INTRODUCTION

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IN MAMMALIAN CARDIAC MUSCLES, an increase in the myoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) precedes contraction, and Ca²⁺ binding to troponin C (TnC) is one of the important steps that triggers cross-bridge attachment to actin filaments (10). However, the attachment of the cross bridges to actin filaments is considered to alter the affinity of TnC for Ca²⁺, which secondarily influences tension development and [Ca²⁺]_i (22). This feedback mechanism from the cross bridge to TnC (tension-dependent change in the affinity of TnC for Ca²⁺) in mammalian cardiac muscles is one of the important factors controlling efficient contraction-relaxation cycles of the heart.

It is reported that the tension-dependent change in the affinity of TnC for Ca²⁺ is reflected in the falling phase of the Ca²⁺ transients in twitch contraction. Therefore, the decay time of the Ca²⁺ transients is significantly altered depending on peak tension (3, 5, 17, 20, 23). However, the falling phase of the Ca²⁺ transients is also influenced by other factors, particularly Ca²⁺ removal mechanisms such as sarcoplasmic reticulum (SR) and/or Na⁺/Ca²⁺ exchange. Therefore, the [Ca²⁺]_i change induced by the tension-dependent change in the affinity of TnC for Ca²⁺ could also be influenced by the activities of the Ca²⁺ removal mechanisms. However, the interaction between the Ca²⁺ removal mechanisms and the tension-dependent change in the affinity of TnC for Ca²⁺ in the Ca²⁺ transient falling phase is not fully understood. Therefore, we intended to investigate the possible contribution of the activity of the Ca²⁺ pump of SR, a major Ca²⁺ removal mechanism under physiological conditions, in the tension-dependent alteration in the decay time of the Ca²⁺ transients.

To increase the rate of the Ca²⁺ uptake by SR, we treated ferrets with thyroxine to induce the hyperthyroid state in the myocardium; this is known to increase the activity of the Ca²⁺ pump in SR (4, 8, 18, 25). We also measured the dependence of the decay time of the Ca²⁺ transients on peak twitch tension in cardiac muscles excised from the thyroxine-treated ferrets. Thyroxine treatment is also known to convert a large portion of the myosin isoforms from V₃ to V₁ and to increase the myofibrillar ATPase activity (4, 13, 18, 25). However, alterations in the properties of the contractile elements in the hyperthyroid myocardium have not been fully investigated. Therefore, we also analyzed the myofibrillar responsiveness to Ca²⁺ and the tension-dependent change in the Ca²⁺ affinity of TnC in the hyperthyroid myocardium. In addition to these changes, other ionic transport mechanisms (Na⁺/H⁺ exchanger and Na⁺-K⁺ pump) are known to be altered in hyperthyroid (33). Therefore, we also discussed the possible involvement of these factors in the present result.

Some of the results were presented at the international Carl Ludwig Symposium (16) and at the Annual Meeting of the Japanese Physiological Society (15).

	MATERIALS AND METHODS

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Animals. We used 3- to 5-mo-old male ferrets and divided them into two groups [euthyroid (Eu) and hyperthyroid (Hy) ferrets]. The ferrets in the Hy group were subcutaneously injected with L-thyroxine (0.3 mg/kg) for 18-21 days. Age-matched ferrets without treatment were used as the Eu group. All animals were kept under the same conditions.

Analysis of myosin isoforms in Eu and Hy hearts. Pyrophosphate polyacrylamide gel electrophoresis (13) was carried out to analyze the myosin isozyme pattern in Eu and Hy myocardium and to ascertain the efficacy of thyroxine treatment. Native myosin from the right ventricle was extracted, and electrophoresis was carried out at 4°C. The gels were fixed and stained with Coomassie brilliant blue and scanned by a laser densitometer (Ultroscan XL, Pharmacia, Uppsala, Sweden).

