Changes In Sarcomere Length Research Articles

Mechanism of the Frank–Starling law—A simulation study with a novel cardiac muscle contraction model that includes titin and troponin I

A stretch-induced increase of active tension is one of the most important properties of the heart, known as the Frank–Starling law. Although a variation of myofilament Ca 2+ sensitivity with sarcomere length (SL) change was found to be involved, the underlying molecular mechanisms are not fully clarified. Some recent experimental studies indicate that a reduction of the lattice spacing between thin and thick filaments, through the increase of passive tension caused by the sarcomeric protein titin with an increase in SL within the physiological range, promotes formation of force-generating crossbridges (Xbs). However, the mechanism by which the Xb concentration determines the degree of cooperativity for a given SL has so far evaded experimental elucidation. In this simulation study, a novel, rather simple molecular-based cardiac contraction model, appropriate for integration into a ventricular cell model, was designed, being the first model to introduce experimental data on titin-based radial tension to account for the SL-dependent modulation of the interfilament lattice spacing and to include a conformational change of troponin I (TnI). Simulation results for the isometric twitch contraction time course, the length-tension and the force–[Ca 2+] relationships are comparable to experimental data. A complete potential Frank–Starling mechanism was analyzed by this simulation study. The SL-dependent modulation of the myosin binding rate through titin's passive tension determines the Xb concentration which then alters the degree of positive cooperativity affecting the rate of the TnI conformation change and causing the Hill coefficient to be SL-dependent.

EFFECT OF FROZEN TEMPERATURE AND STORAGE TIME ON CALPAINS, CATHEPSINS (B, B + L, H AND D) AND THEIR ENDOGENOUS INHIBITORS IN GOAT MUSCLES

The effects of frozen storage on the biochemical properties of myofibrils, muscle proteinases (cathepsins and calpains) and their endogenous inhibitors were investigated. Longissimus dorsi, biceps femoris, semimembranosus and semitendinosus muscles from goat were frozen ([-]15C) and studied up to 120 days. The results showed that the percentage change in sarcomere length was 8.4-13.1. The calpain activity was determined after separation on a diethylaminoethyl-Sephacel column (Sigma, St. Louis, MO). Significantly greater percentage of calpain II activity was recovered when compared to calpain I. There was a 15-25% loss in calpastatin inhibitory activity, and the cystatin level fell by 11-16% after 80 days. Cathepsin B, B [+] L, H and D were very stable when compared to calpains. The calcium concentration may also be the factor for calpain activation. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis result showed the appearance of 55 kDa components. It was concluded that calpains, not cathepsins, play an important role in the proteolysis of myofibrillar proteins at the freezing temperature.

Myocyte‐specific overexpression of NOS3 prevents endotoxin‐induced myocardial dysfunction in mice

To learn the impact of myocardial NOS3 during sepsis, effects of myocyte-specific overexpression of NOS3 (msNOS3TG) on endotoxin (ETX)-induced cardiac dysfunction were studied. In vivo cardiac function was examined with echocardiography after challenge with saline (C) or ETX (L: 0111:B4 E. coli LPS, 50 mg/kg) in wild type mice (WT) and mice with msNOS3TG (TG). Cell shortening (CS) and [Ca2+]i were measured in isolated myocytes. Myofilament sensitivity to Ca2+ was determined in skinned myocytes by measuring the change in sarcomere length (ΔSL) in response to pCa 5,5. Myocardial oxidative stress was estimated using fluoroprobe dihydroethidium (DHE). ETX decreased LV fractional shortening (FS) in WTL but not in TGL. While [Ca2+]i transients were depressed similarly by ETX in both genotypes, %CS was depressed only in WTL myocytes. ΔSL was greater in skinned TGL myocytes than in WTL myocytes suggesting greater myofilament sensitivity to Ca2+ in TGL. A thiol reducing agent, dithiothreitol, augmented the Ca2+ sensitivity in skinned WTL but not in TGL myocytes, suggesting that NOS3 prevents myofilament thiol modification. ETX increased the frequency of DHE stained nuclei in cardiac sections from WTL but not TGL. In conclusion, msNOS3TG prevented ETX-induced myocardial dysfunction by enhancing myofilament sensitivity to Ca2+ possibly via reduction of cardiac oxidative stress. Supported by NIH HL-71987.

