Effects of taurine on Ca²⁺-dependent force development of skinned muscle fibre preparations

Taurine occurs widely in animal tissues, often at concentrations exceeding those of most other free amino acids (Jacobsen and Smith, 1968). The highest concentrations have been detected in excitable tissues such as nerve and muscle (Huxtable, 1980), and a variety of physiological effects have been reported. The most established function is its contribution to osmoregulation in the muscle tissues of many marine animals, especially crustaceans (Shaw, 1958; Allen and Garrett, 1971; Dalla Via, 1989).

Taurine is also present in considerable amounts in heart tissues, constituting up to 50 % of the free amino acid pool in mammals (Awapara et al. 1950). Here, too, taurine has been proposed to maintain cardiac osmolarity (Thurston et al. 1981).

In addition, taurine modulates numerous Ca²⁺-dependent processes in heart and other tissues (Chovan et al. 1980; Kramer et al. 1981; Huxtable, 1989; Dolara et al. 1973). Taurine influences cardiac contractility, inducing antiarrhythmic activity (Read and Welty, 1963) and exerting positive (in low-Ca²⁺ media) as well as negative (in high-Ca²⁺ media) inotropic effects on intact mammalian hearts (Dietrich and Diacono, 1971; Schaffer et al. 1978; Franconi et al. 1982). Furthermore, the positive inotropic effects of cardiac glycosides are potentiated by taurine (Guidotti et al. 1971).

Direct effects of taurine on force generation of the isolated contractile machinery have not yet been reported. We measured the isometric force production of Ca²⁺-activated skinned muscle fibre preparations (see Stephenson, 1981) of pig heart and slow skeletal muscles of crayfish in the presence and absence of physiological concentrations of taurine. Some of our results have been published in abstract form (Galler and Hutzler, 1988).

Pig hearts were obtained from the local slaughterhouse. Crayfish (Pacifastacus leniusculus, Dana) originated from the University of Konstanz. Tissue concentrations of taurine were determined using a slightly modified HPLC technique described earlier (Haller and Lackner, 1987). For mechanical experiments, fibre bundles of pig heart muscle (trabeculae of the right atrium and musculi papillares in the right ventricle) were chemically skinned in a solution containing 50% glycerol, 1% Triton X-100, 2mmol 1⁻¹ dithioerythritol (DTE), 10mmoll⁻¹ NaN₃, 5 mmol I⁻¹ ATP, 5 mmol I⁻¹ MgCl₂, 5 mmol 1⁻¹ EGTAand 20 mmol 1⁻¹ imidazole, pH 7.0 adjusted with KOH. Since preliminary mechanical experiments suggested that there are intracellular Ca²⁺-sequestering vesicles, we extended the skinning procedure to 24 h at a temperature of about 4°C. Such treated preparations did not show any Ca²⁺-translocating activity, but the velocity of force development was generally reduced. Maximum isometric tension was also diminished (about 1.5 Ncm⁻²). We have chosen these preparations to exclude any possible effects of Ca²⁺ translocation due to the activity of sarcoplasmic reticulum.

Skinned skeletal muscle fibres of crayfish were prepared by drying abdominal muscles (using silica gel, at − 20°C) that had been frozen quickly in liquid nitrogen (see Stienen et al. 1983). For mechanical measurements, single fibres of superficial abdominal extensor muscles (Parnas and Atwood, 1966) were used.

