Numerous disturbances in the function and/or expression of proteins involved in the Ca2+ handling have been described in myocytes of failing hearts. The most important seem a decreased expression of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and increased expression of the Na+/Ca2+ exchanger (NCX). These changes may be complicated by diastolic loss of Ca2+ from the SR due to the leaky SR Ca2+ channels (Ryanodine Receptors – RyRs) (1, 2). The mechanism of SR Ca2+ leak is still controversial. RyRs hyperphosphorylation by protein kinase A (PKA) leading to dissociation of FK-506 binding proteins (FKBPs) from the RyRs or by calmodulin-dependent kinase II (CaMKII) has been proposed (3 - 5). Nitrosilation of the tiol side chain of cysteine of RyRs by NO or oxidation of tiols by the reactive oxygen species is also considered (6, 7).

FK-506, an immunosuppressant drug, binds to FKBPs and dissociates them from the RyRs. It has been used to investigate the effect of SR Ca2+ leak on Ca2+ handling and contraction in myocytes isolated from rat, mouse and rabbit hearts (8 - 11). Results of these works show that the diastolic SR Ca2+ leak induced by FK-506 has a potent species-dependent effect on contractile activity in isolated cardiomyocytes. However, in these works Ca2+ leak was induced as an isolated disorder not accompanied by changes in other Ca2+ handling proteins. Therefore, it is important to find out how disorders of other Ca2+ handling proteins found in the failing hearts would modulate the effect of leaky RyRs on myocytes contractile activity. In this work we tested whether modulation of SERCA and NCX activity, which are the most often affected Ca2+ handling proteins during progression of heart failure, influences contractile activity of myocytes in which SR Ca2+ leak has been induced by FK-506.


MATERIALS AND METHODS
Myocytes isolation

Ventricular myocytes of guinea pig hearts were isolated by enzymatic digestion as described in detail elsewhere (12). The myocytes were placed in bathing or in rapid superfusion chamber according to the type of experiment (see Results section). The rapid superfusion method modified from Rich et al. (13) enabled complete exchange of superfusion solutions at controlled temperature within ~ 500 msec. The volume of the temperature controlled bathing chamber was 0.5 ml. The drugs were gently injected under the surface of the bathing solution. The chambers were mounted on the stage of Nikon Diaphot inverted microscope equipped for epifluorescence. All experiments were performed at 37°C.

Recording of cells shortening and Ca2+ transients

Cell shortening was recorded with video edge detector designed and built by J. Parker (Cardiovascular Laboratories, School of Medicine, UCLA).

For recording of Ca2+ transients myocytes were incubated for 15 min with 10 µM Indo 1-acetoxymethyl ester. The ratio of 405 to 495 nm Indo-1 fluorescence for diastolic and systolic intracellular Ca2+ concentration was obtained from the output of Dual Channel Ratio Fluorometer (Biomedical Instrumentation Group, University of Pennsylvania). The difference between the systolic and diastolic Indo-1 ratios was used as a measure of the amplitude of Ca2+ transients.

The fluorescence or cells shortening signals were fed to the computer and stored for further analysis by the ISO2 Multitask-Patch-Clamp Software designed by M. Friedrich and K. Benndorf, University of Dresden.

Estimation of Ca2+ transport by SERCA and NCX

The rate constant (r) of decay of the Ca2+ transient was used as an index of the rate of Ca2+ removal from the sarcoplasm by the Ca2+ transporters. It was estimated by fitting monoexponential curve (described by equation y= Ae-xr, in which r is the rate constant of decay) to the declining phase of the Ca2+ transient.

The rate constant of decay of electrically stimulated Ca2+ transient (r) reflects the rate of Ca2+ removal from sarcoplasm by SERCA, NCX and slow Ca2+ transporters (srcolemmal Ca2+-ATP-ase and mitochondrial uptake). The rate constant of decay of Ca2+ transient in cells superfused with thapsigargin (Tg) (rTg), which blocks SERCA, reflects the rate of Ca2+ removal by NCX and slow transporters. Since transport by NCX accounts for ~ 90% of outward Ca2+ transport (14) rTg may be considered as an index of NCX transporting function (rNCX = rTg). The rate constant of Ca2+ removal by SERCA (rSERCA) was calculated by subtracting rate constant of decay of Ca2+ transient under Tg perfusion (rTg) from rate constant of decay of Ca2+ transient in normal cell (r) (rSERCA = r - rTg) (Fig. 6A). Contribution of SERCA and NCX to relaxation was calculated from the formulas: rSERCA = (r - rTg)/r, rNCX = rTg/r, respectively.

