Franklin Fuchs

Length-dependent Ca 2+ activation in cardiac muscle: some remaining questions

The steep relationship between systolic force and end diastolic volume in cardiac muscle (Frank–Starling relation) is, to a large extent, based on length-dependent changes in myofilament Ca2+ sensitivity. How sarcomere length modulates Ca2+ sensitivity is still a topic of active investigation. Two general themes have emerged in recent years. On the one hand, there is a large body of evidence indicating that length-dependent changes in lattice spacing determine changes in Ca2+ sensitivity for a given set of conditions. A model has been put forward in which the number of strong-binding cross-bridges that are formed is directly related to the proximity of the myosin heads to binding sites on actin. On the other hand, there is also a body of evidence suggesting that lattice spacing and Ca2+ sensitivity are not tightly linked and that there is a length-sensing element in the sarcomere, which can modulate actin–myosin interactions independent of changes in lattice spacing. In this review, we examine the evidence that has been cited in support of these viewpoints. Much recent progress has been based on the combination of mechanical measurements with X-ray diffraction analysis of lattice spacing and cross-bridge interaction with actin. Compelling evidence indicates that the relationship between sarcomere length and lattice spacing is influenced by the elastic properties of titin and that changes in lattice spacing directly modulate cross-bridge interactions with thin filaments. However, there is also evidence that the precise relationship between Ca2+ sensitivity and lattice spacing can be altered by changes in protein isoform expression, protein phosphorylation, modifiers of cross-bridge kinetics, and changes in titin compliance. Hence although there is no unique relationship between Ca2+ sensitivity and lattice spacing the evidence strongly suggests that under any given set of physiological circumstances variation in lattice spacing is the major determinant of length-dependent changes in Ca2+ sensitivity.

Bound calcium and force development in skinned cardiac muscle bundles: Effect of sarcomere length

There is evidence that the steep ascending limb of the force-length curve in cardiac muscle (Frank-Starling relation) is based on a length-dependence of myofilament Ca2+ sensitivity. Previous work from this laboratory has indicated that in the sarcomere length range corresponding to the ascending limb of the cardiac force length curve (1.7 to 2.3 microns) the Ca2+-troponin C affinity is length-dependent. In this study Ca2+ binding to chemically skinned bovine cardiac muscle bundles was measured during ATP-induced force generation with fiber bundles having sarcomere lengths of 2.2 to 2.4 microns and 1.6 to 1.8 microns. A double isotope technique was used to make concurrent determinations of the force-pCa and bound Ca2+-pCa relationships. At the longer sarcomere lengths the fibers bound, at saturation, an amount of Ca2+ equivalent to approximately 3 mol Ca2+/mol troponin C. Force development appeared to be coupled to titration of the single, low-affinity Ca2+-specific site. In the pCa range 7.0 to 6.0 sarcomere length had no effect on Ca2+ binding. In the pCa range 6.0 to 5.0, in which force increased steeply, there was, in addition to a decreased relative force, a significant reduction in bound Ca2+ at the shorter sarcomere length. Thus sarcomere length appears to influence the Ca2+ binding properties of the regulatory site on troponin C. These data provide direct evidence that length-dependent modulation of Ca2+-troponin C affinity may make a major contribution to the force-length relationship in cardiac muscle.

Osmotic compression of skinned cardiac and skeletal muscle bundles: Effects on force generation, Ca2+ sensitivity and Ca2+ binding