Mechanisms causing effects of muscle position on proximo-distal muscle force differences in extra-muscular myofascial force transmission

Certain recent studies showed that extra-muscular myofascial force transmission affects the length–force characteristics of rat extensor digitorium longus (EDL) muscle significantly after distal or proximal lengthening. This suggested that the relative position of a muscle with respect to its surrounding connective tissues is a co-determinant of muscle force in addition to muscle length, and indicated major effects on muscular mechanics. The specific goal of the present study is to investigate such effects by studying: (1) distributions of lengths of sarcomeres within muscle fibres and (2) the relative contributions of muscle fibres and the extra-cellular matrix to muscle total force, using a finite element model. The length of the muscle modelled was kept constant at a high and at a low muscle length whereas the relative position of the muscle was altered exclusively. For both muscle lengths, the forces exerted at distal and proximal tendons were unequal at almost all muscle relative positions. The proximo-distal force difference was enhanced as the muscle was repositioned away from its reference position. This confirmed the role of relative position of a muscle as a co-determinant of muscle force. At higher muscle lengths, distributions of lengths of sarcomeres arranged in series within muscle fibres were substantial. The force transmitted by the muscles’ extra-cellular matrix comprised a sizable part of muscle total force. At lower muscle lengths distribution of sarcomere lengths was relatively limited indicating that the extra-cellular matrix is bearing the extra-muscular force. However, minor sarcomere length changes were shown to accumulate to sizable effects on the summed forces exerted by the muscle fibres. In addition, the extra-muscular load was shown to manipulate the force exerted by the extra-cellular matrix. We conclude that the relative position of a muscle has substantial effects on intra-muscular mechanics and the importance of the role of the extra-cellular matrix in determining the proximo-distal force differences is comparable to that of the intra-cellular domain.

Stimulation of Isolated Ventricular Myocytes Within an Open Architecture Microarray

This paper is concerned with the physiological responses of single heart cells within microfluidic chambers, in response to stimulation by integrated microelectrodes. To enable these investigations, which included the measurement of action potential duration, intracellular Ca2+ and cell shortening, a series of microfluidic chambers (50 microm wide, 180 microm long, 400 microm high, 500 microm pitch) and connecting channels (200 microm wide, 5000 microm long, 50 microm high, 500 microm pitch) were replica-moulded into the silicone elastomer, polydimethylsiloxane (PDMS). The structures were formed against a master of posts and lines, photolithograhically patterned into the high aspect ratio photoresist SU-8. The chambers within the slab of PDMS were aligned against pairs of stimulating gold microelectrodes (50 microm long, 20 microm wide, 0.1-10 microm thick, 180 microm apart) patterned on a microscope coverslip base, thus defining cavities of approximately 4 nL volume. The assembly was filled with physiological saline and single isolated rabbit ventricular myocytes were introduced by micropipetting, thus creating limited volumes of saline above individual myocytes that could be varied between 4 nL and > or = 4 microL. The application of transient current pulses to the cells via the electrodes caused transient contractions with constant amplitude (recorded as changes in sarcomere length), confirming that excitation contraction coupling (EC coupling) remained functional in these limited volumes. Continuous monitoring of the intracellular Ca2+ (using calcium sensitive dyes) showed, that in the absence of bath perfusion, the amplitude of the transients remained constant for approximately 3 min in the 4-nL volume and approximately 20 min for the 4 microL volume. Beyond this time, the cells became unexcitable until the bath was renewed. The action potential duration (APD) was recorded at stimulation frequencies of 1 Hz and 0.5 Hz using potential sensitive dyes and was prolonged at the higher pacing rate. These studies show the prolonged electrical stimulation of isolated adult cardiac myocytes in microchambers with unimpaired EC coupling as verified on optical records of the action potential, Ca2+ transients and cell shortening. The open architecture provided free (pipetting) access for drug dispensation without cross talk between neighboring microwells, and multiplexed optical detection can be realized to study EC coupling on arrays of cells under both control and experimental conditions.