The same calculations were performed in myocytes treated with FK-506 and with FK-506 followed by Tg. The rate constant of Ca2+ transient decay in myocytes treated with FK-506 and Tg was taken as an index of the rate of Ca2+ transport by NCX in myocytes with leaky RyRs (rNCX(FK) = rFK+Tg). Rate of Ca2+ transport by SERCA in these myocytes was calculated by subtracting rFK+Tg from the rate constant of Ca2+ decaying in cells exposed only to FK-506 (rSERCA(FK) = rFK – rFK+Tg) (Fig. 6A). Contribution of SERCA and NCX to relaxation in myocytes exposed to FK-506 was calculated according to formulas: rSERCA(FK) = (rFK- rFK+Tg) / rFK; rNCX(FK) = rFK+Tg / rFK , respectively.

Estimation of NCX inhibition by low [Na+]o

In these experiments the rate of outward Ca2+ transport by NCX was estimated from the rate constants of decay of Ca2+ transients stimulated by caffeine according to Choi and Eisner (15). Myocytes rapidly superfused with Tyrode solution containing 144 mM Na+ or 100 mM Na+ were stimulated at 1Hz. Thereafter stimulation was stopped and 10 mM caffeine dissolved in Tyrode solution was rapidly superfused (Fig. 4). Caffeine releases Ca2+ from the SR and prevents its reaccumulation. Therefore the rate constant of decline of the caffeine-evoked Ca2+ transient reflects the rate of sarcolemmal Ca2+ transport realized in ~90% by NCX.

Solutions

For cells isolation and throughout the experiments we used Tyrode solution of the following composition (inmM): 144 NaCl, 5 KCl, 1 MgCl2, 0.43 NaH2PO4, 10 N-2-hydroxyethylpiperazine-N’-2-etanesufonic acid (HEPES), 11 glucose. pH of Tyrode solution was adjusted with NaOH to 7.3 for myocytes isolation and to 7.4 for experiments. In experiments CaCl2 was added to concentration of 2 mM. In some experiments 44 mM of NaCl in Tyrode solution was replaced by 44 mM of LiCl for partial inhibition of NCX. FK-506, thapsigargin and rapamycin were purchased from Sigma.

Statistical evaluation of the results

The quantitative results are presented as means ±SE. To compare more than two experimental groups two-way analysis of variance (ANOVA) for repeated measures was used. Student’s t-test for unpaired or paired (when appropriate) samples was used to determine the significance of differences between the means. To justify the use of Student’s t-test normal distribution of data was proved by Shapiro-Wilk test. P<0.05 was accepted as a level of significance.

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).


RESULTS
The effect of FK-506 on amplitude of Ca2+ transients and cell shortening

Myocytes were placed in the bathing chamber and stimulated at 1Hz and their Ca2+ transients or shortening recorded. FK-506 was added to the bathing solution to the final concentration of 20 µM. The amplitude of Ca2+ transients and cell shortening decreased to 48.6 ± 6.9% and 55.9 ± 4.6% of control, respectively (Fig. 1A and 1B). These effects stabilized within ~10 min.

Fig. 1. The effect of FK-506 on Ca2+ transients and cell shortening. (A): Left panel: representative traces of the steady state Ca2+ transient in guinea pig ventricular myocyte stimulated at 1 Hz before (control) and 15 min after addition of 20 µM FK-506 to the bathing solution; right panel: average values of Ca2+ transient amplitude in myocytes exposed to FK-506 expressed in % of control (mean±SE, n=8, *P<0.05 vs. control). (B): Left panel: representative traces of shortening of myocyte stimulated at 1Hz before (control) and 15 min after addition of 20 µM FK-506 to the bathing solution; right panel: average values of cell shortening in myocytes exposed to 20 µM FK-506 or 10 µM rapamycin (Rap) expressed in % of control (mean±SE, n=7-22, *P<0.05 vs. control). Difference between the effects of FK-506 and rapamycin on amplitude of cell shortening is not significant (P>0.05).

