Introduction

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The frequency-dependent changes in force of contraction of isolated myocardium is present in most mammalian species, but also seems to be species dependent (Buckley et al., 1972). Whereas in specimens like guinea-pig, rabbit and human myocardium an increase of the frequency of stimulation is followed by an increase in developed tension, also called positive FFR or "Bowditch-Treppe," a negative FFR is present in the rat. It is widely accepted that the FFR and the kinetics of the myocardial contraction cycle strongly depend on the intracellular Ca⁺⁺ homeostasis (Gwathmey et al., 1990; Pieske et al., 1995). The Ca⁺⁺-ATPase of the sarcoplasmic reticulum and the Na⁺-Ca⁺⁺ exchanger of the sarcolemma seem to be the most important mechanisms to extrude Ca⁺⁺ from the cytosol during diastole and lower high systolic intracellular Ca⁺⁺ concentrations initiating an effective relaxation (Bassani et al., 1994). The contribution of the SR-Ca⁺⁺-ATPase and the Na⁺-Ca⁺⁺ exchanger in the regulation of the intracellular Ca⁺⁺ during contraction and relaxation is species-dependent. In rat myocardium, the SR-Ca⁺⁺-ATPase predominates in the function of lowering intracellular Ca⁺⁺, whereas the contribution of the Na⁺-Ca⁺⁺ exchanger seems to be negligible. In contrast, in rabbit myocardium, Ca⁺⁺ extrusion via the Na⁺-Ca⁺⁺ exchanger has a more important contribution on Ca⁺⁺ extrusion out of the cytosol besides the function of the SR-Ca⁺⁺-ATPase compared with rat myocardium (Bassani et al., 1994). The relative contribution of the SR-Ca⁺⁺-ATPase to the provision of the Ca⁺⁺ involved in contractile activation and relaxation in human myocardium is still a matter of debate. Only indirect data are presently available (Hasenfuss et al., 1994).

To evaluate the role of the SR-Ca⁺⁺-ATPase for the regulation of contraction-twitch intracellular Ca⁺⁺-transients and the force-frequency relationship in human myocardium, we used the highly specific inhibitor of the SR-Ca⁺⁺-ATPase CPA. CPA, a mycotoxin produced by various fungi of Aspergillus and Penicillium species, has been reported to be a selective inhibitor of the SR-Ca⁺⁺-ATPase expressed in fast-twitch muscle (Seidler et al., 1989), smooth muscle (Uyama et al., 1992) and also cardiac muscle (Takahashi et al., 1995; Yard et al., 1994). It has been reported to be without effect on F-type ATPase, such as mitochondrial H⁺,K⁺ -ATPase and on several other P-type ATPases, such as Na⁺,K⁺-ATPase of the kidney and brain and the Ca⁺⁺-ATPase of the plasma membrane (Seidler et al., 1989). Even at high concentrations, CPA has no effect on Ca⁺⁺ sensitivity of the contractile apparatus (Takahashi et al., 1995), Ca⁺⁺ currents (Bonnet et al., 1994; Badaoui et al., 1995) and the Na⁺-Ca⁺⁺ exchanger (Yard et al., 1994) besides its inhibitory action on the SR-Ca⁺⁺-ATPase in cardiac muscle. Thus, because of its high specificity CPA is a useful pharmacological tool to investigate the predominant contribution of the SR-Ca⁺⁺-ATPase to the intracellular Ca⁺⁺ homeostasis and the kinetics of contraction at different frequencies of stimulation in human myocardium.

	Materials and Methods

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Human myocardium. Nonfailing human myocardium was obtained from seven donors who were brain dead as a result of traumatic injury. These nonfailing hearts could not be used for transplantation for technical reasons. Patient history of the organ donors (age: 45 ± 6 years) revealed no evidence of heart disease.