Preparations. Ferrets were anesthetized with pentobarbital sodium (intraperitoneal injection, 100 mg/kg). At the beginning of the present study, a blood sample of ~5 ml was taken from the left atria or left ventricle for the measurement of free 3,3',5'-triiodothyronine (T₃) and free thyroxine with a conventional radioimmunoassay method, and then the heart was quickly removed. After the blood was washed by retrograde perfusion of normal Tyrode solution through the aorta, thin papillary muscles or trabeculae were dissected from the right ventricle in a bath continuously perfused with normal Tyrode solution at 30 ± 0.5°C. Both ends of the preparation were tied with silk threads. One end of the preparation was connected to the lever of a motor (JCCX-101A, General Scanning, Watertown, MA) that was used to alter muscle length, and the other end was connected to the arm of a tension transducer (BG-10, Kulite, Semiconductor Products, Leonia, NJ; compliance, 2.5 µm/g; unloaded resonant frequency, 1 kHz). The preparation was mounted horizontally in an experimental chamber with a pair of platinum black electrodes placed parallel to the preparation for electrical stimulation. The preparation was stimulated with a rectangular pulse at 1.2-fold threshold with a 5-ms duration. The stimulation frequency was 0.2 Hz unless otherwise mentioned. Before the experiment was started, the preparation was slowly stretched to L_max, the length at which developed tension reached maximum. The muscle lengths of the Hy and Eu preparations were 3.52 ± 0.19 mm (n = 22) and 4.16 ± 0.18 mm (n = 21), and the diameters of the Hy and Eu preparations were 0.660 ± 0.045 mm (n = 22) and 0.686 ± 0.032 mm (n = 21), respectively.

Measurement of the intracellular Ca²⁺ transients with aequorin. A glass micropipette (resistance, 20-40 M) was filled with the calcium-sensitive photoprotein aequorin, which was dissolved in 150 mM KCl and 5 mM HEPES solution with a final aequorin concentration of 50-100 µM. Aequorin was injected into ~150 superficial cells of the preparation by monitoring the membrane potential. Aequorin light signal was detected with a photomultiplier (EMI 9789A, Ruislip, Middlesex, UK) mounted in a small housing [for details, see Allen and Kurihara (3)]. All data were stored in a computer (PC-9801, NEC, Tokyo, Japan) for later analysis. To improve the signal-to-noise ratio, 64 signals were averaged. In the experiments, using ryanodine and caffeine, 150 signals were averaged.

Solutions for intact preparations. The composition of the normal Tyrode solution used for dissection and for the injection of aequorin was as follows (in mM): 135 Na⁺, 5 K⁺, 2 Ca²⁺, 1 Mg²⁺, 102 Cl, 20 HCO₃, 1 HPO²₄, 1 SO²₄, 20 acetate, 10 glucose, and 5 U/l insulin, pH 7.35 at 30°C when equilibrated with 5% CO₂-95% O₂. In most experiments, Tyrode solution buffered with HEPES was used (HEPES-Tyrode solution) with the following composition (in mM): 128 Na⁺, 5 K⁺, 2 Ca²⁺, 1 Mg²⁺, 117 Cl, 1 SO²₄, 5 HEPES, 20 acetate, 10 glucose, and 5 U/l insulin; pH was adjusted to 7.40 with NaOH at 30°C. In some experiments, the extracellular Na⁺ was completely replaced by Li⁺ (Li-HEPES-Tyrode solution) to block the Na⁺/Ca²⁺ exchange. The solution was equilibrated with 100% O₂. When the concentration of Ca²⁺ was altered, the osmotic pressure of the solution was not adjusted, and CaCl₂ was added to or removed from the solution. The temperature of the solution was continuously monitored with a thermocouple and was maintained at 30 ± 0.5°C.

Experimental protocol. The peak tension was varied by changing the extracellular Ca²⁺ concentration ([Ca²⁺]_o) and/or muscle length. Tension and the Ca²⁺ transient were measured after both signals reached a steady state. The decay time of Ca²⁺ transient (DT) and the relaxation time of contraction (RT) were plotted against the relative peak tension, and the regression lines were drawn. We considered that the slopes of the regression lines represent the dependency of DT and RT on relative peak tension (20).

Measured parameters. The following parameters were measured: peak [Ca²⁺]_i, the magnitude of Ca²⁺ transient which was converted to [Ca²⁺]_i; time to peak light (TPL), the time for aequorin light to reach the peak from the onset of stimulus; DT, the time for aequorin light to decay from 75 to 25% of the peak; time to peak tension (TPT), the time measured from the onset of stimulus to the peak; relaxation time, the time for tension to decrease from the peak to 50%. In the experiments using ryanodine and caffeine, the time course of aequorin light was slow and the peak of aequorin light became flat. When the time course of Ca²⁺ transient was slow, the magnitude of the Ca²⁺ transient was substantially influenced by a change in the peak developed tension. Therefore, the change in the Ca²⁺ affinity of TnC is reflected not only in the decay phase of Ca²⁺ transient but also in the early phase near the peak of Ca²⁺ transient. Thus we measured the time for aequorin light to decline from the point that corresponds to the peak light measured in the solution with 2 mM [Ca²⁺]_o without ryanodine or caffeine and at L_max, to 25% of the peak in the presence or absence of the drugs.