FK-506 binds to FKBPs and dissociates them from RyRs. This increases Ca2+ sensitivity of RyRs and induces diastolic SR Ca2+ leak (8, 16). However, the complexes of FK-506 and FKBPs also inhibit calcineurin activity (17). In order to check which of these effects is responsible for the negative inotropic effect of FK-506, 10 µM rapamycin was added to the bathing solution and cell shortening recorded. Rapamycin dissociates FKBPs from RyRs, but complexes of rapamycin and FKBPs do not affect calcineurin activity (18). As shown in Fig. 1B rapamycin had similar effect on cells shortening as FK-506. Thus, the negative inotropic effect of FK-506 does not result from its effect on calcineurin activity as proved already in mice and rabbit myocytes by Su et al. (9).

The mechanism of negative effect of FK-506 on cell shortening

The myocytes were placed in bathing chamber with normal Tyrode solution (144 mM Na+), stimulated at 1Hz and Ca2+ transients recorded. Thereafter stimulation was stopped and diastolic fluorescence recorded (Fig. 2A). Over 30 s of rest the diastolic fluorescence declined in normal myocytes by 0.026±0.011 units (Fig. 2A and B). In myocytes exposed to FK-506 fluorescence declined by 0.102±0.025 units, which suggests increased Ca2+ loss from the cells presumably due to diastolic SR Ca2+ leak and Ca2+ extrusion by NCX (Fig. 2A and B). In order to check whether this was the case, Na+ concentration in the Tyrode solution bathing the myocytes exposed to FK-506 has been decreased from 144 mM to 100 mM. In these myocytes diastolic decline of fluorescence was completely inhibited (Fig. 2A and B). Moreover, in other series of experiments we found that 20 µM FK-506 added to low [Na+] Tyrode solution instead of decreasing, increased the amplitude of cells shortening to 117.7 ± 3.6% of pre-FK-506 control (Fig. 3).

Fig. 2. Inhibition of FK-506 induced decline of the diastolic Ca2+ concentration by lowering of [Na+] in the bathing solution from 144 to 100 mM. (A): representative traces of systolic and diastolic Indo-1 fluorescence in control myocytes and in myocytes exposed for 15 min to 20 µM FK-506. [Na+] in Tyrode solution bathing the myocytes was 144 mM or 100 mM. (B): decline of the diastolic Ca2+ concentration expressed as the difference between the diastolic Indo-1 fluorescence levels measured at the beginning and after 30 s of rest (F) (mean±SE, n=9-25). * P<0.05 vs. control, # P<0.05 vs. the effect of FK-506 at 144 mM [Na+]o. f.u. – fluorescence units.

Fig. 3. The effect of FK-506 on cell shortening depends on extracellular Na+ concentration. Left panel: representative traces of shortening of guinea pig ventricular myocytes stimulated at 1 Hz and bathed in Tyrode solution containing 144 mM or 100 mM before (control) and 15 min after addition of 20 µM FK-506 to the bathing solution. Right panel: average values of shortening of myocytes exposed to 20 µM FK-506 expressed in % of control (mean±SE, n=12 for 100 mM [Na+]o and n=23 for 144 mM [Na+]o). * P<0.05 vs. control.

In order to check the degree of inhibition of NCX by low [Na+]o we measured the rate of decline of Ca2+ transients evoked by caffeine superfusion (Fig. 4). The rate constant of Ca2+ transient decay in myocytes superfused with normal Tyrode solution ([Na+]=144 mM) was 5.2 ± 0.3 s-1 whereas in the myocytes superfused with the low Na+ solution ([Na+]=100 mM) the rate constant was 2.9 ± 0.4 s-1. Thus lowering of [Na+]o decreased the rate of Ca2+ transport by about 45%.