Effect of heat treatment on changes in texture, structure and properties of Thai indigenous chicken muscle

Changes in texture, microstructure, colour and protein solubility of Thai indigenous and broiler chicken Pectoralis muscle stripes cooked at different temperatures were evaluated. The change in shear value of both chicken muscles was a significant increase from 50 to 80 °C but no change from 80 to 100 °C. A significant decrease in fibre diameter was obtained in samples heated to an internal temperature of 60 °C and the greatest shrinkage of sarcomeres was observed with internal temperatures of 70–100 and 80–100 °C for broiler and indigenous chicken muscles, respectively ( P < 0.05). Cooking losses of indigenous chicken muscles increased markedly in the temperature range 80–100 °C and were significantly higher than those of the broiler ( P < 0.001). With increasing temperature, from 50 to 70 °C, cooked chicken muscle became lighter and yellower. Relationships between changes in sarcomere length, fibre diameter, shear value, cooking loss and solubility of muscle proteins were evaluated. It was found that the solubility of muscle protein was very highly correlated with the texture of cooked broiler muscle while sarcomere length changes and collagen solubility were important factors influencing the cooking loss and texture of cooked indigenous chicken muscle.

STEP SIZE IN ACTIVATED RABBIT SARCOMERES IS INDEPENDENT OF FILAMENT OVERLAP

Investigations carried out on single cardiac and bumblebee myofibrils have shown stepwise sarcomere-length change of ~2.7 nm.1 We have carried out parallel measurements on single myofibrils from rabbit psoas muscle. Activated specimens were released or stretched using a motor-imposed ramp. With a high-resolution algorithm, we found that step sizes were always integer multiples of 2.7 nm, whether the length change was positive or negative, and independent of ramp velocity. Also, the influence of initial sarcomere length was studied, and found to be negligible. The value 2.7 nm, seen consistently, is equal to the linear repeat of actin monomers along the thin filament, a result that ties dynamical events to molecular structure, and places narrow constraints on any proposed molecular mechanism.

Factors regulating lamb longissimus tenderness are affected by age at slaughter

The objective of this experiment was to determine age-related changes in collagen concentration, sarcomere length, calpain (μ- and m-) and calpastatin activities, postmortem proteolysis and Warner–Bratzler shear force (WBSF) in ovine longissimus thoracis et lumborum. Rambouillet lambs were slaughtered at 2, 4, 6, 8 and 10 months of age and samples of longissimus were collected at 0, 2 and 10 days postmortem. Collagen concentration and sarcomere lengths were determined from the cores used for WBSF measurements and reflected changes in the background toughness. Longissimus collagen concentration did not change ( P>0.05) due to lamb age. Sarcomere lengths also showed age-related changes, increasing ( P<0.05) from 1.35 μm at 6 months to 1.48 and 1.55 μm at 8 and 10 months, respectively. The extent of calpain mediated proteolysis determines the improvement in meat tenderness with postmortem storage. The most notable change in the calpain proteolytic system was the decline ( P<0.05) in calpastatin activity from 4.18 to 1.91 U/g muscle between 2 and 10 months. The activity of μ-calpain showed a 16% increase ( P<0.05) from 4 to 6 months, before it dropped again at 8 and 10 months. There was a gradual decline ( P<0.05) in m-calpain activity with age, and by 10 months m-calpain activity had reduced to 80% of 2 months levels. The ratio of μ-calpain to calpastatin activities increased ( P<0.05) from 2 to 6 months (from 0.31 to 0.56) with no further changes ( P>0.05) at 8 or 10 months. There were no age-related changes ( P>0.05) in desmin degradation at day 2, however, examination of day 10 samples showed increased ( P<0.05) degradation from 2 to 6 months. Thus, the changes observed in the ratio of μ-calpain to calpastatin activities are reflected in the extent of postmortem proteolysis. Meat tenderness was measured using WBSF at 2 and 10 days postmortem. Because little proteolysis had taken place at 2 days postmortem, the decline in day 2 WBSF from 6 to 8 months could be explained by changes in sarcomere length. However, at 10 days postmortem, where WBSF was shown to decrease from 2 to 8 months, the improvement in tenderness could be explained by the amount of postmortem proteolysis. The data presented in this paper show evidence that sarcomere length is the main determinant of background toughness in ovine longissimus, and that postmortem proteolysis, resulting from μ-calpain activity regulated by calpastatin, is the main determinant of ovine longissimus tenderization during aging. Thus, lamb longissimus tenderness after refrigerated storage is determined by postmortem proteolysis and its interaction with sarcomere length.

