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Cardiovascular Research 2000 48(2):323-331; doi:10.1016/S0008-6363(00)00191-7
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Altered interaction of FKBP12.6 with ryanodine receptor as a cause of abnormal Ca2+ release in heart failure

Kaoru Ono, Masafumi Yano, Tomoko Ohkusa, Masateru Kohno, Takayuki Hisaoka, Taketo Tanigawa, Shigeki Kobayashi, Michihuro Kohno and Masunori Matsuzaki*

Second Department of Internal Medicine, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi, 755-8505, Japan

* Corresponding author. Tel.: +81-836-22-2248; fax: +81-836-22-2246 masunori{at}po.cc.yamaguchi-u.ac.jp

Received 28 April 2000; accepted 10 July 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Little information is available as to the Ca2+ release function of the sarcoplasmic reticulum (SR) in heart failure. We assessed whether the alteration in this function in heart failure is related to a change in the role of FK binding protein (FKBP), which is tightly coupled with the cardiac ryanodine receptor (RyR) and recently identified as a modulatory protein acting to stabilize the gating function of RyR. Methods: SR vesicles were isolated from dog LV muscles [normal (N), n = 6; heart failure induced by 3-weeks pacing (HF), n = 6]. The time course of the SR Ca2+ release was continuously monitored using a stopped-flow apparatus, and [3H]ryanodine-binding and [3H]dihydro-FK506-binding assays were also performed. Results: FK506, which specifically binds to FKBP12.6 and dissociates it from RyR, decreased the polylysine-induced enhancement of [3H]ryanodine-binding by 38% in N (P<0.05) but it had no effect in HF. In HF, the rate constant for the polylysine-induced Ca2+ release from the SR was 61% smaller than in N. FK506 decreased the rate constant for the polylysine-induced Ca2+ release by 67% in N (P<0.05) but had no effect in HF. The [3H]dihydro-FK506-binding assay revealed that the number (Bmax) of FKBPs was decreased by 83% in HF (P<0.05), while the Kd value was unchanged. FK506 did not significantly change SR Ca2+.-ATPase activity in either N or HF. Conclusions: In HF, the number of FKBPs showed a tremendous decrease; this may underlie the RyR-channel instability and the impairment of the Ca2+ release function of RyR seen in the failing heart.

KEYWORDS SR, sarcoplasmic reticulum; RyR, ryanodine receptor; E–C coupling, excitation–contraction coupling; FKBP, FK binding protein; PMSF, phenylmethanesulfonyl fluoride; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-[N-morpholino]propanesulfonic acid; EGTA, ethylene glycol bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; BSA, bovine serum albumin

This article is referred to in the Editorial by C.M.N. Terracciano (pages 191–193) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
An abnormal regulation of intracellular Ca2+ by the sarcoplasmic reticulum has been shown to be involved in the mechanism underlying contractile and relaxation dysfunction in heart failure. Indeed, several investigators have demonstrated decreases in Ca2+-ATPase activity or the Ca2+ uptake function of the sarcoplasmic reticulum (SR) in association with a decreased density of Ca2+-ATPase in cardiac hypertrophy and/or failure [1–8].

Previously [9], we demonstrated that in a canine model of pacing-induced heart failure (i) the SR Ca2+ release induced by polylysine (a RyR-specific Ca2+-release trigger) showed a significant decrease in rate and (ii) for both the initial rate of Ca2+ release and the [3H]ryanodine binding, the polylysine concentration-dependence showed a shift towards a lower concentration of polylysine. Moreover, of the total amount of Ca2+ uptake, the polylysine-releasable fraction of sequestered Ca2+ was significantly larger in failing SR vesicles than in normal SR vesicles [9]. These findings suggested that the gating function of the SR Ca2+ release channel is altered in heart failure.

In skeletal muscle, it has been shown that an associate protein, FKBP12, is both copurified with RyR during sucrose-density-gradient centrifugation and colocalized with RyR to the terminal cisternae of the SR, and also that FKBP12 antibody can immunoprecipitate RyR [10]. The physiological function of FKBP12 is modulation of RyR-1, the skeletal muscle isoform of the Ca2+-release channel, a role it may play by enhancing cooperation among the four subunits of RyR-1 [10–12]. Recently, a novel FKBP with a different electrophoretic mobility (FKBP12.6) was found to be specifically associated with RyR-2, the cardiac muscle isoform [13,14]. The stoichiometry of binding is approximately four FKBPs per RyR complex, i.e. one FKBP to one RyR monomer [13]. However, in contrast to the general concensus as to effects of FKBP12 on RyR-1, there is controversy as to the modulatory influence exerted by FKBP12.6 over RyR-2. Kaftan et al. [15] found that rapamycin, a drug that inhibits the prolyl isomerase activity of FKBP12.6 and dissociates this protein from RyR-2, increases the open probability and reduces the current amplitude of cardiac Ca2+-release channels. On the other hand, Timerman et al. [14] showed that removal of FKBP12.6 from the canine RyR-2 (by means of FK590, an FK506 analogue) did not alter the sensitivity of the channel to Ca2+-induced activation.

Very recently we proposed a novel mechanism of cardiac dysfunction on the basis of our finding that in a canine model of heart failure a prominent abnormal Ca2+ leak occurs through RyR, presumably following a partial loss of RyR-bound FKBP12.6 and the resultant conformational change in RyR [16]. This abnormal Ca2+ leak might possibly cause Ca2+ overload and consequent diastolic dysfunction, as well as systolic dysfunction.

In the present study, we set out to assess whether FKBP12.6 is involved in the abnormality of the Ca2+-release function seen in heart failure. To this end, we investigated whether an alteration of interaction between FKBP12.6 and RyR-2 might be involved in the pathogenesis of the abnormal channel-gating function of RyR-2 we earlier observed in the failing heart.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
The investigation conforms with 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).