Skinned preparations. Thin trabeculae (diameter, 0.1-0.2 mm) were dissected from the right ventricle of the same ferrets as those used for the measurements of Ca²⁺ transient and tension. The preparations were immersed in the relaxing solution containing 1% Triton X-100 for 60 min. After treatment of the preparation with Triton X-100, the preparation was washed with the relaxing solution without Triton X-100. The preparation was then immersed in the relaxing solution containing 50% glycerol and kept at 10°C before use. The mean diameter of the Hy preparations (0.15 ± 0.01 mm, n = 9) was not significantly different from that of the Eu preparations (0.13 ± 0.01 mm, n = 8). The preparation was cut into a small bundle (length, 1-1.5 mm) in the relaxing solution and used for the experiments. Both ends of the preparation were tied with silk monofilaments and then the preparation was transferred carefully to a muscle chamber of the same design as that reported by Horiuti (14). One end of the preparation was fixed to a tungsten wire (diameter, 0.1 mm) extending from the fixed arm, and the other end was attached to the arm of a tension transducer (BG-10, Kulite, Semiconductor Product). The sarcomere length of the preparations was adjusted to 2.3 µm by measuring the first order of laser diffraction lines.

Solutions and procedures for skinned preparations. The composition of the relaxing solution was as follows (in mM): 88.6 potassium methanesulfonate, 4.5 ATP, 5.2 magnesium methanesulfonate, 10 EGTA, 20 PIPES, 0.5 dithiothreitol, and 10 IU/ml creatine phosphokinase. pH was adjusted to 7.1 with KOH at 20°C. Free Ca²⁺ concentration of the solution was calculated using the binding constant of each ion for each ligand (27). The calculated apparent dissociation constant of EGTA for Ca²⁺ was 407 nM. The concentrations of free Mg²⁺ and Mg-ATP were kept at 1.0 and 3.5 mM, respectively. The ionic strength was maintained at 0.2 M. The solutions of various pCa (=log [Ca²⁺]) were made by mixing the relaxing solution and the solution at a pCa of 4.0. The temperature of the solution was kept at 20 ± 0.5°C throughout the experiment.

Chemicals. L-Thyroxine was purchased from Nakarai Tesque (Kyoto, Japan). Aequorin was purchased from Dr. J. R. Blinks (Friday Harbor, WA). Caffeine (Sigma Chemical, St. Louis, MO), with a desired concentration, was dissolved directly in HEPES-Tyrode solution before use. Ryanodine was purchased from AgriSystem (Wind Gap), and 1 mM stock solution was prepared by dissolving it in warmed double-distilled water that was stored at 0°C. 2,5-Di(tert-butyl)-1,4-benzohydroquinone (Aldrich Chemical, Milwaukee, WI) was dissolved in DMSO (Wako Pure Chemical, Tokyo, Japan) as the stock solution and diluted with HEPES-Tyrode solution before use. Na₂ATP was purchased from Boehringer Mannheim (Mannheim, Germany). EGTA was purchased from Wako Pure Chemical. PIPES and phosphocreatine disodium salt were purchased from Nakarai Tesque (Kyoto, Japan). Methanesulfonic acid and calcium methanesulfonate were from Tokyo Kasei (Tokyo, Japan). Creatine phosphokinase and dithiothreitol were from Sigma.

Statistics. The measured values were expressed as means ± SE. Unpaired Student's t-test was used, and statistical significance was verified at P < 0.05. Correlations between the developed tension and DT and RT were evaluated by testing the correlation of these parameters.

	RESULTS

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Effects of thyroxine on various parameters related to the treatment. To confirm that our thyroxine treatment was effective, we compared the following parameters in both preparations. The wet heart weight-to-body weight ratios in Eu and Hy ferrets were 0.0046 ± 0.002 (n = 7) and 0.0067 ± 0.002 (n = 23) (significant change, P < 0.001). Serum-free T₃ and free thyroxine concentrations in Hy were 18.1 ± 6.9 pg/ml and 7.4 ± 0.6 ng/ml (n = 5), respectively, which were significantly higher than those in Eu (free T₃, 3.7 ± 2.6 pg/ml, n = 3; free thyroxine, 0.92 ± 0.07 ng/ml, n = 3) (P < 0.05 for T₃ and P < 0.001 for thyroxine).

