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J W Krueger

Publications and source records attributed to J W Krueger.

17 recordsLinked to original sources
PubMed1992

High resolution measurement of striation patterns and sarcomere motions in cardiac muscle cells.

We describe an extension of the method of Myers et al. (1982) to measure with high precision the uniformity of contractile motions that occur between sarcomeres in the isolated cardiac muscle cell (guinea pig and rat). The image of the striations, observed with modulation contrast microscopy, was detected by a linear array of photodiodes. Sarcomere length was measured greater than 500/s from the frequency of the array's video signal at two selectable regions of the cell. A precision test grating demonstrated that method resolves known differences in the spacing between two contiguous striations to +/- 0.01 micron and that the effects of image translation and microscopic resolution are minor. The distribution of striation spacing appears to be discrete in isolated segments of the cell, and patches of fairly uniform length can be identified that are laterally contiguous. When electrically triggered, contraction is synchronous and the sarcomeres shorten and relengthen smoothly. The contrast between the striations is transiently enhanced during relengthening, an indication that the contracting cell can not be treated as a simple grating. Pauses that occur during late in relengthening (and transient contractile alternans) are characterized by very synchronized activity. These forms of irregular contractile behavior are not explained by desynchronization of a mechanism of release of intracellular calcium. A companion article describes application of the technique to study the nonuniform motions that occur between sarcomeres.

Animals↗
PubMed1992

Nature of motions between sarcomeres in asynchronously contracting cardiac muscle cells.

We observed the asynchronized motions that occur between the sarcomeres during spontaneous contractions to study the mechanical nature of the intact cardiac muscle cell (guinea pig, rat). The cell's striated image is detected by a photodiode array, and sarcomere length is measured very precisely 526/s in two separate, selected fixed regions of the image from the localized frequency of the array's video signal (Krueger and Denton, 1992, Biophys. J., 61:129-144, second of two companion manuscripts). An extension of this approach is described here in which the spatial variation of sarcomere length is visualized by scanned sampling, i.e., displacing the first window along the length of the cell, and nonuniform strain is deduced from the histograms of sarcomere length. The nature of asynchronous motion that was obtained from both fixed sampling of the sarcomere's dynamics and by scanned sampling of sarcomere length was consistent. In spontaneously active cells, sarcomeres lengthen approximately 0.1 micron beyond their rest length before the arrival of the propagated wave of contraction. Such prelengthening extends in a nonuniform fashion for approximately 10 to 15 microns in the unattached cell. Shortening and lengthening motions, being in proportion for both large and small displacements, are well coupled. Lifting the cell from the substrate showed that the force that sustains prelengthening arises within the cell. Differences in the sarcomere's dynamics in synchronous and asynchronous contractions corroborate that asynchrony imposes an additional internal restoring force. The extra force estimated to account for prelengthening (0.5-0.7 mN/mm2) has little effect on the velocity of shortening, and so the true intracellular restoring force must be correspondingly larger. The intracellular restoring force may contribute significantly to the rapid 'diastolic' recoil of the heart muscle at short sarcomere lengths.

Animals↗
PubMed1991

Influence of the cardiac myosin hinge region on contractile activity.

The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myosin, and had no effect on ATPase activity of purified S1 and myofibrils. However, it completely suppressed the movement of actin filaments in in vitro motility assays and reduced active shortening of sarcomeres of skinned cardiac myocytes by half. Suppression of motion by the anti-hinge antibody may reflect a mechanical constraint imposed by the antibody upon the mobility of the S2 region of myosin. The results suggest that the steps in the mechanochemical energy transduction can be separately influenced through S2.

Amino Acid Sequence↗
PubMed1991

A compact and stable hydraulic micromanipulator patterned after a Huxley-style approach.

I describe a remote-controlled micromanipulator platform that is stable, durable, precise, and easy to construct. Small metallic bellows are used for hydraulic control, where all fluid connections are made by standard 1/16-in. high-performance liquid chromatography fittings. Inspired by the parallelogram suspension utilized in the larger Huxley-style micromanipulator (A. F. Huxley. J. Physiol. Lond. 157: 6-5P, 1961), the device is a compact cradle suspension of folded lever arms that creates vertical motions which have minimal cross-coupled horizontal error. A simple arrangement for securing the bellows in the remote controller counteracts the vertical cross-coupling error that arises in the parallelogram suspension so that the position of the microtool more faithfully corresponds to the micrometer settings. Being compact, the micromanipulator can be mounted on a microscope stage to eliminate the microscope's resonance as a source of vibration. This feature also reduces the cantilevering of the microtool that 1) is a source of parasitic vibrations and 2) limits the load bearing in larger devices which can only be placed alongside the microscope. The device has a good dynamic response, and one design suits both right- and left-handed use.

Chromatography, High Pressure Liquid↗
PubMed1988

A simple hydraulic drive for remote control of the Huxley-style micromanipulator.