Passive stiffness of Drosophila IFM myofibrils: a novel, high accuracy

As the smallest muscle-cell substructure that retains the intact contractile apparatus, the single myofibril is considered the optimal specimen for muscle mechanics, although its small size also poses some technical difficulties. Myofibrils from Drosophila indirect flight muscle (IFM) are particularly difficult to study because their high passive stiffness makes them hard to handle, and too resistant to stretch to produce enough elongation for the accurate measurement of sarcomere length change. In this study, we devised a novel method for accurate stiffness measurement of single relaxed myofibrils using microfabricated cantilevers and phase contrast microscopy. A special experimental protocol was developed to minimize errors, and some data analysis strategies were used to identify and exclude spurious data. Remarkably consistent results were obtained from Drosophila IFM myofibrils. This novel, high accuracy method is potentially an effective tool for detecting small passive stiffness change in muscle mutants.

Asynchronous functional, cellular and transcriptional changes after a bout of eccentric exercise in the rat.

Thirty eccentric contractions (ECs) were imposed upon rat dorsiflexors (n = 46) by activating the peroneal nerve and plantarflexing the foot ~40 deg, corresponding to a sarcomere length change over the range 2.27-2.39 microm for the tibialis anterior and 2.52-2.66 microm for the extensor digitorum longus. Animals were allowed to recover for one of 10 time periods ranging from 0.5 to 240 h, at which time muscle contractile properties, immunohistochemical labelling and gene expression were measured. Peak isometric torque dropped significantly by ~40 % from an initial level of 0.0530 +/- 0.0009 Nm to 0.0298 +/- 0.0008 Nm (P < 0.0001) immediately after EC, and then recovered in a linear fashion to control levels 168 h later. Immunohistochemical labelling of cellular proteins revealed a generally asynchronous sequence of events at the cellular level, with the earliest event measured being loss of immunostaining for the intermediate filament protein, desmin. Soon after the first signs of desmin loss, infiltration of inflammatory cells occurred, followed by a transient increase in membrane permeability, manifested as inclusion of plasma fibronectin. The quantitative polymerase chain reaction (QPCR) was used to measure transcript levels of desmin, vimentin, embryonic myosin heavy chain (MHC), myostatin, myoD and myogenin. Compared to control levels, myostatin transcripts were significantly elevated after only 0.5 h, myogenic regulatory factors significantly elevated after 3 h and desmin transcripts were significantly increased 12 h after EC. None of the measured parameters provide a mechanistic explanation for muscle force loss after EC. Future studies are required to investigate whether there is a causal relationship among desmin loss, increased cellular permeability, upregulation of the myoD and desmin genes, and, ultimately, an increase in the desmin content per sarcomere of the muscle.

Troponin I in the murine myocardium: influence on length-dependent activation and interfilament spacing.