Cardiac muscle strip preparation and measurement of force of contraction. Immediately after excision, the papillary muscles were placed in ice-cold preaerated Tyrode's solution (for composition see below) and delivered to the laboratory within 10 min. From each native myocardial tissue sample papillary muscle strips were prepared (less than 0.8 mm width and 8-10 mm length) with muscle fibers running in parallel to the length of the strips. Connective tissue was carefully trimmed away. The muscles were suspended in an organ bath (25 ml) at 37°C containing a modified Tyrode's solution of the following composition (in mM): NaCl,119.8; KCl, 5.4; MgCl₂, 1.05; CaCl₂, 1.8; NaHCO₃, 22.6; NaH₂PO₄, 0.42; glucose, 5.05; ascorbic acid, 0.28; Na₂EDTA, 0.05. The bathing solution was continuously aerated with 95% O₂ and 5% CO₂. The muscles were stimulated by two platinum electrodes by field stimulation from a Grass S 88 stimulator (frequency 1 Hz; duration 5 ms; intensity 10-20% above threshold). Preparations were allowed to equilibrate for at least 90 min, with the bathing solution being changed once after 45 min. Isometric force of contraction was measured with an inductive force transducer (W. Fleck, Mainz, Germany or Föhr Medical Instruments GmbH, Egelsbach, Germany) attached to a Hellige Helco scriptor (Hellige, Freiburg, Germany) or Gould recorder (Gould Inc., Cleveland, OH). Concentration-dependent mechanical effects were obtained, i.e., developed tension, +T, T, TPT, T1/2T and T95T. After complete mechanical stabilization, the force-frequency relationship was studied starting with a rate of 0.5 Hz up to 3 Hz. Control strips showed no changes in base-line isometric tension during the time necessary to complete pharmacological testing. Experiments were performed as described previously in detail (Schwinger et al., 1993).

Isolation of vesicles from the SR. The SR was prepared according to the method of Sitsapesan and Williams (1990). The preparation was carried out at 4°C. Myocardial tissue was chilled in ice-cold homogenization buffer with the following composition (in mM): sucrose, 300; phenylmethylsulfonyl fluoride, 1; piperazine-N,N-bis[2-ethanesulfonic acid] (PIPES), 20, pH 7.4. Connective tissue was trimmed away, and myocardial tissue was homogenized with a motor-driven homogenizer (Braun, Berlin, Germany). The homogenate was spun at 8,000 rpm (Beckman JA20, Beckman, Munich, Germany) for 20 min. The supernatant was filtered through four layers of gauze, and the pellet was discarded. The supernatant was centrifuged at 35,000 rpm (Sorvall A 641, Sorvall, Bad Homburg, Germany). The pellet containing SR-membrane vesicles was resuspended in a buffer containing (in mM): sucrose, 400; (N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic] acid (HEPES), 5; 2-amino-2-(hydroxy-methyl)-1,3-propandiol (TRIS), 5, pH 7.2, and were frozen fractionated in liquid nitrogen and stored at 80°C until use. Protein concentration was measured according to Lowry et al. (1951).

Measurement of Ca⁺⁺-ATPase activity. The reaction was carried out as described previously (Schwinger et al., 1995) based on the following coupled enzyme reactions:

1.	ATP  ADP + Pi (This reaction was catalyzed by the SR-Ca++-ATPase.)
2.	ADP + phosphoenolpyruvate  ATP + pyruvate (The reaction was catalyzed by the pyruvate kinase.)
3.	Pyruvate + NADH  lactate + NAD+ (The reaction was catalyzed by the lacate dehydrogenase.)

Measurement of the Ca⁺⁺-transient in electrically stimulated multicellular muscle preparations. Electrically driven muscle strips from human nonfailing myocardium (left ventricular papillary muscle strips, n = 3) were used to study the influence of CPA (30 µM) on Ca⁺⁺-transients simultaneously with force generation by use of the fura-2 ratio method (Grynkiewicz et al., 1985). Intracellular Ca⁺⁺ was measured by the fluorescence indicator fura-2 (Grynkiewicz et al., 1985). To facilitate cell loading fura-2 was used as AM ester. These AM esters passively cross the plasma membrane, and once inside the cell, they are cleaved to cell-impermeant products by intracellular esterases. For the initial control measurement of force of contraction, one end of the muscle strips was clamped at a muscle holder and the other end was attached to a force transducer (Scientific Instruments, Heidelberg, Germany). The muscle fibers were superfused with an oxygenated (95% O₂, 5% CO₂) Tyrodes solution (in mM): NaCl, 119.8; KCl, 5.4; MgCl₂, 1.05; CaCl₂, 0.9; NaHCO₃, 22.6; NaHPO₄, 0.42; glucose, 5.05; ascorbic acid, 0.28; Na₂EDTA, 0.05; 37°C, pH 7.40). The muscles were stimulated by a pulse generator (Föhr Medical Instruments GmbH, Egelsbach, Germany) with a square wave pulse of 10 ms duration 10% above threshold voltage at a frequency of 1 Hz. Muscle strips with an adequate mechanical performance were incubated for 4 hr in darkness to avoid photobleaching of the dye in an oxygenated (95% O₂, 5% CO₂) Ringer's solution (in mM: NaCl, 147; KCl, 4; CaCl₂, 2.2) at 22°C, pH 7.4, containing 5 µmol/l of the fura-2-AM. Experiments were performed as described previously (Vahl et al., 1994).

