PubMed Health⌕ Search

Biomedical subjects

F Fabiato

Publications and source records attributed to F Fabiato.

At least 19 recordsLinked to original sources
PubMed1979

Use of chlorotetracycline fluorescence to demonstrate Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum of skinned cardiac cells.

It has been proposed that the trans-sarcolemmal influx of Ca2+ occurring during the plateau of the mammalian cardiac action potentials is insufficient in itself to activate the myofilaments, but can trigger a release of Ca2+ from the sarcoplasmic reticulum (SR) which is sufficient for activation. The demonstration of this Ca2+-induced release of Ca2+ relied entirely on experiments in which the tension developed by the myofilaments was used as a sensor of the changes of myoplasmic free Ca2+ concentration ([free Ca2+]) in segments of single cardiac cells from which the sarcolemma had been removed by microdissection (skinned cardiac cells). The small size of these preparations has previously prevented the use of more direct methods for the detection of myoplasmic Ca2+ movements. The present study is a direct demonstration of Ca2+-induced release of Ca2+ from the SR of skinned cardiac cells treated with chlorotetracycline (CTC), a fluorescent chelate probe which enables changes in the amount of Ca2+ bound to a variety of biological membranes or micelles to be monitored. The fluorescence increases when more Ca2+ is bound.

Animals↗
PubMed1978

Cyclic AMP-induced enhancement of calcium accumulation by the sarcoplasmic reticulum with no modification of the sensitivity of the myofilaments to calcium in skinned fibres from a yeast skeletal muscle.

In the presence of low concentrations of total EGTA (5 . 10(-4) M) and free Mg2+ (3.16 . 10(-5) M) and in the presence of caffeine (8 . 10(-3) M), cyclic AMP (5 . 10(-6) M) produces a relaxation of the tension developed by skinned fibres from cat caudo-femoralis. The relaxation can be attributed to an enhancement of the Ca2+ accumulation by the sarcoplasmic reticulum, since cyclic AMP does not modify the sensitivity of the myofilaments of Ca2+. These results are similar to those previously reported for the effect of cyclic AMP on skinned cardiac cells in the presence of a higher free Mg2+ concentration and in the absence of caffeine. This similarity suggests that the mode of action of cyclic AMP on the sarcoplasmic reticulum is not fundamentally different in cardiac and fast skeletal muscles.

Animals↗
PubMed1978

Myofilament-generated tension oscillations during partial calcium activation and activation dependence of the sarcomere length-tension relation of skinned cardiac cells.

During partial Ca2+ activation, skinned cardiac cells with sarcoplasmic reticulum destroyed by detergent developed spontaneous tension oscillations consisting of cycles (0.1-1 Hz) of rapid decrease of tension corresponding to the yield of some sarcomeres and slow redevelopment of tension corresponding to the reshortening of these sarcomeres. Such myofilament-generated tension oscillations were never observed during the full activation induced by a saturating [free Ca2+] or during the rigor tension induced by decreasing [MgATP] in the absence of free Ca2+ or when the mean sarcomere length (SL) of the preparation was greater than 3.10 microm during partial Ca2+ activation. A stiff parallel elastic element borne by a structure that could be digested by elastase hindered the study of the SL--active tension diagram in 8-13-microm-wide skinned cells from the rat ventricle, but this study was possible in 2-7-microm-wide myofibril bundles from the frog or dog ventricle. During rigor the tension decreased linearly when SL was increased from 2.35 to 3.80 microm. During full Ca2+ activation the tension decreased by less than 20% when SL was increased from 2.35 to approximately 3.10 microm. During partial Ca2+ activation the tension increased when SL was increased from 2.35 to 3.00 microm. From this observation of an apparent increase in the sensitivity of the myofilaments to Ca2+ induced by increasing SL during partial Ca2+ activation, a model was proposed that describes the tension oscillations and permits the derivation of the maximal velocity of shortening (Vmax). Vmax was increased by increasing [free Ca2+] or decreasing [free Mg2+] but not by increasing SL.

Animals↗
PubMed1978

Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiace and skeletal muscles.