For heart muscle preparations, the relaxation solution contained Mopso [3-(N-morpholino)-2-hydroxypropanesulphonic acid] (20 mmol 1⁻¹), K₂H₂EGTA (10 mmol 1⁻¹), Na₂H₂ATP (10 mmol 1⁻¹), sodium phosphocreatine (10 mmol I⁻¹), magnesium propionate (13.4mmoll⁻¹) and taurine (0 or 5mmoll⁻¹). The maximal activating solution had the same composition, except that K₂H₂EGTA was substituted by CaH₂EGTA (10 mmol 1⁻¹) and the magnesium propionate concentration was 12.9 mmol I⁻¹. The ionic strength of the solutions was 0.11 mol 1⁻¹. For the crayfish slow muscle preparation, relaxation and maximal activating solutions (ionic strength, 0.22 mol 1⁻¹; solutions modified after Moisescu and Thieleczek, 1979) both contained Mopso (60 mmol 1⁻¹), Na₂H₂ATP (8mmoll⁻¹), sodium phosphocreatine (10mmoll⁻¹), caffeine (15mmoll⁻¹) and taurine (0 or 5 mmol 1⁻¹). In addition, the relaxation solution contained K₂H₂EGTA (50 mmol 1⁻¹) and magnesium propionate (8.3 mmol I⁻¹) and the maximal activating solution contained CaH₂EGTA (50 mmol I⁻¹) and magnesium propionate (7.4 mmol I⁻¹). In all cases, pH was adjusted to 6.70 at a temperature of 22±0.5°C.

In preliminary experiments, different ion compositions of the bath solutions were tested to obtain optimal conditions for reproducible force values of the skinned muscle fibre preparations. A relatively low pH of 6.70 appeared most appropriate. In addition, the adjustment of the required free Ca²⁺ concentrations in the EGTA-buffered solutions is facilitated at this relatively low pH. For crayfish fast skeletal muscle fibre preparations no condition could be detected in which force transitions were reproducible enough to study effects on myofibrillar Ca²⁺ sensitivity. Thus, taurine effects could not be investigated in crayfish fast muscle fibres.

To obtain solutions with different Ca²⁺ concentrations, relaxing and maximal activating solutions were mixed in different ratios. Creatine kinase (50i.u. ml⁻¹) was added to all solutions immediately before the mechanical experiments. Precise concentrations of free calcium and magnesium ions in all bath solutions were achieved with an iterative computer program based on the equilibrium constants listed by Martell and Smith (1977). The free magnesium ion concentration in the bath solutions was 1 mmol I⁻¹ in the case of crayfish skeletal muscle fibres and 3 mmol 1⁻¹ in the case of the pig heart preparations. Free Ca²⁺ concentrations of the activating solutions with and without taurine were measured with a Ca²⁺-selective electrode (Schefer et al. 1986) calibrated with the solutions described by Tsien and Rink (1980) (ionic strength, 0.13 mol I⁻¹). Differences, due to different ionic strengths, of the activity coefficients of Ca²⁺ in the calibration and experimental solutions were corrected by applying the Debye Hückel formalism as described by Meier et al. (1980).

The skinned fibres were mounted horizontally between a fixed glass needle and a force transducer (AE801, SensoNor, Horton, Norway) with nitrocellulose dissolved in acetone. The preparations were 0.5–2 mm long with diameters of 70–200 μm. After mounting, the fibres were immediately incubated in the relaxing solution. To improve the pattern of cross-striation, length changes of about 1 % of total fibre length were induced for about 5 min by moving the glass needle sinusoidally with a vibrator (Ling V101). Sarcomere length of the muscle fibres.was measured with a laser beam (He-Ne laser from Spectra physics, model 102; 4 mW) according to the method described by Zite-Ferenczi and Rüdel (1978).

Sarcomere length of the heart preparations was set at 2.1 μm; that of crayfish slow skeletal muscle fibres was not changed after incubating the stiff dried fibres mounted at the apparatus for force measurements. It ranged from 7 to 10μm.

The skinned fibre preparations were activated by incubation in solutions with different Ca²⁺ concentrations. An automatic cuvette transporting system provided a rapid change of bath solutions. Force signals were measured with a bridge amplifier connected to a chart recorder (Gould, Brush 220).

Concentrations of taurine in different tissues and animals are listed in Table 1. The amount of taurine in atrial muscles of pig is smaller than in ventricular muscles (P<0.05). In crayfish, slow abdominal extensor muscles contain up to seven times more taurine than do fast abdominal extensor muscles. The ratio varies considerably in different animals.

Maximal activating solutions with or without taurine were applied with or without intermittent relaxation to test the effects of taurine on maximum isometric force development. Up to concentrations of 20 mmol 1⁻¹, no significant influence of taurine on maximum isometric force could be detected.