Electrodeposited metal bellows permit the construction of simple, reliable, and inexpensive hydraulic drives for the Huxley-style micromanipulator. This approach enables precise remote control of micromanipulation without introducing conductive leads or other potential sources of electrical noise near the experimental system. Moreover, the bellows arrangement preserves a stability and reproducibility in the positioning of the heavier transducers that is not matched by other hydraulic micromanipulators. The small size of the hydraulic coupler enables easy installation, and it can be adapted to preexisting manipulators. Importantly, the manipulator's original micrometer control is preserved, a feature that allows the investigator to tailor the apparatus for novel arrangements for micromanipulation.

Electrophysiology↗
PubMed1988

A "give" in tension and sarcomere dynamics in cardiac muscle relaxation.

Isometric relaxation in cardiac sarcomeres is characterised by an early, very slow phase of tension fall which is terminated by a 'give' in tension. A 'give' which occurs during relaxation in cardiac muscle can not be attributed to decrease in myofilament overlap. After the 'give' asynchronous motion occurs between sarcomeres, but the duration and extent of their displacement is limited. Intriguingly, the effect of isotonic displacements on the early fall in the velocity of sarcomere shortening indicates that an internal resistance increases near the peak of contraction. The complex shape of the sarcomere's complete force-velocity relation, with lengthening motions in particular, was consistent with an idealized model of cross-bridge cycling. The sarcomere's resistance to stretch is high at low velocity, but it diminishes to reveal yielding at larger velocities. Relative to tension, the resistance to yielding does not decrease during relaxation, and it may actually increase. The decay of isometric tension after a controlled stretch also slows during relaxation. Consequently, cycling slows in those cross-bridges which form (or persist but produce less force) later in contraction. Changes in cross-bridge properties may restrict sarcomere shortening, prolong activation, but promote a disequilibrium which favors rapid relaxation in cardiac muscle.

Animals↗
PubMed1986

Contraction in voltage-clamped, internally perfused single heart cells.

We studied contraction in single voltage-clamped, internally perfused myocytes isolated from guinea pig ventricles. The microscopic appearance of the cell was observed and recorded with a television system, while contractile shortening was measured 1,000 times/s using a linear photodiode array. Uniform, synchronous sarcomere shortening occurred in response to depolarizations that triggered a slow inward current (Isi). Changes in Isi caused by altering the amplitude of the voltage step, the extracellular [Ca2+], or the holding potential were accompanied by immediate parallel changes in the extent and velocity of shortening. In particular, twitch shortening during depolarization was immediately decreased when large voltage steps decreased Isi, and was eliminated by depolarizations that exceeded +75 mV, the apparent reversal potential for Ca2+. In these cases, shortening was associated with the tail current during repolarization. Increases in the amplitude, duration, and the rate of the depolarizing step increased the extent and speed of sarcomere shortening over the course of four to five contractions without a simultaneous parallel increase of Isi. Large prolonged depolarizations caused an asynchronous, nonuniform, oscillatory shortening of the cell and potentiated future twitch contractions. Increases in the duration of the depolarizing step immediately prolonged contraction; otherwise, interventions that altered the extent, velocity, and time course of shortening in intact, nonperfused cells did not affect the time course of the contraction in the internally perfused single cells. Our results provide direct support for the hypothesis that Isi both induces and grades the size of the Ca2+ release from the sarcoplasmic reticulum of intact cardiac muscle. In addition, a separate, depolarization-dependent process unrelated to Isi grades the size of contraction, presumably by modulating Ca2+ accumulation in the intracellular stores, and affects its time course.

Animals↗
PubMed1984

Contraction bands: differences between physiologically vs. maximally activated single heart muscle cells.

High resolution interference and phase microscopy were used to inspect the striations' appearance in shortening rat heart cells. Isolated cells were treated with detergent so that shortening could be graded by addition of calcium. Upon activation sarcomeres shortened to form (a) contraction densities in the middle of the A band at 1.7 micrometer (b) disappearance of the I bands and (c) phase brightening of the A bands at 1.6 micrometer and (d) dense Cz contraction bands at shorter lengths. These changes are totally consistent with the uniform sliding of myofilaments of previously accepted fixed dimensions. However, the striated patterns differed significantly in intact cells which were electrically stimulated to shorten. Here individual A bands remained distinct, without phase brightening or contraction band formation despite sarcomere shortening to less than the length of the A band as measured in the unstimulated cell. Maximal activation of intact cells by barium contracture elicited the full sequence of striation changes (a-d) seen in the chemically skinned cells. Light diffraction analysis gave comparable interpretation, i.e., the protein within the shortened sarcomere in the physiologically activated cardiac cell is more narrowly distributed than expected for thick filaments of fixed dimensions. These optical differences may reflect the restricted presence of the globular myosin heads at the ends of the cardiac sarcomere. This situation would explain the narrow range of the cardiac length-tension relation.

Animals↗
PubMed1981

Isolated heart myocytes: ultrastructural case study technique.

We have developed a method for performing case studies of heart muscle cells enzymatically isolated from the ventricular walls of rats that is a simple and inexpensive adaptation of procedures developed for the examination of monolayers of attached, cultured cells. The technique represents a marked departure from published accounts of electron microscopic studies of pellets or monolayers from a population of potentially heterogeneous isolated myocytes. Here we report the method, which we have used under controlled conditions with 0 mmol and 1 mmol added CaCl2, to correlate sarcomere length and electrical stimulatibility in the living state with ultrastructural features that include the relative disposition of myofilaments and the integrity of the cell coat. The degree of shrinkage during the preparative steps in less than 5%, as directly determined from photographs of striations in the living, fixed, and embedded states.