1. The effects of decreasing pH from 7.40 to 6.20 on the tension developed by direct activation of the myofilaments and by Ca2+ release from the sarcoplasmic reticulum were studied comparatively in segments of single cells of skeletal muscle (frog semitendinosus) and cardiac muscle (rat ventricle) from which the sarcolemma had been removed by micro-dissection (skinned muscle cells). 2. The concentration of free Ca2+ in the solutions was buffered with ethylene glycol-bis (beta-aminoethylether N,N'-tetraacetic acid (EGTA). The change of the buffer capacity of a given [total EGTA] caused by varying pH and the uncertainty about the value of the equilibrium constant for Ca-EGTA have been taken into account in the interpretation of the results. 3. Decreasing pH from 7.40 to 6.20 produced an increase in the [free Ca2+] required for the myofilaments to develop 50% of the maximum tension by a factor of about 5 in skinned cardiac cells but of only 3 in skeletal muscle fibres. In addition, acidosis depressed the maximum tension developed in the presence of a saturating [free Ca2+] by approximately the same amount in the two tissues. 4. The pH optimum for loading the sarcoplasmic reticulum of skinned fibres from skeletal muscle decreased when the pCa (-log [free Ca2+]) in the loading solution decreased. The optimum was pH 7.40-7.00 for a loading at pCa 7.75, pH 7.00-6.60 at pCa 7.00 and pH 6.60-6.20 at pCa 6.00. 5. The pH optimum for loading the sarcoplasmic reticulum of skinned cardiac cells with a solution at pCa 7.75 was about pH 7.40 as in skeletal muscle fibres. But the cardiac sarcoplasmic reticulum could not be loaded with a [free Ca2+] much higher than pCa 7.75 because a higher [free Ca2+] triggered a Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum. 6. The pH optimum of about 7.40 for the loading of the cardiac sarcoplasmic reticulum was also optimum for the Ca2+-induced release of Ca2+ from it. 7. It was concluded that the effects of acidosis on the cardiac sarcoplasmic reticulum accentuate the depressive action of decreasing pH on the myofilaments. This may explain the pronounced depression of contractility observed during acidosis in cardiac muscle. In contrast, a moderate acidosis causes an effect on skeletal muscle sarcoplasmic reticulum that could compensate for the depressive action on the myofilaments, which is, in addition, less pronounced than in cardiac muscle.

Animals↗
PubMed1976

Dependence of calcium release, tension generation and restoring forces on sarcomere length in skinned cardiac cells.

The ascending limb of the length-tension diagram was studied in single skinned (sarcolemma-free) cardiac cells of rat ventricle. The free (Ca2+) in the perfusing solutions was buffered with ethyleneglycol-bis(beta-aminoethyl)N,N'-tetraacetic acid (EGTA). Tension was recorded with a photo-diode force transducer (detection limit: 1 mug, compliance 2-3 mum/mg) and was expressed as a function of the sarcomere length (SL) measured during contraction with a high-speed movie camera. When the SL was decreased by a brief (1-2 sec) exposure to high free (Ca2+), a restoring force qas observed upon return of the cell to a relaxing solution. The restoring force was comprised of two components: (1) a rapid elongation to 1.57 identical to 0.06 mum developing a negative tension (relaxing force) as large as 4% of the maximum positive tension, which was observed in both skinned cells and in single myofibrils, and (2) a slow elongation from 1.57 identical to 0.06 to 1.93 identical to 0.14 mum, which was observed in skinned cells but not in single myofibrils. When the SL was kept extremely short by continuous Ca2+ activation for more than 30 sec, a shortening of the A band much below 1.0 mum persisted for several minutes after imposed re-elongation of the sarcomers (delta state). The amplitude of the tonic tension developed by direct activation of the myofilaments in the presence of high total (EGTA) was maximum for 2.20 mum SL and decreased very little when the SL was decreased. However, the decrease of tension became more pronounced at SL shorter than 1.55 mum when polyvinylpyrrolidone was added to the solutions at a concentration reducing the swelling of the myofilament lattice. This finding imposes some caution in applying the results obtained in skinned cardiac cells to intact tissue. The SL-tension diagram of the phasic contractions induced by Ca2+-triggered of Ca2+ in the presence of a slight EGTA buffering was similar to the SL-tension diagram of the intact rat ventricular muscle when the latter was expressed as a function of the active SL. In contrast, the SL-tension diagram of caffeine-induced phasic contractions was similar to that of the tonic tension produced by direct activation of the myofilaments. Decreasing SL results therefore in a partial inhibition of the Ca2+ triggered release process. It was concluded that Starling's Law may correspond to a length-dependence of several mechanisms including Ca2+-triggered release from the SR, interaction between thick and thin filaments and restoring forces.

Calcium↗
PubMed1976

Techniques of skinned cardiac cells and of isolated cardiac fibers with disrupted sarcolemmas with reference to the effects of catecholamines and of caffeine.