Successive activations of crayfish slow skeletal muscle fibres by addition of Ca²⁺ were interrupted by total relaxation, whereas in pig heart preparations, Ca²⁺ concentration was raised gradually without intermittent relaxations. Activating solutions with and without taurine were applied alternately (Fig. 1).

In several experiments, the preparations were preincubated in a taurine-containing relaxation solution for different times (0–30 min) prior to the submaximal activations in the presence of taurine. In other experiments, crayfish slow muscle preparations were incubated continuously in taurine-containing media, but at the end of the experiments submaximal activations were applied in taurine-free media. Here, preincubation in taurine-free relaxation solution lasted 1 min. In all these cases of different incubation procedures, similar taurine effects were observed.

In most of the experiments on crayfish slow muscle, preparations were kept in taurine-free media for about 15 min during the first incubations in relaxation and activation solutions. Pig heart preparations were kept in taurine-free media for more than 24 h during the skinning procedure and the first mechanical experiments. When changing from taurine-free to taurine-containing media or vice versa, preincubations lasting from about 20s (crayfish preparations) to 2 min (pig heart preparations) in the corresponding relaxation solutions were applied.

Submaximal forces were enhanced by taurine in both cardiac and crayfish slow muscles. At about half-maximal activation the observed increase was 15.2±9.7 % (S.D.) of maximal force in atrial preparations (N=8), 17.9±13.0% (S.D.) in ventricular preparations (N=10), and 13.2±6.5% (S.D.) in crayfish slow skeletal muscle fibres (N=12) in the presence of 5 mmol I⁻¹ taurine. Relative force plotted against pCa of the activating solutions and fitted with a Hill equation (Altringham and Johnston, 1982) revealed a shift of the curve towards lower Ca²⁺ values in the presence of taurine (Fig. 2). The increase of pCa₅₀ (the negative logarithm of Ca²⁺ concentration for half-maximal activation) is significant (paired t-test, P<0.001) and similar for different preparations: pig atrial fibres, 0.13±0.09pCaunits (±S.D., N=7); pig ventricular fibres, 0.15±0.11 pCa units (N=10); crayfish slow muscle fibres, 0.10±0.04pCa units (N=12). Taurine did not significantly change the slope of the pCa-force curve in any preparation.

Our measurements of taurine concentrations in different muscle tissues correspond with the findings in skeletal muscles of chicken (Airaksinen and Partanen, 1985) and fish (Haller and Lackner, 1987), where higher concentrations were found in the slow muscles than in the fast muscles. The higher taurine levels in pig ventricular muscles compared to those in atrial muscles also fit the above pattern, since ventricular muscle is considered to be slow and atrial muscle to be fast (Morano et al. 1988).

Physiological concentrations of taurine in pig heart and crayfish slow skeletal muscle fibres shift the pCa-force curve to lower Ca²⁺ concentrations without affecting the maximal force. These findings cannot be explained by a change of Ca²⁺ activity within the muscle fibre preparations as a result of Ca²⁺ movement through the sarcoplasmic reticulum. Pig heart preparations were treated with the non-ionic detergent Triton X-100, which removes membranes of sarcoplasmic reticulum (Meisheri and Rüegg, 1983). In the case of crayfish slow skeletal muscle fibres, all solutions for mechanical experiments contained 15 mmol I⁻¹ caffeine, which prevents accumulation of Ca²⁺ in the sarcoplasmic reticulum (Nagasaki and Kasai, 1983). In addition, a constant Ca²⁺ level was maintained using high concentrations of the Ca²⁺ buffer EGTA(50mmol 1⁻¹). The possibility, therefore, that taurine acts via a change of Ca²⁺ concentration mediated through the sarcoplasmic reticulum can be excluded. We assume instead that taurine increases the Ca²⁺ sensitivity of the force-generating structures in pig heart and crayfish slow skeletal muscles.