Fig. 4. Inhibition of Na+/Ca2+ exchanger by decrease of extracelullar Na+ concentration. The monoexponentials were fitted to decaying phase of Ca2+ transients (normalized) evoked by rapid superfusion of 10 mM caffeine in myocytes superfused with Tyrode solution containing 144 mM or 100 mM Na+. Since caffeine precludes the Ca2+ uptake by SERCA, Ca2+ transient decline is due to sarcolemmal Ca2+ transport depending in ~90% on Na+/Ca2+ exchanger (see also Material and Methods) (n=7 in both groups). r144 mM [Na+]o, r100 mM [Na+]o - the rate constants of decay of Ca2+ transients evoked in myocytes superfused with Tyrode solution containing 144 mM or 100 mM Na+, respectively.

Results of experiments with low [Na+]o show that negative effect of FK-506 on amplitude of contractions and Ca2+ transients results from enhanced diastolic Ca2+ loss. Preventing of this loss by decrease of the rate of Ca2+ transport by NCX reverses the effect of FK-506 on myocyte contractions.

The effect of thapsigargin on cell shortening in normal myocytes and in myocytes pretreated with FK-506

Myocytes were placed in the bathing chamber and stimulated at 1 Hz to record cell shortening. After attending steady state 20 µM FK-506 was added to the Tyrode solution. This reduced the amplitude of cells shortening by 45.1% of control. When the negative effect of FK-506 stabilized, 10-7M Tg was added to the bath. The amplitude of cells shortening significantly increased, being now by only 25.9% less then in controls (Fig. 5).

Fig. 5. The effect of 10-7M Tg on shortening of normal myocytes and of myocytes exposed to FK-506. (A): Top panel: representative traces of shortening of guinea pig myocyte stimulated at 1Hz before (control), 10 min after addition of 20 µM FK-506 to the bathing solution and 5 min after subsequent addition of 10-7M Tg to the bathing solution still containing 20 µM FK-506; bottom panel: shortening of guinea pig myocyte before (control) and 5 min after addition of 10-7M Tg to the bathing solution. (B): average shortening of myocytes stimulated at 1 Hz and exposed to 20 µM FK-506, 10-7M Tg or to 20 µM FK-506 followed by 10-7 M Tg expressed as % of control (mean±SE, n=7). * P<0.05 vs. control; #P<0.05 vs. FK-506.

In normal myocytes 10-7M Tg tended to decrease amplitude of cell shortening but this effect was not significant (Fig. 5).

The effect of thapsigargin on amplitude of Ca2+ transients in normal myocytes and in myocytes pretreated with FK-506

Myocytes loaded with Indo-1 and preincubated for at least 15 min with 20 µM FK-506 were placed in the rapid superfusion system, immediately superfused with Tyrode solution containing 20 µM FK-506 and field stimulated at 1Hz. The amplitude of their Ca2+ transients was 54.5% of that of control group (Fig. 6A and B). 10-7M Tg added to the FK-506 containing superfusion solution increased the amplitude of Ca2+ transients to 77.3% of that of control group within 3-5 min (Fig. 6A and B).

Fig. 6. The effect of 10-7M Tg on amplitude and decay of Ca2+ transients in control myocytes and in myocytes exposed to FK-506. (A): representative records of Ca2+ transient in control myocyte, in myocyte superfused with 10-7M thapigargin (Tg), myocyte superfused with 20 µM FK-506 and in myocyte superfused with 20 µM FK-506 followed by 10-7M Tg. The cells were stimulated at 1 Hz. The monoexponential curves were fitted to decaying part of Ca2+ transients in order to calculate the rate constant (r) of decay. (B), (C): average values of Ca2+ transients amplitude and rate of Ca2+ transients decay in control, myocytes, myocytes superfused with 10-7M Tg, with 20µM FK-506 or with FK-506 followed by Tg expressed as % of control (mean±SE, n=6-15, *P<0.05 vs. control; #p<0.05 vs. FK-506). (D): The effect of FK-506 on the relative contribution of SERCA and Na+/Ca2+ exchanger (NCX) to relaxation calculated from rate constants of Ca2+ transients decay (panel (A)) according formulas shown in Methods (mean±SE, n=6-15, *P<0.05 vs. control).