Materials. ATP, pyruvate kinase/lactate dehydrogenase mixture and phosphoenolpyruvate were obtained from Boehringer (Mannheim, Germany). Isoprenaline, phenylmethylsulfonyl fluoride, Ca⁺⁺-Ionophore 23187 and CPA were purchased from Sigma (Deisenhofen, Germany). Fura-2-AM was obtained from Molecular Probes (Eugene, OR). A stock solution of fura-2-AM (10 mM) was dissolved in dimethyl sulfoxide and stored at 20°C as described by Vahl et al. (1994). All other chemicals were of analytical grade or the best grade commercially available. Only deionized and double-distilled water was used throughout.

Statistics. The data shown are means ± S.E.M. For comparison within one group, the paired t test was applied. Otherwise, statistical significance was analyzed by use of the Student's t test for unpaired observations or by ANOVA; P < .05 was considered significant.

	Results

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Effect of CPA on Ca⁺⁺-ATPase activity. To demonstrate that CPA is able to inhibit SR-Ca⁺⁺-ATPase specifically in human nonfailing myocardium, the SR-Ca⁺⁺-ATPase activity was measured with use of isolated vesicle preparations from nonfailing human myocardium (n = 7). CPA (0.01-10 µM) concentration-dependently depressed Ca⁺⁺-ATPase activity as shown in figure 1. The activity of the SR-Ca⁺⁺-ATPase in nonfailing human myocardium in the absence of CPA was 242 ± 20 nmol/mg protein × min (+ A 23187; free Ca⁺⁺, 35 µM). The EC₅₀ value for the inhibitory effect of CPA on SR-Ca⁺⁺-ATPase was 0.14 µM (95% confidence limits, 0.12-0.16 µM). Therefore, CPA inhibited SR-Ca⁺⁺-ATPase activity in isolated vesicles from human nonfailing myocardium concentration-dependently.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effect of CPA (0.01-10 µmol/l) on SR Ca++-ATPase in vesicles of SR from nonfailing human myocardium (n = 7). CPA concentration-dependently inhibits SR-Ca++-ATPase activity. Ca++-ATPase activity in the absence of CPA was 242 ± 20 nmol/mg protein per min. Changes of Ca++-ATPase activity are shown as percent of the initial value.

Effects of CPA on the force-frequency relationship. Figure 2 shows the influence of the SR-Ca⁺⁺-ATPase inhibitor CPA (30 µM) on the force-frequency relationship (0.5-3 Hz) in electrically driven left ventricular papillary muscle strips from nonfailing human hearts (n = 7). In the absence of CPA (control) an increase in the frequency of stimulation was followed by a significant increase in the developed tension from 0.5 Hz up to 2 Hz (4.1 ± 0.7 mN vs. 5.4 ± 0.6 mN; 6.3 ± 0.7 mN/mm² vs. 8.3 ± 0.3 mN/mm² after normalization of developed force to cross-sectional area of the muscle strips, P < .05, fig. 2A). In the presence of CPA the developed tension at 0.5 Hz was decreased compared with control (3.2 ± 0.7 mN; 5.0 ± 1.0 mN/mm²). In contrast to control, the increase in the frequency of stimulation in the presence of CPA (30 µM) was not accompanied by a significant increase in force of contraction (0.5 Hz: 3.2 ± 0.7 mN vs. 2 Hz: 3.5 ± 0.6 mN; 5.0 ± 1.0 mN/mm² vs. 5.4 ± 0.6 mN/mm²). Especially at higher frequencies of stimulation (above 2 Hz), the increase in force of contraction was significantly depressed after inhibition of the SR-Ca⁺⁺-ATPase by CPA (30 µM) compared with control, as marked in figure 2. Therefore, in the presence of CPA an increase in frequency of stimulation was not accompanied by an increase in force of contraction.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Force-frequency relationship (0.5-3 Hz) of human nonfailing myocardium (electrically driven papillary muscle strips) in the absence and presence of 30 µmol/l CPA (n = 7). (A) Ordinate, change in force of contraction in mN/mm2; abscissa, frequency in Hz. In the presence of CPA (30 µmol/l) the former positive force-frequency relationship worsened especially at frequencies above 2 Hz. Significant differences of increase in force of contraction between control condition and CPA treatment were indicated (*control vs. CPA, P < .05). (B) Ordinate, diastolic tension in mN/mm2; abscissa, frequency in Hz.