Cyclic AMP-dependent protein kinase (PKA) targets contractile proteins, troponin-I (TnI) and myosin binding protein C (MyBP-C) in the heart and induces a decrease in myofilament Ca2+ sensitivity. Yet, the effect of sarcomere length (SL) change on Ca2+ sensitivity (length-dependent activation: LDA) following PKA-dependent phosphorylation is not clear. To clarify the role of PKA-dependent phosphorylation of TnI and MyBP-C on LDA in the heart, we examined LDA in skinned myocytes from a non-transgenic (NTG) and a transgenic murine model in which the native cardiac isoform (cTnI) was completely replaced by the slow skeletal isoform of TnI (ssTnI-TG) lacking the phosphorylation sites for PKA, while retaining PKA sites on MyBP-C. In NTG myocytes, PKA treatment decreased Ca2+ sensitivity at each SL, but enhanced the impact of SL change on Ca2+ sensitivity. Despite a greater sensitivity to Ca2+ and a reduction in LDA, neither Ca2+ responsiveness nor LDA was affected by PKA treatment in ssTnI-TG myocytes. To determine whether the above observations could be explained by the lateral separation between thick and thin filaments, as suggested by others, we measured interfilament spacing by X-ray diffraction as a function of SL in skinned cardiac trabeculae in the passive state from both NTG and ssTnI-TG models before and following treatment with PKA. Phosphorylation by PKA increased lattice spacing at every SL in NTG trabeculae. However, the relationship between SL and myofilament lattice spacing in ssTnI-TG was markedly shifted downward to an overall decreased myofilament lattice spacing following PKA treatment. We conclude: (1) PKA-dependent phosphorylation enhances length-dependent activation in NTG hearts; (2) replacement of native TnI with ssTnI increases Ca2+ sensitivity of tension but reduces length-dependent activation; (3) MyBP-C phosphorylation by PKA does not alter calcium responsiveness and induces a decrease in myofilament lattice spacing at all sarcomere lengths and (4) length-dependent activation in the heart cannot be entirely explained by alterations in myofilament lattice spacing.

Modulation of physiological angiogenesis in skeletal muscle by mechanical forces: involvement of VEGF and metalloproteinases.

Growth factors are involved in physiological angiogenesis in female reproductive organs but their role in capillary growth in skeletal muscles during activity or exercise training is not proven. Evidence suggests that increases in muscle blood flow and accompanying capillary shear stress and/or wall tension, or mechanical stress due to sarcomere length changes during contraction/relaxation cycles are closely linked with angiogenesis. Time-dependent studies of rat muscles in models with increased shear stress (chronic vasodilator treatment with alpha(1) antagonist prazosin), altered sarcomere length (stretch-induced overload with no increase in blood flow), or both (chronic electrical muscle stimulation) showed a similar increase in capillary supply in all models but by different modes of growth. With prazosin, it occurred by intra-luminal splitting of vessels, with stretch by abluminal sprouting, and in stimulated muscles by both methods. Whole muscle matrix metalloproteinase-2 (MMP-2) was elevated during sprouting growth induced by extravascular tensile forces but not during splitting growth induced by shear. Vascular endothelial growth factor (VEGF) protein was elevated at capillary sites in all three models but with different time courses. With shear as the stimulus, the increase occurred early although there was little capillary proliferation; it matched the rise in proliferation in stretched muscles but lagged behind proliferation in stimulated muscles. Mechanical forces therefore influence MMP and VEGF expression and capillary growth patterns in skeletal muscle differentially depending upon whether they act intra- or ab-luminally. In exercise-trained muscles, the type of capillary growth remains to be determined but the most likely stimuli for angiogenesis are increased blood flow and shear forces to vessel supplying the active fibres, probably linked with metabolic factors.

Inter-sarcomere dynamics in muscle fibres. A neglected subject?

The sarcomere is the functional unit of muscle, and all sarcomeres are connected in series in myofibrils within a muscle fibre. From this point of view of the structure a single model consisting of a contractile, a series and a parallel element can not account for the description of a real muscle fibre. Additionally, the titin protein filament needs to be considered as a passive visco-elastic element in parallel with the contractile apparatus. Therefore, the structure of a single muscle fibre is complex due mechanical elements ("motors") operating in series and in parallel. Moreover, variability does exist in the mechanical properties along a fibre and hence a multi-segmental model is more realistic and would give rise to many new insights. By attributing a segment model to each half-sarcomere, a fibre can be constructed through rigorous coupling of these units in series and parallel. The dynamics of such a multi-segmental model is much more complex, but it can explain a variety of effects reported in standard classical mechanics experiments. With a relatively simple mechanistic description we can show that the dynamics of such multi-sarcomere systems exhibit a variety of effects (relaxation phenomena, permanent extra-tension, biphasic force-velocity relation) and should therefore not be neglected in muscle fibre modelling. We have observed in single skinned fibre experiments that non-uniformities in sarcomere length changes are prominent during activation and relaxation.

