Inotropic interventions do not change the resting state of myosin motors during cardiac diastole

When striated (skeletal and cardiac) muscle is in its relaxed state, myosin motors are packed in helical tracks on the surface of the thick filament, folded toward the center of the sarcomere, and unable to bind actin or hydrolyze ATP (OFF state). This raises the question of whatthe mechanism is that integrates the Ca2+-dependent thin filament activation, making myosin heads available for interaction with actin. Here we test the interdependency of the thin and thick filament regulatory mechanisms in intact trabeculae from the rat heart. We record the x-ray diffraction signals that mark the state of the thick filament during inotropic interventions (increase in sarcomere length from 1.95 to 2.25 µm and addition of 10-7 M isoprenaline), which potentiate the twitch force developed by an electrically paced trabecula by up to twofold. During diastole, none of the signals related to the OFF state of the thick filament are significantly affected by these interventions, except the intensity of both myosin-binding protein C- and troponin-related meridional reflections, which reduce by 20% in the presence of isoprenaline. These results indicate that recruitment of myosin motors from their OFF state occurs independently and downstream from thin filament activation. This is in agreement with the recently discovered mechanism based on thick filament mechanosensing in which the number of motors available for interaction with actin rapidly adapts to the stress on the thick filament and thus to the loading conditions of the contraction. The gain of this positive feedback may be modulated by both sarcomere length and the degree of phosphorylation of myosin-binding protein C

Infrared spectroscopy of NGC 1068: Probing the obscured ionizing AGN continuum

The ISO-SWS 2.5-45 um infrared spectroscopic observations of the nucleus of the Seyfert 2 galaxy NGC 1068 (see companion paper) are combined with a compilation of UV to IR narrow emission line data to determine the spectral energy distribution (SED) of the obscured extreme-UV continuum that photoionizes the narrow line emitting gas in the active galactic nucleus. We search a large grid of gas cloud models and SEDs for the combination that best reproduces the observed line fluxes and NLR geometry. Our best fit model reproduces the observed line fluxes to better than a factor of 2 on average and is in general agreement with the observed NLR geometry. It has two gas components that are consistent with a clumpy distribution of dense outflowing gas in the center and a more extended distribution of less dense and more clumpy gas farther out that has no net outflow. The best fit SED has a deep trough at ~4 Ryd, which is consistent with an intrinsic Big Blue Bump that is partially absorbed by ~6x10^19 cm^-2 of neutral hydrogen interior to the NLR.Comment: 15 pp, 4 figures, ApJ accepte

The coronal line regions of planetary nebulae NGC6302 and NGC6537: 3-13um grating and echelle spectroscopy

We report on advances in the study of the cores of NGC6302 and NGC6537 using infrared grating and echelle spectroscopy. In NGC6302, emission lines from species spanning a large range of ionization potential, and in particular [SiIX]3.934um, are interpreted using photoionization models (including CLOUDY), which allow us to reestimate the central star's temperature to be about 250000K. All of the detected lines are consistent with this value, except for [AlV] and [AlVI]. Aluminium is found to be depleted to one hundredth of the solar abundance, which provides further evidence for some dust being mixed with the highly ionized gas (with photons harder than 154eV). A similar depletion pattern is observed in NGC6537. Echelle spectroscopy of IR coronal ions in NGC6302 reveals a stratified structure in ionization potential, which confirms photoionization to be the dominant ionization mechanism. The lines are narrow (< 22km/s FWHM), with no evidence of the broad wings found in optical lines from species with similar ionization potentials, such as [NeV]3426A. We note the absence of a hot bubble, or a wind blown bipolar cavity filled with a hot plasma, at least on 1'' and 10km/s scales. We also provide accurate new wavelengths for several of the infrared coronal lines observed with the echelle.Comment: Accepted for publication in MNRA

The mechanism of the force response to stretch in human skinned muscle fibres with different myosin isoforms

Force enhancement during lengthening of an active muscle, a condition that normally occurs during locomotion in vivo, is attributed to recruitment of myosin heads that exhibit fast attachment to and detachment from actin in a cycle that does not imply ATP splitting. We investigated the kinetic and mechanical features of this cycle in Ca(2+) activated single skinned fibres from human skeletal muscles containing different myosin heavy chain (MHC) isoforms, identified with single-fibre gel electrophoresis. Fibres were activated by using a new set-up that allows development of most of the tension following a temperature jump from 0-1 degrees C to the test temperature (approximately 12 degrees C). In this way we could prevent the development of sarcomere non-uniformity and record sarcomere length changes with a striation follower in any phase of the mechanical protocol. We found that: (i) fibres with fast MHC isoforms develop 40-70% larger isometric forces than those with slow isoforms, as a result of both a larger fraction of force-generating myosin heads and a higher force per head; (ii) in both slow and fast fibres, force enhancement by stretch is due to recruitment of myosin head attachments, without increase in strain per head above the value generated by the isometric heads; and (iii) the extent of recruitment is larger in slow fibres than in fast fibres, so that the steady force and power output elicited by lengthening become similar, indicating that mechanical and kinetic properties of the actin-myosin interactions under stretch become independent of the MHC isoform

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin.

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor

The mechanism of the force response to stretch in human skinned muscle fibres with different myosin isoforms

Force enhancement during lengthening of an active muscle, a condition that normally occurs during locomotion in vivo, is attributed to recruitment of myosin heads that exhibit fast attachment to and detachment from actin in a cycle that does not imply ATP splitting. We investigated the kinetic and mechanical features of this cycle in Ca(2+) activated single skinned fibres from human skeletal muscles containing different myosin heavy chain (MHC) isoforms, identified with single-fibre gel electrophoresis. Fibres were activated by using a new set-up that allows development of most of the tension following a temperature jump from 0-1 degrees C to the test temperature ( approximately 12 degrees C). In this way we could prevent the development of sarcomere non-uniformity and record sarcomere length changes with a striation follower in any phase of the mechanical protocol. We found that: (i) fibres with fast MHC isoforms develop 40-70% larger isometric forces than those with slow isoforms, as a result of both a larger fraction of force-generating myosin heads and a higher force per head; (ii) in both slow and fast fibres, force enhancement by stretch is due to recruitment of myosin head attachments, without increase in strain per head above the value generated by the isometric heads; and (iii) the extent of recruitment is larger in slow fibres than in fast fibres, so that the steady force and power output elicited by lengthening become similar, indicating that mechanical and kinetic properties of the actin-myosin interactions under stretch become independent of the MHC isoform