Inotropic and lusitropic effects of CPA. Figure 3A compares time to peak tension at 0.5 Hz and 2 Hz under control conditions and in the presence of CPA (30 µmol/l). Under both conditions, control and CPA, time to peak tension was diminished significantly at 2 Hz compared with 0.5 Hz (control: 167 ± 4 ms vs. 208 ± 13 ms, CPA: 190 ± 7 ms vs. 226 ± 15 ms; P < .05). However, CPA increased TPT tension significantly compared with control conditions at the low (0.5 Hz) and the high rate (2 Hz) of stimulation (P < .05). Similarly, T95T was diminished while increasing frequency from 0.5 to 2 Hz in the control (430 ± 54 ms vs. 244 ± 7 ms, P < .05) and the CPA group (484 ± 58 ms vs. 273 ± 7 ms, P < .05) (fig. 3B). At 0.5 Hz CPA had no additional influence on T95T as compared with control (484 ± 58 vs. 430 ± 54 ms), whereas at 2 Hz CPA significantly prolonged T95T compared with control (273 ± 7 vs. 244 ± 7 ms). In contrast, T1/2T was not significantly prolonged in the presence of CPA, neither at 0.5 Hz (control: 143 ± 5 ms vs. CPA: 137 ± 12 ms) nor at 2 Hz (control: 112 ± 6 ms vs. CPA: 127 ± 11 ms).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Influence of CPA (30 µmol/l) on time to peak tension (A) and time to 95% tension decay (B) in human nonfailing myocardium (electrically driven left ventricular muscle strips, n = 7) at 0.5 Hz and 2 Hz. Significant differences between control condition and CPA treatment and between 0.5 Hz and 2 Hz in each group were indicated (#P < .05, 0.5 Hz vs. 2 Hz at control condition and after CPA treatment; * P < .05, control condition vs. CPA treatment).

Effects of CPA on the intracellular Ca⁺⁺-transient. Figure 4 gives an original tracing of simultaneous measurements of the Ca⁺⁺-transient with the fura-2 ratio method and the twitch of contraction with isolated papillary muscle strips from nonfailing human myocardium to demonstrate the effects of CPA on Ca⁺⁺-transients. In the presence of CPA the developed tension and the systolic light emission (R_340/380) was decreased by 45.68 ± 3.73% and 28.80 ± 6.90% compared with control. TPT and IPI were prolonged in the presence of CPA by 26% and 45% compared with control, but CPA has only small effects on parameters of relaxation such as T1/2T and I1/2I, which were increased in the presence of CPA. Additionally, CPA increased the diastolic tension as well as the diastolic light emission as shown in figure 4.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Representative original registration of the contractile twitch and the intracellular Ca++-transient of human left ventricular myocardium measured by fura-2. CPA (30 µmol/l) diminished developed tension and the systolic light emission. CPA increased the diastolic tension and diastolic Ca++ levels as well. FOC, force of contraction.

	Discussion

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The role of the SR-Ca⁺⁺-ATPase in initiating a positive force-frequency relation in human myocardium can be concluded only from the indirect data available (Hasenfuss et al., 1994). Thus, direct evidence on the contribution of the SERCA on parameters of contraction and relaxation, [Ca⁺⁺]_i transients and the FFR in human myocardium are still lacking. In addition, the function of the SERCA does not necessarily correlate with reduced protein or mRNA levels (Schwinger et al., 1995). Furthermore, the reduced activity of the SERCA described in failing human hearts is not followed by the expected prolongation of the parameters of relaxation or an increase in diastolic tension in measurements of isolated myocardial tissue, even at higher frequencies of stimulation (Schwinger et al., 1993; Hasenfuss et al., 1994), as well. Because species differences may exist, the role of the SERCA in contraction and relaxation must be studied in human myocardium. The contribution of the SERCA or the Na⁺/Ca⁺⁺ exchanger to initiate the decline of the [Ca⁺⁺]_i transient is 92% and 7% in rat myocardium, but 70% to 28% in rabbit myocardium, respectively (Bassani et al., 1994). The high specificity of CPA on inhibition of the SERCA in skeletal (Seidler et al., 1989; Goeger and Riley, 1989) and smooth muscle (Uyama et al., 1992) has been confirmed in cardiac muscle as well (Takahashi et al., 1995, Bonnet et al., 1994, Badaoui et al., 1995, Agata et al., 1993; Yard et al., 1994). Thus, CPA was used in this study to characterize the importance of the SERCA activity on regulation of contraction and relaxation (twitch kinetics, [Ca⁺⁺]_i transients, force-frequency relationship) in human myocardium.