Length-dependence of myofilament Ca2+ sensitivity is now considered to be an important component of the steep relationship between active force and sarcomere length along the ascending limb of the cardiac force-length curve. Studies with skinned cardiac muscle preparations have demonstrated that Ca(2+)-troponin C affinity is significantly increased as sarcomere length is increased over the range 1.7-2.3 microns. Increase in sarcomere length is accompanied by a reduction in interfilament spacing. In skinned fiber preparations from both cardiac and skeletal muscle osmotic compression of the filament lattice enhances myofilament Ca2+ sensitivity. This study was undertaken to evaluate the hypothesis that a change in filament separation may contribute to the length-dependent activation seen in cardiac muscle. Moderate reduction in interfilament spacing caused by exposure to Dextran T-500 (5-10%) produced an increase in force generation in both maximally activated and partially activated preparations of skinned bovine ventricular muscle. With fiber bundles of mean sarcomere length 1.7 microns the addition of 5% Dextran T-500 produced an increase in Ca2+ sensitivity of about 0.25 pCa units and a significant increase in Ca2+ binding in the pCa range (6.0-5.0) in which the single regulatory site of cardiac troponin C is titrated. This concentration of Dextran T-500 produced a reduction in fiber width equivalent to that produced by stretching fibers from sarcomere length 1.7 microns to sarcomere length 2.3 microns Osmotic compression of skinned rabbit psoas muscle fibers also enhanced Ca2+ sensitivity but there was no significant change in Ca(2+)-troponin C affinity. These data suggest that 1) an important component of length-dependent Ca2+ sensitivity in both cardiac and skeletal muscle is the change in interfilament spacing, and 2) in cardiac muscle a reduction in spacing, like increase in length, leads to a specific increase in Ca(2+)-troponin C affinity. Thus both filament overlap and filament separation contribute to the length dependence of Ca2+ sensitivity and Ca2+ binding in cardiac muscle.

Inhibition of sarcotubular calcium transport by caffeine: Species and temperature dependence

Abstract The effect of caffeine on calcium transport by isolated sarcotubular vesicles from rabbit and frog skeletal muscle was compared. At 25° the calcium uptake by rabbit vesicles which sedimented at 1500–10000 × g was only slightly inhibited by caffeine (1–10 mM). Increasing the temperature to 37° caused a marked increase in the caffeine sensitivity of the rabbit vesicles. The lighter vesicular fraction (10000–30000 × g ) was relatively insensitive to caffeine at both temperatures. Frog vesicles at 25° were as sensitive to caffeine as the rabbit vesicles at 37°. This difference corresponds to the known differential sensitivity of amphibian and mammalian muscle to caffeine contracture. These results are consistent with the hypothesis that caffeine produces contracture through an inhibition of sarcotubular calcium transport.

Ion exchange properties of the calcium receptor site of troponin

Abstract 1. The exchangeability of troponin-bound 45Ca2+ with various cations was investigated, using a gel filtration technique. 2. Bound 45Ca2+ was completely exchangeable with non-radioactive Ca2+ and partially exchangeable with Cd2+, Sr2+, Pb2+, and Mn2+, in the order given. There was no significant exchange with Mg2+, Ba2+, Ni2+, Zn2+, and Co2+. The affinity of divalent cations for the receptor site of troponin was closely related to ionic radius, with 1 A (Ca2+) providing the most favorable fit. 3. The trivalent lanthanide ions, La3+, Ce3+, Nd3+, Sm3+, and Dy3+ could partially displace 45Ca2+ from troponin but the affinity of these ions for the receptor site of troponin was nevertheless lower than that of Ca2+ despite their similarity to Ca2+ in size and chemical properties. 4. Elevation of the ionic strength to 0.5 with KCl or NaCl caused no dissociation of Ca2+ from troponin.

Cooperative interactions between calcium-binding sites on glycerinated muscle fibers the influence of cross-bridge attachment

A double isotope technique and EGTA buffers were used to measure the binding of Ca2+ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca2+-binding sites in the intact filament lattice. In the presence of 5 mM MgCl2 (no ATP), reduction of filament overlap was associated with a reduced binding of Ca2+ over the entire range of free Ca2+ concentrations (5.10(-8)-2.10(-5) M). With maximum filament overlap (sarcomere length 2.1-2.2 micrometer) the maximum bound Ca2+ was equivalent to 4 mol Ca2+/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8-4.4 micrometer) the maximum bound Ca2+ was equivalent to 3 mumole Ca2+/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca2+ binding site appear, but cooperative properties are imposed upon the binding sites.