In normal myocytes stimulated at 1Hz addition of 10-7M Tg to supefusion solution decreased the amplitude of Ca2+ transients by 5.2% (not significant) (Fig. 6A and B). The small effects of Tg on amplitude of Ca2+ transients is consistent with the results obtained previously by Lewartowski et al. (12) and Janiak et al. (19) in guinea pig myocytes and by Davia et al. (20) in human myocytes.

The effect of FK-506 on SERCA and NCX contribution to relaxation

In order to evaluate the effect of FK-506 on the rate of Ca2+ removal from sarcoplasm by SERCA and by NCX we estimated the rate constants of the decaying part of Ca2+ transients in normal myocytes (r), myocytes treated with Tg (rTg) or FK-506 (rFK) and in myocytes treated with FK-506 followed by Tg (rFK+Tg). (Fig. 6A)

FK-506 decreased rate constant of Ca2+ transient decay from 7.06 ± 0.54 s-1 to 4.26 ± 0.23 s-1 (slowing of relaxation by 39.9%). Blocking of SERCA in myocytes superfused with FK-506 by Tg decreased further the rate constant to 3.35 ± 0.24 s-1. Tg alone decreased the rate constant to 4.17 ± 0.29 s-1 (Fig. 6C).

Since Tg inhibits SERCA the rTg and rFK+Tg was taken as an index of the rate of Ca2+ transport by NCX in normal and in FK-506 treated myocytes, respectively (rNCX=rTg, rNCX(FK)=rFK+Tg). The rate of Ca2+ transport by SERCA was evaluated by subtraction of rTg from r and rFK+Tg from rFK respectively (rSERCA=r-rTg, rSERCA(FK)=rFK-rFK+Tg). Contribution to relaxation of the transporters was calculated by division of rSERCA and rNCX by r and rSERCA(FK) and rNCX(FK) by rFK (see also Materials and Methods).

The relative contribution of SERCA to relaxation was 40.9 ± 7.6% in normal myocytes and 21.4 ± 5.4% in myocytes superfused with FK-506. Thus the relative contribution of the remaining transporters was 59.1 ± 4.2% and 78.7 ± 5.7%, respectively. Since NCX provides for ~90% of outward Ca2+ transport this means that FK-506 increased significantly the relative contribution of NCX to relaxation at the expense of SERCA (Fig. 6D).


DISCUSSION
The major findings of this work are that inhibition of SERCA by Tg increased the amplitude of contractions and Ca2+ transients previously diminished by FK-506 and that the effect of FK-506 could be reversed by partial inhibition of Ca2+ transport by NCX.

FKBPs, accessory proteins of RyRs play an important role in stabilization of the closed state of the channels. FK-506 binds to FKBPs and induces their dissociation from the RyRs resulting in increase of sensitivity of RyRs to Ca2+ and enhancement of diastolic Ca2+ leak from the SR (8). Thus, FK-506 imitates the SR Ca2+ leak present in cardiomyocytes of failing heart due to RyRs hyperphosphorylation.

FK-506 decreased amplitude of Ca2+ transients and contractions in isolated myocytes of guinea pig heart. This result is consistent with that of Su et al. (9), who reported that FK-506 decreased amplitude of Ca2+ transients in myocytes of rabbit heart, the properties of which are similar to those of guinea pig myocytes. In contrast, FK-506 was reported to increase amplitude of Ca2+ transients and/or contractions in mouse and rat cardiomyocytes (8 - 10, 12 and our unpublished results).

We found that FK-506 largely promoted decline of diastolic Ca2+ concentration and this effect was blocked by partial inhibition of NCX by low [Na+]o. Moreover, FK-506 increased instead of decreasing amplitude of shortening of myocytes superfused with low [Na+]o solution. These results strongly suggest that decrease in the amplitude of Ca2+ transients and cell shortening by FK-506 was due to diastolic Ca2+ leak from the RyRs and subsequent Ca2+ extrusion by NCX. Thus, change in NCX activity and/or expression could modulate the effect of FK-506 on myocyte contraction. Therefore, it is conceivable that increased expression of NCX observed in the failing hearts may enhance the effect of SR Ca2+ leak on myocyte contraction.