As shown in figure 1 CPA concentration-dependently inhibits the activity of the SERCA in myocardial vesicle preparations. Inhibition is complete at a concentration of 10 µM. As described by Baudet et al. (1993), a complete inhibition of the SERCA by CPA in multicellular preparations is not seen, even at high concentrations. This requirement of higher concentrations of CPA to achieve effects on contractility (compared with effects on isolated SR vesicles) may be caused by compartmentalization (Langer, 1992) or diffusion (higher diffusion distance, intracellular target) of CPA in multicellular preparations. To achieve the inhibitory action of CPA on SERCA, a concentration of 30 µM was used. As described by several authors, even at concentrations up to 30 µM, CPA has no effect on the Ca⁺⁺ sensitivity of the myofilaments (Takahashi et al., 1995; Bonnet et al., 1994), on Ca⁺⁺ currents (Bonnet et al., 1994; Badaoui et al., 1995), on the peak inward and steady state membrane currents (Takahashi et al., 1995) and on Na⁺/Ca⁺⁺ exchange in cardiac muscle (Yard et al., 1994).

As shown in this study, inhibition of the SERCA by CPA is predominantly followed by alterations in the parameters of muscle contraction. The time to peak tension was significantly prolonged in the presence of CPA at low and high frequencies of stimulation; the developed tension and peak rate of tension rise was significantly reduced by CPA at high frequencies of stimulation. Additionally, the maximal amplitude of the [Ca⁺⁺]_i transient was reduced and time to the peak [Ca⁺⁺]_i transient was prolonged in the presence of CPA. In contrast, the effects of CPA on muscle relaxation were less pronounced than its effects on muscle contraction. CPA did not have an effect on the peak rate of tension decay or the time to half-relaxation. These findings were independent from the stimulation frequency used. In contrast, the time to 95% relaxation and the increase in diastolic tension was augmented at higher frequencies in the presence of CPA compared with control. Consistent with these effects on muscle contraction and relaxation, the decay of intracellular Ca⁺⁺ was not affected. However, some increase of diastolic Ca⁺⁺ levels occurred.

It seems possible that the degree of SERCA inhibition in the multicellular preparations is lower than that observed in the isolated SR preparations of the same hearts. A small inhibitory action on the SERCA activity might lead to a reduced force of contraction because of a stronger competition of the Na⁺/Ca⁺⁺ exchanger with the SERCA. This may lead to a reduced force generation but could be less effective in slowing relaxation. Thus, the impaired SERCA activity in the present study is followed predominantly by alterations of inotropic and, to a lesser extent, lusitropic function. This may be supported by the finding that thapsigargin (another specific inhibitor of SERCA activity) caused remarkably slow and incomplete SR Ca⁺⁺ depletion in multicellular preparations of rabbit myocardium (Baudet et al., 1993) in contrast to observations in single myocytes. With consistent use of rapidly cooling contractures to assess the SR load in multicellular rabbit papillary muscle strip preparations, CPA was only effective in decreasing SR Ca⁺⁺ content by 59% (Baudet et al., 1993) and reducing twitch force by 40%. These authors concluded that complete blockade of SR Ca⁺⁺ uptake by CPA or thapsigargin in multicellular muscle preparations cannot be assumed (in rabbit myocardium). Both agents seemed to shift the balance of Ca⁺⁺ fluxes in favor of Ca⁺⁺ extrusion by the sarcolemmal Na⁺/Ca⁺⁺ exchanger (Baudet et al., 1993). It seems that a modest decline in SR Ca⁺⁺ content can have a disproportionately large effect on the twitch amplitude and SR Ca⁺⁺ release (Bassani et al., 1995) (e.g., measured as caffeine-induced Ca⁺⁺ release). Thus, slowing of relaxation by CPA may be modest (because of incomplete blockade of the SERCA in multicellular preparations) in the present experiments even though force development is decreased significantly. However, the inhibitory action of CPA on SERCA activity largely affects the frequency-dependent force generation in humans.