Animals↗
PubMed1980

Uniform sarcomere shortening behavior in isolated cardiac muscle cells.

We have observed the dynamics of sarcomere shortening and the diffracting action of single, functionally intact, unattached cardiac muscle cells enzymatically isolated from the ventricular tissue of adult rats. Sarcomere length was measured either (a) continuously by a light diffraction method or (b) by direct inspection of the cell's striated image as recorded on videotape or by cinemicroscopy (120--400 frames/s). At physiological levels of added CaCl2 (0.5--2.0 mM), many cells were quiescent (i.e., they did not beat spontaneously) and contracted in response to electrical stimulation (less than or equal to 1.0-ms pulse width). Sarcomere length in the quiescent, unstimulated cells (1.93 +/- 0.10 [SD] micrometers), at peak shortening (1.57 +/- 0.13 micrometers, n = 49), and the maximum velocity of sarcomere shortening and relengthening were comparable to previous observations in intact heart muscle preparations. The dispersion of light diffracted by the cell remained narrow, and individual striations remained distinct and laterally well registered throughout the shortening-relengthening cycle. In contrast, appreciable nonuniformity and internal buckling were seen at sarcomere lengths < 1.8 micrometers when the resting cell, embedded in gelatin, was longitudinally compressed These results indicate (a) that shortening and relengthening is characterized by uniform activation between myofibrils within the cardiac cell and (b) that physiologically significant relengthening forces in living heart muscle originate at the level of the cell rather than in extracellular connections. First-order diffracted light intensity, extremely variable during sarcomere shortening, was always greatest during midrelaxation preceding the onset of a very slow and uniform phase of sarcomere relengthening.

Animals↗
PubMed1978

Sarcomere relaxation in intact cardiac muscle.

The process of tension decay in the absence of sarcomere motion was studied in intact heart muscle isolated from rats. Sarcomere length, observed by an infrared light diffraction technique, was controlled and the effect on force dissipation measured. Distribution of the diffracted light suggested that sarcomere length was uniform immediately after contraction than prior to the next. When sarcomeres - as distinct from the total muscle length - are kept isometric, the rate of tension decay (a) reaches its maximum sooner, and (b) appears to be constant throughout relaxation. Slow stretch (about 3% sarcomere length) prolonged cardiac muscle tension during early relaxation without direct evidence of sarcomere 'yielding'. The dynamics of isometric relaxation and the effect of stretch were qualitatively the same at external calcium concentrations of 0.6 and 1.9 mM. For a specified sequence of sarcomere length changes during relaxation, the dynamics of tension decay were independent of preloaded sarcomere length when the sarcomeres are kept isometric. The data suggest that lowering contractile tension enhanced relative relaxation rate (sec-1) at a sarcomere length of 2.0 micron. The results clarify the physiological mechanism regulating the dynamics of myocardial fiber relaxation.

Animals↗
PubMed1976

Sarcomere dynamics in intact cardiac muscle.

Elastic properties, length-tension relations, and some characteristics of unloaded shortening were measured at the sarcomere level in rat papillary muscles. Muscle length during contraction was controlled by a servo system, while instantaneous sarcomere length was measured with a light diffraction technique. Muscles quick-released to zero load recoiled by 6% of their length; of this, sarcomere shortening amounted to only 1.6%, the remainder of the series elastic recoil occurring outside the striated region of the muscle, i.e. at the damaged ends of the specimen adjacent to the mounting clips. The length-tension relation was obtained with the sarcomere length maintained constant during contraction. Peak isometric tension increased linearly with sarcomere length from 1.6 to 2.1 mum; but between 2.1 and 2.3 mum tension appeared to be constant. The velocity of sarcomere shortening in an unloaded contraction bore a functional relation to sarcomere length which was similar to that of isometric tension. Both isometric tension and velocity of unloaded shortening reached their peak values relatively early in the contractile cycle.

Animals↗
PubMed1975

Myocardial sarcomere dynamics during isometric contraction.

1. Sarcomere lengths were measured during rest and throughout the time course of isometric contractions in thin, isolated rat papillary muscles using light diffraction techniques. 2. Shortening of the sarcomere length occurred upon contraction at all muscle lengths, averaging 7% at optimal length and more at shorter lengths. Relative to the narrow range of sarcomere lengths spanning the length--tension curve, this degree of shortening was considerable. 3. Local changes of sarcomere length were quantitatively paralleled by local changes of tissue segment length, the latter demarcated by microspheres lodged within the muscle tissue. At all but the shortest muscle lengths, sarcomere shortening was fully accounted for by equivalent lengthening of the non-striated regions near the clamped ends of the preparation. 4. It seems likely that these regions near the clamped ends of the preparation. 4. It seems constitute the source of the large series elasticity characteristic of isolated papillary muscle preparations such as this.

Animals↗