As to the mechanism of action of taurine, its dipolar character may be responsible for the effects mentioned above, since taurine weakens charge dependent protein-protein interactions. The troponin-I-actin binding which inhibits the force-generating myosin-actin interaction (El-Saleh et al. 1986) could theoretically be weakened by taurine. This would lead to the observed calcium-sensitizing effect (Rüegg, 1987).

The positive inotropic effect of taurine on living mammalian hearts (e.g. Dietrich and Diacono, 1971; Dolara et al. 1973) is usually thought to be caused by an increase in intracellular Ca²⁺ concentration mediated by taurine. Franconi et al. (1982) measured the force and tissue Ca²⁺ concentrations of superfused guinea pig ventricular strips at different external CaCl₂ concentrations in the presence and absence of taurine. They found only a coarse positive correlation between the taurine-mediated change in contractility and the taurine-induced change in the Ca²⁺ concentration of the heart tissue at different external CaCl₂ concentrations. The maxima of the two parameters appeared at different external CaCl₂ concentrations. The maximal taurine-mediated increase of internal Ca²⁺ concentration occurred at an external CaCl₂ concentration of 1.8 mmol I⁻¹, whereas the maximal taurine-induced positive inotropic effect appeared at an external CaCl₂ concentration of 0.9 mmol I⁻¹. Thus, in the presence of taurine, the increase of force is not always strictly correlated with an increase of internal Ca²⁺ concentration. Therefore, the positive inotropic effect of taurine may not be an exclusive effect of increased intracellular Ca²⁺ in intact heart muscle fibres; our study suggests that it may be partially due to enhanced Ca²⁺ sensitivity of force generating myofilaments.

The potentiation of the positive inotropic effect of cardiac glycosides on intact mammalian hearts by taurine (Guidotti et al. 1971) may also be explained by the Ca²⁺-sensitizing action. For these reasons, further attention should be paid to taurine as a potential moderate cardiotonic drug.

Since changes in taurine concentration appear in mammalian hearts and in crustacean skeletal muscles in different physiological states, the Ca²⁺-sensitizing effect of taurine could have physiologically important implications. Congestive heart failure in humans (Huxtable and Bressler, 1974) and dogs (Peterson et al. 1973) is accompanied by an increased taurine concentration in the heart tissues affected. Probably taurine is increased because of its Ca²⁺-sensitizing effects on myofibrils.

In some crustacean species (e.g. Carcinus, Eriocheir and Palaemon; see Shaw, 1958; Allen and Garrett, 1971; Dalla Via, 1989) changes in sarcoplasmic taurine concentration were found to be dependent on extracellular osmolarity. Implications for muscle contraction are still unknown.

Apart from the naturally occurring sarcoplasmic imidazoles carnosine and N-acetyl histidine (Harrison et al. 1986), no endogenous Ca²⁺ sensitizers have yet been found. Non-endogenous Ca²⁺-sensitizing drugs (submazole, Herzig et al. 1981; caffeine, Wendt and Stephenson, 1983) are imidazole derivatives. With taurine, a Ca²⁺ sensitizer with different chemical properties has been found. It is possible that other sarcoplasmic amino acids and peptides (e.g. glutamine and glutathione) may also have modulatory effects on the Ca²⁺ sensitivity of myofibrils.

This work is dedicated to Professor Dr W. Wieser (Institut für Zoologie, Universitat Innsbruck) on the occasion of his 65^th birthday. The authors wish to thank Professor J. C. Rüegg (Heidelberg) and his group for a gentle introduction into the skinned fibre technique and their encouragement. Our thanks are also due to Professor W. Rathmayer (Konstanz) for generous and helpful support, Professor W. Wieser (Innsbruck) for reading the manuscript and D. Günzel and S. Lanka (Konstanz) for valuable assistance and support. J. Vogt, W. Kühnel and S. Hahn (Konstanz) are acknowledged for constructing our mechanical apparatus and Drs D. Ammann and T. Bührer (Zürich) for providing some Ca²⁺-selective membranes. This work was supported by the DFG (SFB156) and by the FWF (P-7162/BIO).