The negative inotropic effect of CPA may be explained by an increased Ca⁺⁺ extrusion by the sarcolemmal Na⁺/Ca⁺⁺ exchanger and a reduced SR Ca⁺⁺ load. At higher frequencies of stimulation the negative inotropic effects by inhibition of the SERCA (e.g., by CPA) may be augmented as the time of diastole is shortened. The effects of CPA on parameters of relaxation (lusitropy) are present consistently at high frequencies of stimulation. CPA prolonged time to 95% relaxation and increased diastolic tension. Thus, the Na⁺/Ca⁺⁺ exchanger cannot completely compensate the increased diastolic Ca⁺⁺ levels after the CPA inhibitory action on SERCA activity at high frequencies of stimulation. On the other hand, the Na⁺/Ca⁺⁺ exchanger works most effectively when intracellular Ca⁺⁺ is high. Thus, as intracellular Ca⁺⁺ declines during relaxation this system is less able to extrude Ca⁺⁺, especially with frequency-dependent rising intracellular Na⁺. In addition, the inhibited SR Ca⁺⁺ pump also cannot lower intracellular Ca⁺⁺ very rapidly when blocked by CPA. Consequently, CPA may still create a diastolic as well as systolic dysfunction without significantly slowing relaxation.

As discussed by Langer (1992), the Na⁺/Ca⁺⁺ exchanger may reduce intracellular Ca⁺⁺ load. A fast relaxation can be arranged by action of the Na⁺-Ca⁺⁺ exchanger and the SERCA in rabbit myocardium (Bers and Bridge, 1989). In human myocardium an increased activity and protein expression of the Na⁺/Ca⁺⁺ exchanger has been reported in heart failure (Studer et al., 1994; Reinecke et al., 1996; Flesch et al., 1996) and has been discussed to be compensatory for the altered SERCA activity. This might be supported by the finding that parameters of relaxation (Schwinger et al., 1993) and diastolic tension (Hasenfuss et al., 1994) are not altered in failing compared with nonfailing myocardium. However, this holds true only for low frequencies of stimulation. Intracellular Na⁺ has been reported to increase with increasing frequency of stimulation. In various species Na⁺/Ca⁺⁺ exchange has been shown to play a role in the frequency-dependent increase in force of contraction by inhibition of Ca⁺⁺ extrusion secondary to an increase in Na⁺ (Cohen et al., 1982; Frampton et al., 1991; Shouten and Ter Keurs, 1991). In addition, Bassani et al. (1995) conclude from their findings that a rise in SR Ca⁺⁺ at higher intracellular Na⁺ concentration could sensitize the Ca⁺⁺ release mechanism, enhancing the efficacy of Ca⁺⁺ entry via the Na⁺/Ca⁺⁺ exchanger as a potential activator of SR Ca⁺⁺ release. This mechanism may potentiate changes on Ca⁺⁺ handling after increased intracellular Na⁺. In human heart failure, i.e., in a condition with reduced SERCA activity, as well as after blockade of the SERCA by CPA, the force-frequency relationship is negative. Thus, the altered SERCA activity affects the SR Ca⁺⁺ load and the interaction of SERCA activity and Na⁺/Ca⁺⁺ exchange. Because the direction of Ca⁺⁺ movement through Na⁺/Ca⁺⁺ exchanger depends on the membrane potential, an elevation of average intracellular Ca⁺⁺ could increase the operation of the Na⁺-Ca⁺⁺ exchanger in the net Ca⁺⁺ efflux mode because of a less positive Ca⁺⁺ equilibrium potential (Langer, 1992). However, this assumption needs to be proven.

In summary, the present study provides evidence that an altered SR-Ca⁺⁺-ATPase function in human myocardium may lead to an impaired inotropic and lusitropic function and to a negative force-frequency relationship in humans. Thus the SR-Ca⁺⁺-ATPase activity is important in initiating the activation and relaxation of human muscle contraction. Further studies are needed to focus on the complex interaction of SR-Ca⁺⁺-ATPase and Na⁺/Ca⁺⁺ exchanger on the regulation of contraction, relaxation and the force-frequency relationship, especially in humans.