Single cardiac cells with disrupted sarcolemmas were obtained by homogenization in low free [Ca2+] medium. Skinned cardiac cells were obtained by microdissection of remaining pieces of sarcolemmas. In both preparations the buffers bathing the cells were in contact with the structures controlling activation: myofilaments, sarcoplasmic reticulum, and mitochondria. Tension was recorded with a highly sensitive force transducer. The use of either high or low total [EGTA] permitted the study of the effect of free [Ca2+] on either the myofilaments or the internal stores. At physiological levels of myoplasmic free [Ca2+], the internal stores of Ca2+ were located mostly in the sarcoplasmic reticulum, whereas mitochondria were involved at higher free [Ca2+]. Single cardiac cells with simply disrupted sarcolemmas responded to variations of perfusion media in a manner similar to the skinned cardiac cells. However, the remaining pieces of sarcolemma represented a limit to the intracellular diffusion of externally applied media and might contain receptor sites for catecholamines. In multicellular fragments, evidence of surface membrane activity (resting and action potentials) were found during certain ionic modifications.

Adenosine Triphosphate↗
PubMed1975

Contractions induced by a calcium-triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells.

1. Fragments of single cardiac cells were obtained by homogenization of ventricular tissue from adult rats. Remaining pieces of sacrolemma were removed by micro-dissection. Tension was recorded from the ends of the skinned (sarcolemma-free) cells with a photodiode force transducer. 2. In the presence of a strong buffering of the free [Ca2+] with 4-0 mM total EGTA, a tonic tension was obtained that increased according to t sigmoid curve when the free ([Ca2+] was increased from 10(-6-75)M to 10(-5-0)M. This curve was not modified by the destruction of the sarcoplasmic reticulum (SR) by the detergent Brij 58. Therefore, the tonic tension corresponded to the direct effect of the free [Ca2+] present in the buffer on the myofilaments. 3. In the presence of a slight buffering of the free [Ca2+] with 0-050 mM total EGTA, cyclic contractions were observed that were attributed to cyclic releases and re-sequestrations of Ca2+ by the SR. The absence of effect of azide and ruthenium red on the cyclic contractions obtained at a free [Ca2+] lower than 10(-6-50)M demonstrated that the mitochondria played no role in the triggering of these contractions. 4. Cyclic contractions were induced by a slight variation of free [Ca2+] in the buffer from 10(-7-65)M to 10(-7-40)M. Their amplitude at 10(-7-40)M free Ca2+ was equal to the tonic tension developed by a free [Ca2+] 20 times higher applied to the myofilaments when the SR was destroyed by detergent or functionally inhibited by high total [EGTA]. It was concluded that these cyclic contractions corresponded to a Ca2+-triggered release of Ca2+ from the SR. 5. The cyclic contractions were induced by the filling of the SR with Ca2+ to a critical level at which it released a fraction of the Ca2+ it contained. Each contraction was followed by a re-sequestration of Ca2+, the kinetics of which conditioned the duration of the cycles. 6. The amplitude of the cyclic contractions increased when the free [Ca2+] that triggered them was increased. This gradation was deemed incompatible with a simple regenerative process, which should produce an all-or-nothing response. Additional process, such as a modulation of the Ca2+ release by free [Mg2+] and [ADP] may help to explain the gradation of the contractions. 7. It was concluded that a Ca2+-triggered release of Ca2+ from the SR of rat ventricular cells may amplify the Ca2+ flux crossing the sarcolemma during the plateau of the action potential, thereby permitting the activation of the myofilaments.

Animals↗
PubMed1975

Effects of magnesium on contractile activation of skinned cardiac cells.

1. In the presence of a slight buffering of the free [Ca2+] with 0.050 mM total EGTA cyclic contractions were induced by a Ca2+-triggered release of Ca2+ on skinned (sarcolemma-free) segments of single cardiac cells from rat ventricle. The threshold of the free [Ca2+] trigger was elevated when the free [Mg2+] was increased. 2. At a suprathreshold free [Ca2+] increasing the free [Mg2+] resulted in a decrease in frequency and in an increase in amplitude of the phasic contractions. Addition of caffeine at a specified interval after a cyclic contraction produced a larger contraction when free [Mg2+] was higher. It was concluded that an increase of free [Mg2+] increased the capacity and the rate of binding for Ca2+ by the sarcoplasmic reticulum (SR). 3. Small skinned fibres of skeletal muscle which were perfused with 10 mM caffeine yielded results similar to those obtained in skinned cardiac cells. It was concluded that the mechanism of action of free Mg2+ was similar in both preparations, but that the SR of skeletal muscle had a higher capacity and rate of binding for Ca2+ than the cardiac SR. 4. With a strong buffering of the free [Ca2+] with 4-0 mM total EGTA, a smaller tonic tension was developed for a given pCa in the presence of a higher free [Mg2+]. This result was nearly identical in skinned cells from cardiac and skeletal muscle tissue. 5. A decrease of the [MgATP2-] produced a tension in the skinned cardiac cells that were perfused in Ca2+ free media. The maximum tension was observed for [MgATP2-] 10(-5-50)M as in skinned fibres of skeletal muscle. A further decrease of [MgATP2-] resulted in a decrease of tension.

Animals↗