A Non-Cross-Bridge Stiffness in Activated Frog Muscle Fibers

Force responses to fast ramp stretches of various amplitude and velocity, applied during tetanic contractions, were measured in single intact fibers from frog tibialis anterior muscle. Experiments were performed at 14°C at ∼2.1 μm sarcomere length on fibers bathed in Ringer’s solution containing various concentrations of 2,3-butanedione monoxime (BDM) to greatly reduce the isometric tension. The fast tension transient produced by the stretch was followed by a period, lasting until relaxation, during which the tension remained constant to a value that greatly exceeded the isometric tension. The excess of tension was termed “static tension,” and the ratio between the force and the accompanying sarcomere length change was termed “static stiffness.” The static stiffness was independent of the active tension developed by the fiber, and independent of stretch amplitude and stretching velocity in the whole range tested; it increased with sarcomere length in the range 2.1–2.8 μm, to decrease again at longer lengths. Static stiffness increased well ahead of tension during the tetanus rise, and fell ahead of tension during relaxation. These results suggest that activation increased the stiffness of some sarcomeric structure(s) outside the cross-bridges.

The elementary force generation process probed by temperature and length perturbations in muscle fibres from the rabbit.

Single chemically permeabilized fibres from rabbit psoas muscle were activated maximally at 5-6 degrees C and then exposed to a rapid temperature increase ('T-jump') up to 37 degrees C by passing a high-voltage pulse (40 kHz AC, 0.15 ms duration) through the fibre length. Fibre cooling after the T-jump was compensated by applying a warming (40 kHz AC, 200 ms) pulse. Tension and changes in sarcomere length induced by the T-jumps and by fast length step perturbations of the fibres were monitored. In some experiments sarcomere length feedback control was used. After T-jumps tension increased from approximately 55 kN m(-2) at 5-6 degrees C to approximately 270 kN m(-2) at 36-37 degrees C, while stiffness rose by approximately 15 %, suggesting that at a higher temperature the myosin head generates more force. The temperature-tension relation became less steep at temperatures above 25 degrees C, but was not saturated even at near-physiological temperature. Comparison of tension transients induced by the T-jump and length steps showed that they are different. The T-jump transients were several times slower than fast partial tension recovery following length steps at low and high temperature (phase 2). The kinetics of the tension rise after the T-jumps was independent of the preceding length changes. When the length steps were applied during the tension rise induced by the T-jump, the observed complex tension transient was simply the sum of two separate responses to the mechanical and temperature perturbations. This demonstrates the absence of interaction between these processes. The data suggest that tension transients induced by the T-jumps and length steps are caused by different processes in myosin cross-bridges.

Spasticity causes a fundamental rearrangement of muscle-joint interaction.

Sarcomere length was measured in flexor carpi ulnaris (FCU) muscles from patients with severely spastic wrist flexion contractures (n = 6), as well as from patients with radial nerve injury and a normally innervated FCU (n = 12). Spastic FCU muscles had extremely long sarcomere lengths with the wrist fully flexed (3.48 +/- 0.44 microm) compared to the FCU muscles of patients with radial nerve injury (2.41 +/- 0.31 microm). In three of the patients with spastic wrist flexion contractures, the slope of the FCU sarcomere length-joint angle relationship was measured and found to be, essentially, normal (0.017 +/- 0.005 microm/degree, n = 3) suggesting that serial sarcomere number (and therefore muscle fiber length) was unchanged in spite of the dramatic absolute sarcomere length change. These results indicate that spasticity results in a major alteration of normal muscle-joint anatomical relationships that has not previously been recognized to our knowledge. We hypothesize that the results are explained either by the inability of muscle fibers to add serial sarcomeres in response to growth, or the selective loss of FCU muscle length secondary to the central nervous system lesion.