The binding of calcium to detergent-extracted rabbit psoas muscle fibres during relaxation and force generation

SummaryRigor complexes between actin and myosin have been shown to cause increased binding of Ca2+ to troponin C. A similar effect of force-generating crossbridges has been suggested as an explanation for the coupling between load and activation which has been observed in skeletal and cardiac muscle. The goal of this study was to test the hypothesis that Ca2+-troponin affinity during crossbridge cycling is load-dependent. Ca2+-binding to detergent-extracted rabbit psoas fibres was measured during ATP-induced force generation and in the relaxed state. To compare Ca2+ binding in the latter two states it was necessary to establish conditions in which ATP-induced force could be regulated independently of free Ca2+ concentration. Such conditions were obtained by the use of either the ATPase inhibitor sodium vanadate or the substitution of MgITP for MgATP as an energy source. This study showed that in the presence of MgATP (or MgITP) the amount of Ca2+ bound to the myofilaments at a given free Ca2+ concentration was independent of the force generated. Thus forceper se is not a determinant of Ca2+-troponin affinity.

A study on the mechanism of phalloidin-induced tension changes in skinned rabbit psoas muscle fibres.

SummaryThe time course of phalloidin induced changes in isometric tension of partially activated skinned rabbit psoas fibres was studied as a function of both phalloidin concentration and time of pre-incubation with phalloidin. Upon addition of phalloidin to non-pretreated (control) fibres there was a fall in tension folllowed by an increase in tension. The latency of both parts of the response was inversely related to the phalloidin concentration in the range 40–130 μm phalloidin. By pre-incubating the fibres with phalloidin for varying periods of time it was possible to obtain responses which appeared to represent later portions of the control response. Thus after pre-treatment with 40 μm phalloidin in either rigor or relaxing solution for 5 min (the time corresponding to minimal tension in the control response) the tension response resembled that of the control, beginning from the vicinity of the minimum. The pattern of staining of the fibres by rhodamine-phalloidin was analysed by laser confocal microscopy to relate the mechanical response to phalloidin localization. If fibres were treated with rhodamine-phalloidin for 20–25 min there was a labelling of the I-Z-I segment with intense peaks of fluorescence at the Z-line and the ends of the I filaments. If fibres were pre-incubated for 5 min with phalloidin and then labelled with rhodamine-phalloidin the fluorescence at the Z-line and at the ends of the I filaments was suppressed and the peak of the fluorescence intensity was shifted toward the middle part of the I filament. The data indicate that the decrease in tension caused by phalloidin was associated with binding of phalloidin to the pointed ends of actin filament and the Z-line region, whereas the increase in tension occurred when phalloidin was bound along entire length of the actin filament.

Parallel measurements of bound calcium and force in glycerinated rabbit psoas muscle fibers

A simple double-isotope procedure has been developed for making simultaneous measurements of bound Ca2+ and relative force in glycerinated rabbit psoas bundles containing two fibers. With this preparation it is possible to study Ca2+-troponin interactions coincident with MgATP-induced force development. Over the free [Ca2+] range 6 . 10(-8)--1.2 . 10(-5) M the bound Ca2+ varied from 0.25 to 1.65 mumol/g protein. The free [Ca2+] at half-maximal Ca2+ saturation was 2 . 10(-7) M while that a half-maximal force was 5 . 10(-7) M. Half-maximal Ca2+ saturation was associated with 20% maximal force. The force-[Ca2+] saturation curve showed a steep rise in slope at greater than half saturation. The observed relationship was consistent with a model in which multiple occupancy of troponin Ca2+-binding sites is essential for initiation of cross-bridge cycling.

On the relation between filament overlap and the number of calcium-binding sites on glycerinated muscle fibers.

The formation of rigor complexes between the thick and thin filaments of glycerinated rabbit psoas muscle fibers causes the fibers to bind more calcium at any given level of free calcium. I studied the maximum amount of calcium bound as a function of filament overlap under rigor conditions. Fibers stretched to zero filament overlap (sarcomere length greater than 3.8 micron) bound exactly 75% as much calcium as fibers with maximum overlap. Between these extremes a linear relationship was found between maximum bound calcium and the length of the overlap zone. The results support the hypothesis that in the intact filament lattice one of the four calcium-binding sites of troponin depends for its existence on attachment between myosin and actin. In addition, the linear relation between maximum bound calcium and filament overlap is consistent with the assumption that the cooperative effect of rigor complex formation on calcium binding is limited to the binding site in the immediate vicinity of the rigor complex.