Su et al. (9) proposed that differences in the pattern of NCX function might be responsible for the diverse effects of FK-506 on myocytes contraction in various species. Indeed, Lewartowski and Zdanowski (21) found that there is a net loss of Ca2+ from the resting myocytes of guinea pig heart, which results in rest decay, whereas there is a net Ca2+ gain in resting rat cells resulting in long lasting post- rest potentiation. As suggested by Shattock and Bers (22) this difference results from higher sarcoplasmic Na+ concentration in rat myocytes. This may decrease relative contribution of NCX to relaxation, and reverse NCX in resting myocytes of this species to “Ca2+ in” mode. Thus, in the rat myocytes Ca2+ leaking from the RyRs is retained within a cell, may be retaken by the SR and used for activation of the next contraction as suggested by Su et al. (9) for the mice myocytes. Partial inhibition of NCX in our experiments rendered the myocytes of guinea pig heart similar with this respect to the rat or mice myocytes.

The diastolic leak of Ca2+ from the RyRs induced by FK-506 significantly increased relative contribution of NCX to relaxation at the expense of the SERCA. This does not necessarily mean that SERCA pumps less Ca2+ into the SR. Rather, Ca2+ pumped into the SR is released again to sarcoplasm through the leaky RyRs, which slows relaxation, and is partly transported out of a cell by NCX. Thus, less Ca2+ is available for activation of contraction upon the next myocyte stimulation. This results in decrease of amplitude of Ca2+ transients and contractions.

The amount of Ca2+ lost by myocytes due to the SR Ca2+ leak depends on the rate of Ca2+ flux along the route: SERCA, leaky RyRs, NCX (19). Blocking of one of these proteins connected functionally in series can diminish diastolic Ca2+ loss. In our experiments we affected two of these proteins: NCX and SERCA. As already mentioned, decrease of activity of NCX reversed the effect of FK-506 on amplitude of cell shortening. Apparently, Ca2+ released from the RyRs at diastole was then taken up by the SR and released upon cell stimulation to activate contraction.

Elimination of SERCA by Tg resulted in myocytes pretreated with FK-506 in increase of amplitude of Ca2+ transients and contractions, whereas in normal cells the amplitudes slightly (not significantly) decreased. Tg by blocking SERCA depletes the SR Ca2+ rendering activation of contraction dependent solely on sarcolemmal Ca2+ influx. The reason why in normal cells the amplitude of Ca2+ transients and contractions is little affected by Tg is not clear. One mechanism might be an increase in Ca2+ influx due to the delay in inactivation of the sarcolemmal L-type Ca2+ channels resulting from lower subsarcolemmal Ca2+ concentration (23). Moreover, the Ca2+ current may be more effective in direct activation of contraction due to elimination of competition for sarcolemmal Ca2+ between SERCA and contractile proteins.

In myocytes pretreated with FK-506 Tg stopped the Ca2+ flux through the SR thereby stopping diastolic SR Ca2+ leak despite still leaky RyRs. Thus Tg rendered Ca2+ turnover in the cells pretreated with FK-506 similar to that in normal myocytes treated with Tg.

Positive effect of Tg on contractions in myocytes pretreated with FK-506 is consistent with the results of Chiesi et al. (24), Lewartowski et al. (12) and Janiak et al. (19) who found that Tg inhibits the negative effect of ryanodine on amplitude of contractions and Ca2+ transients of guinea pig heart. Ryanodine in concentrations <10 µM locks RyRs in subconductance state, which results in the diastolic SR Ca2+ leak (25).

In conclusion, the effect of FK-506 on myocytes contraction is strictly dependent on SERCA and NCX activity. Our results show that in myocytes with SR Ca2+ leak induced by FK-506 complete elimination of SR function by SERCA inhibition is advantageous for myocyte contraction. Whether decreased function of SERCA coexisting in some models of heart failure with the SR Ca2+ leak may protect contractile activity of myocytes remains the matter of further investigation.

Acknowledgements: This work has been supported by the CMKP Grant 501-1-1-27-13/03. An expert and devoted technical contribution of Ms Alicja Protasowicka is gratefully acknowledged.


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