Changes in the Ca²⁺ transients and tension in Hy cardiac muscles. Table 1 shows the summarized data of the intracellular Ca²⁺ transients and tension measured in the solution with 2 mM [Ca²⁺]_o and at L_max in Eu and Hy myocardium. The peak [Ca²⁺]_i and peak twitch tension (tension) did not significantly differ between the two groups. However, the absolute values of the developed tension in both groups were much higher compared with those reported by MacKinnon et al. (25). Highly significant differences of the time courses of the Ca²⁺ transients (11, 25) and tension (9, 11, 18, 25, 26) were observed between Eu and Hy cardiac muscles; however, no such significant difference was seen with the TPL.

Tension-dependent change in DT and RT in Eu and Hy myocardium. To investigate the possible contribution of the enhanced Ca²⁺ removal by SR to the tension-dependent change in DT, we measured the Ca²⁺ transients and tension when developed tension in Eu and Hy myocardium was varied. Developed tension was altered by changing the [Ca²⁺]_o in solution and/or the muscle lengths. Figure 1 is an example of measured records (9 experiments) at different [Ca²⁺]_o and at different muscle lengths in Hy myocardium. At shorter muscle lengths and lower [Ca²⁺]_o, the peaks of the Ca²⁺ transients and tension were significantly lower than those measured at longer lengths and at higher [Ca²⁺]_o. When the developed tension was larger, DT was slightly but significantly shortened (Fig. 1C; pooled data are shown in Fig. 2A), and the RT was significantly prolonged (Fig. 1D; pooled data are shown in Fig. 2B).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effects of changing developed tension on Ca2+ transients and tension in hyperthyroid (Hy) preparations. Ca2+ transients (faster signals) and tension (slower signals) were measured at different extracellular Ca2+ concentrations ([Ca2+]o) and initial muscle lengths. A: 2 mM [Ca2+]o (2Ca) and 84% maximum muscle length (Lmax). B: 8 mM [Ca2+]o (8Ca) and Lmax. Peak intracellular Ca2+ concentration and developed tension significantly increased in B compared with those in A. To observe differences in time courses of Ca2+ transients and tension, peaks of Ca2+ transients and tension were normalized to match their peaks and superimposed (C and D, respectively). In C, a and b correspond to Ca2+ transient record in A and B, respectively. In D, a and b correspond to tension record in A and B, respectively. Time to peak light and decay time were slightly but significantly shortened in B. Time to peak tension and relaxation time were significantly prolonged in B.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Tension-dependent changes in decay time of Ca2+ transients (DT) (A) and relaxation time of tension (RT) (B) in euthyroid (Eu; ) and Hy () myocardium. DT and RT were plotted against relative peak tension [tension (relative)] (each measured value was normalized to that measured at 2 mM [Ca2+]o and at Lmax in each preparation). Regression lines were drawn and fitted by equation labeled in graphs. Peak tension was altered as described in MATERIALS AND METHODS. Both DT and RT were significantly dependent on relative peak tension in Eu and Hy. Equations indicate linear correlation between x-axis (relative developed tension) and y-axis (DT and RT). Sample correlation coefficients (R) and levels of significance are also shown under equations. Slopes of 2 regression lines of Eu and Hy in A and B were significantly different (P < 0.001 in A and P < 0.001 in B). Data are pooled from 13 experiments for Eu and 18 experiments for Hy.