The elementary force generation process probed by temperature and length perturbations in muscle fibres from the rabbit

Single chemically permeabilized fibres from rabbit psoas muscle were activated maximally at 5–6 °C and then exposed to a rapid temperature increase (‘T-jump’) up to 37 °C by passing a high-voltage pulse (40 kHz AC, 0.15 ms duration) through the fibre length. Fibre cooling after the T-jump was compensated by applying a warming (40 kHz AC, 200 ms) pulse. Tension and changes in sarcomere length induced by the T-jumps and by fast length step perturbations of the fibres were monitored. In some experiments sarcomere length feedback control was used. After T-jumps tension increased from ∼55 kN m−2 at 5–6 °C to ∼270 kN m−2 at 36–37 °C, while stiffness rose by ∼15 %, suggesting that at a higher temperature the myosin head generates more force. The temperature-tension relation became less steep at temperatures above 25°C, but was not saturated even at near-physiological temperature. Comparison of tension transients induced by the T-jump and length steps showed that they are different. The T-jump transients were several times slower than fast partial tension recovery following length steps at low and high temperature (phase 2). The kinetics of the tension rise after the T-jumps was independent of the preceding length changes. When the length steps were applied during the tension rise induced by the T-jump, the observed complex tension transient was simply the sum of two separate responses to the mechanical and temperature perturbations. This demonstrates the absence of interaction between these processes. The data suggest that tension transients induced by the T-jumps and length steps are caused by different processes in myosin cross-bridges.

High-throughput assessment of calcium sensitivity in skinned cardiac myocytes.

Isolated permeabilized cardiac myocytes have been used in the study of myofilament calcium sensitivity through measurement of the isometric force-pCa curve. Determining this force-pCa relationship in skinned myocytes is relatively expensive and carries a high degree of variability. We therefore attempted to establish an alternative high-throughput method to measure calcium sensitivity in cardiac myocytes. With the use of commercially available software that allows for precise measurement of sarcomere spacing, we measured sarcomere length changes in unloaded skinned cardiac myocytes over a range of calcium concentrations. With the use of this technique, we were able to accurately detect acute increases or decreases in myofilament calcium sensitivity after exposure to 10 mM caffeine or 5 mM 2,3-butanedione monoxime, respectively. This technique allows for the simple and rapid determination of myofilament calcium sensitivity in cardiac myocytes in a reproducible and inexpensive manner and could be used for high-throughput screening of pharmacological agents and/or transgenic mouse models for changes in myofilament calcium sensitivity.

Quantal Sarcomere-Length Changes in Relaxed Single Myofibrils

We carried out experiments on single isolated myofibrils in which thin filaments had been functionally removed, leaving the connecting (titin) filaments as the sole agent taking up the length change. With technical advances that gave sub-nanometer detectability we examined the time course of single sarcomere-length change when the myofibril was ramp-released or ramp-stretched by a motor. The sarcomere-length change was stepwise. Step sizes followed a consistent pattern: the smallest was ∼2.3 nm, and others were integer multiples of that value. The ∼2.3-nm step quantum is the smallest consistent biomechanical event ever demonstrated. Although the length change must involve the connecting filament, the size of the quantum is an order of magnitude smaller than anticipated from folding of Ig- or fibronectin-like domains, implying either that folding occurs in sub-domain units or that other mechanisms are involved.

Length-dependence of Cross-bridge Mediated Activation of the Cardiac Thin Filament

Length-dependent Ca2+activation of the thin filament plays a critical role in the steep force–length relationship of cardiac muscle (Frank-Starling relation). Recent evidence indicates that the increase in myofilament Ca2+sensitivity and Ca2+-troponin C affinity that occurs with increase in sarcomere length results from a cooperative activation of the thin filament by attached cross-bridges. At short sarcomere length the Ca2+sensitivity is lower because the access of cross-bridges for actin is reduced. The aim of this study was to determine the length-dependence of myosin-mediated thin filament activation in skinned bovine ventricular muscle, as assayed by the generation of force with progressive reduction of MgATP concentration in the absence of Ca2+. If the interaction between myosin and actin is weaker at short sarcomere length there should be a lower MgATP concentration needed to maintain the relaxed state. Contrary to expectation, the force–pMgATP relationship was not significantly influenced by change in sarcomere length. However this relationship became length-sensitive in the presence of phosphate analogs which stabilize weak-binding cross-bridges. We suggest that sarcomere length modulates Ca2+sensitivity by controlling the size of the population of thin filament regulatory units in the weakly-bound state.