contraction, the sarcomeres in the regions with the longest lengths are the most susceptible to contraction-induced injury.