Tension-dependent changes in DT and RT in Hy myocardium in the presence of 3 mM caffeine. To test the Ca²⁺ removal hypothesis mentioned above, we tried to measure the tension dependence of DT and RT by inhibiting the enhanced SR Ca²⁺ uptake in Hy myocardium.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effects of changing developed tension on Ca2+ transients and tension in Hy in presence of caffeine (Caff) (3 mM) (B and C). Ca2+ transients and tension were measured at Lmax (B) or at 84% Lmax (C) in 2 mM [Ca2+]o solution. Ca2+ transients and tension measured in absence of caffeine [Caff ()] are shown in A for comparison. Peaks of Ca2+ transients and tension in A-C were normalized to match their peaks and superimposed (D and E, respectively). In D: a, b, and c correspond to Ca2+ transient record in A, B, and C, respectively. In E, a, b, and c correspond to tension record in A, B, and C, respectively. In presence of caffeine, peak of Ca2+ transient became flat and peak was obscure. Measurement of decay time is as described in MATERIALS AND METHODS.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Dependence of DT and RT on relative peak developed tension [tension (relative)] in absence () and presence () of caffeine (Caff; 3 mM) in Hy myocardium. DT and RT were plotted against relative peak tension (each value was normalized to that at 2 mM [Ca2+]o and at Lmax in absence of caffeine). Equations, correlation coefficients, and levels of significance are shown in graphs. In A and B, slopes of regression lines measured in presence or absence of caffeine were significantly different (P < 0.005 in A and P < 0.02 in B). Data are pooled from 6 experiments.

Contribution of the Na⁺/Ca²⁺ exchanger to the lesser tension dependence of DT in Hy myocardium. Although the alteration in the expression of the mRNA of the Na⁺/Ca²⁺ exchanger in Hy ventricular muscles is controversial (8, 33), we tested the contribution of Ca²⁺ removal by the Na⁺/Ca²⁺ exchanger in the lesser tension dependence of DT in Hy myocardium. For this purpose, we measured the tension dependence of DT and RT in the Li-HEPES-Tyrode solution (2 experiments) (see MATERIALS AND METHODS).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Dependence of DT and RT on relative peak developed tension in HEPES-Tyrode solution (Na-HEPES) () and in Li-HEPES-Tyrode solution (Li-HEPES) () in Hy myocardium. DT and RT were plotted against relative peak tension (each measured value was normalized to that at 2 mM [Ca2+]o and at Lmax in HEPES-Tyrode solution). Equations, correlation coefficients, and levels of significance are shown in graphs. No significant difference was observed in slopes of lines in A and B measured in presence or absence of Na+. Data are pooled from 2 experiments.

Extra Ca²⁺ in Eu and Hy myocardium. When the muscle length is quickly shortened, [Ca²⁺]_i transiently increases (extra Ca²⁺, the arrow in Fig. 6A). The extra Ca²⁺ is considered to reflect Ca²⁺ dissociated from the Ca²⁺-bound form of TnC that is due to the tension-dependent change in the affinity of TnC for Ca²⁺ (3, 5, 19, 23, 24). We measured the extra Ca²⁺ to compare the amount of Ca²⁺ binding to TnC and the responsiveness of the contractile elements to the change in active tension in both groups.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   A: experimental data of effects of a quick muscle length change on Ca2+ transient and tension in Hy preparation. First trace, muscle length; second trace, intracellular Ca2+ concentration ([Ca2+]i). Arrow indicates extra Ca2+. Third trace, tension; fourth trace, stimulus; fifth trace, difference of [Ca2+]i between 2 signals (extra Ca2+). B: relation between amount of extra Ca2+ and tension reduction in Eu () and Hy () myocardium. Magnitude of extra Ca2+ normalized to [Ca2+]i immediately before length change (extra Ca2+/[Ca2+]i) (ordinate) was plotted against magnitude of tension reduction. These experiments were carried out in solution with 2 mM [Ca2+]o. To minimize disparity of developed tension among all preparations, magnitude of tension reduction was normalized with developed tension at Lmax [tension reduction (relative)] (abscissa). Equations, correlation coefficients, and levels of significance are shown in graphs. Two regression lines in Eu and Hy are not statistically different, although normalized extra Ca2+ showed a dependence on tension reduction in each preparation. Data were pooled from 6 experiments for Eu and 7 experiments for Hy.

pCa-tension relation in skinned preparation in Eu and Hy myocardium. To investigate the Ca²⁺ responsiveness of the contractile elements in Eu and Hy myocardium in the steady state, we measured the pCa-tension relation using thin skinned trabeculae. In addition, we also tested the length-dependent change in the Ca²⁺ sensitivity of the contractile elements in Eu and Hy myocardium.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   pCa-tension relation in skinned preparations in Eu (solid line) and Hy (dashed line) myocardium at long (2.3 µm) and short (1.9 µm) sarcomere lengths. Curves are normalized and fitted with Hill equation. Tension at each pCa is expressed as relative values to maximum tension obtained at pCa 5.05. pCa50 values at longer () and shorter () sarcomere lengths in Hy were similar to those at longer () and shorter () sarcomere lengths in Eu. Hill coefficients in Hy were slightly but significantly lower than those in Eu at each length (P < 0.02 at longer length, P < 0.05 at shorter length).

	DISCUSSION

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Relevance of thyroxine treatment. The thyroxine treatment in the present study is considered to be effective, because the measured parameters, such as body weight, heart weight-to-body weight ratio, serum free T₃ and free thyroxine levels, and myosin isoform were all changed as expected from previous reports (4, 8, 9, 13, 18, 25, 26, 33). In accordance with the changes of these parameters, the time courses of tension and Ca²⁺ transient were shortened, which was essentially similar to the report by MacKinnon et al. (25) except for the TPL. We noticed a slightly shorter TPL signal in Hy, but the change was not statistically significant. Because in Hy hearts the Ca²⁺ current (26) and the expression of the ryanodine receptor mRNA (4) are increased, these factors might be responsible for the faster TPL. However, the reason for the different results is not clear at present. The other quantitative difference was found in the peak developed tension in both reports; peak twitch tension of Eu and Hy in the present study, under conditions comparable to their study, was much larger than those of their preparations. This might be due to the different aequorin loading methods in these studies; microinjection was used in the present study and chemical loading in their study. The hyperpermeable treatment for chemical loading might deteriorate the preparation, although this is just speculation.

Tension-dependent change in DT in Hy myocardium. DT showed a significant dependence on the peak tension in twitch contraction in Eu and Hy as previously reported (20). When developed tension was increased or decreased by alteration of the [Ca²⁺]_o and/or the muscle length, DT was changed in accordance with the developed tension. A significant dependence of RT on developed tension was also observed in Eu and Hy (Figs. 2 and 3), but it was an inverse relation compared with that between DT and tension. Therefore, DT is not only determined by the Ca²⁺ removal mechanism, but it is also influenced by other factors that lower the myoplasmic Ca²⁺ concentration. Although DT is known to be influenced by the magnitude of the Ca²⁺ transients (6), this was not a major factor in the present study, because the tension-dependent change in DT is reported to be observed at different muscle lengths that do not significantly alter the peak of the Ca²⁺ transients (3, 5, 17, 20, 23).

Tension-dependent change in DT in the caffeine-treated Hy myocardium. Caffeine (3 mM) prolonged the time course of the Ca²⁺ transients and tension in Hy as in Eu; this was due to the regenerative Ca²⁺ release from the SR, and thus Ca²⁺ uptake was apparently inhibited (Fig. 3) (2). If the Ca²⁺ uptake by SR was inhibited by caffeine, then the dependency of DT and RT on peak twitch tension became more significant (Fig. 4). Similar results were obtained when the Hy preparation was treated with ryanodine (unpublished data) as mentioned before. These results further support the Ca²⁺ removal hypothesis that faster Ca²⁺ uptake by SR substantially influences the tension dependence of DT.

Properties of the contractile elements of Hy myocardium in intact and skinned preparations. When muscle length is quickly shortened, the active cross bridges are detached, the affinity of TnC for Ca²⁺ decreases according to the tension-dependent feedback mechanism, and the myoplasmic Ca²⁺ concentration transiently increases (extra Ca²⁺) (3, 5, 19, 23, 24). The previous report indicates that the extra Ca²⁺ is a function of the amount of Ca²⁺ bound to TnC and the magnitude of tension reduction that works as a trigger for the decrease in the affinity of TnC for Ca²⁺ (23). A similar tension-dependent change in the affinity of TnC for Ca²⁺ has been reported using different preparations and a different Ca²⁺ indicator (5, 12, 17). Therefore, the tension-dependent change in the affinity of TnC for Ca²⁺ is a common mechanism in mammalian cardiac muscles. The normalized extra Ca²⁺ in Hy showed a similar dependence on tension reduction to that in Eu. The result indicates that sufficient Ca²⁺ is on the binding sites of TnC in Hy as it is in Eu, and the intrinsic mechanism that is responsible for the tension-dependent change in the Ca²⁺ affinity of TnC works similarly in Eu and Hy. Thus the lesser dependence of DT on developed tension in Hy is not due to the properties of the contractile elements; rather, it is mainly due to the faster Ca²⁺ uptake by SR.