2.1 Experimental model and instrumentation
Heart failure was induced in beagle dogs of either sex by 21 days of rapid right-ventricular pacing at a rate of 250 bpm using an externally programmable miniature pacemaker (Medtronic, Minneapolis, USA) as described previously [9]. Each dog was sedated with morphine sulfate (15 mg, s.c.) and cromazine maleate (10 mg, s.c.), then, anesthetized with isoflurane (2%, 1.5 l/min) and a mixture of nitrous oxide and oxygen (50:50), intubated with a cuffed endotracheal tube and ventilated at a tidal volume of 22 ml/kg and a respiratory rate of 15 breaths per minute. A bipolar pacing lead was fixed to the endocardial RV surface and its distal ends were tunneled to a subcutaneous pocket constructed on the animal's back, where they were connected to a pacemaker (Medtronic). modified to pace at 250 bpm. The thoracotomy was then closed in layers. Cefazolin (1 g, i.v.) was administered before and after surgery.

After allowing 1 week for recovery, the pacemaker was programmed to pace at 250 bpm. Dogs were monitored daily for clinical signs of heart failure. Approximately 1 h after the termination of the rapid RV pacing, LV pressure was measured using a 7-F micromanometer (Millar, Texas, USA) inserted percutaneously via the carotid artery, and two-dimensional short-axis echocardiograms were obtained at the level of the head of the papillary muscle.

The care of the animals and the protocols used were in accord with guidelines laid down by the Animal Ethics Committee of Yamaguchi University School of Medicine.

2.2 Preparation of SR vesicles
SR vesicles were prepared as described previously [9,17], according to the method of Kranias et al. [18]. Left ventricles were homogenized in a solution containing 30 mM Tris–malate, 0.3 M sucrose, 5 mg/l leupeptin and 0.1 mM PMSF, at pH 7.0 (Solution I). The homogenate was centrifuged at 5500 g for 10 min and the resultant supernatant was filtered through four layers of cheesecloth before centrifugation at 12 000 g for 20 min. The supernatant was again filtered through cheesecloth and centrifuged at 143 000 g for 30 min. The pellet was resuspended in a solution containing 0.6 M KCl, 30 mM Tris–malate, 0.3 M sucrose, 5 mg/l leupeptin and 0.1 mM PMSF, at pH 7.0 (Solution II). This suspension was centrifuged at 143 000 g for 45 min. The pellet was resuspended again in Solution II, homogenized and centrifuged at 143 000 g as described above. The pellet was suspended in Solution I and centrifuged at 143 000 g. The resultant pellet, which represents the microsomal fraction rich in SR vesicles, was suspended in a solution containing 0.1 M KCl, 20 mM Tris–malate, 0.3 M sucrose, 5 mg/l leupeptin and 0.1 mM PMSF, at pH 7.0, to give a final concentration of 10–20 mg protein/ml. This fraction was rapidly frozen in liquid nitrogen and stored at –80°C. An aliquot was retained for determination of protein concentration by the method of Lowry et al. [19].

2.3 Enhancement of [3H]ryanodine binding by polylysine
To assess the effect of FK506 on the polylysine-induced enhancement of [3H]ryanodine binding, binding assays were carried out according to the method of Lu and Meissner [20]. Cardiac microsomes (0.1 mg/ml) were incubated in 1.0 ml of a reaction solution containing 5 nM [3H]ryanodine (68.3 Ci/ml, Dupont NEN), 0.1 M NaCl and 20 mM Na–Hepes, pH 7.2. The [Ca2+] was kept at 3 µM using an EGTA–calcium buffer (0.974 mM CaC12, 1 mM EGTA, 20 mM Hepes, pH 7.2). The above incubation was carried out for 120 min at 36°C in the absence or presence of 0.37 µM polylysine plus various concentrations of FK506 (1–30 µM). The incubated reaction mixture was filtered through Millipore filters (type HA, pore size 0.45 µm) and washed twice with 5 ml of the same reaction solution devoid of microsomes and [3H]ryanodine. The specific binding was calculated as the difference between the binding in the absence (total binding) and in the presence (non-specific binding) of 10 µM unlabelled ryanodine. Each datum point was obtained by averaging values from duplicate experiments. The molecular structures of ryanodine and FK506 are shown in Fig. 1.


Figure 1
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  		Fig. 1 Molecular structures of ryanodine and FK506.

 
2.4 Ca2+-release assay
The Ca2+-release assay was performed as described previously [9,17]. SR vesicles (0.5 mg/ml) were incubated in a solution containing 0.15 M KC1, 10 mM NaN3, 2.5 mM MgATP and 20 mM MES, pH 6.8 (adjusted using KOR) (Solution A) for 5 min to load the SR with Ca2+. Then, 1 volume of Solution A was mixed with 1 volume of Solution B (containing 0.15 M KC1 and 20 mM MES; adjusted to pH 6.8 using KOH). Polylysine (0.74 µM; Mr 27 000) was added to Solution B when needed. The final concentration of polylysine after mixing was 0.37 µM, at which a half-maximal stimulation of Ca2+ release was obtained [9]. The Ca2+ concentration in each of the two solutions was buffered at 3 µM using an EGTA–calcium buffer (0.212 mM CaC12, 0.25 mM EGTA, pH 6.8). In the presence or absence of 30 µM FK506, the time course of the polylysine induced Ca2+ release was monitored using a stopped-flow apparatus (Unisoku RSP-6015, Osaka), with 5 µM arsenazoIII as a Ca2+ indicator [9,17,21–23]. All the reactions mentioned above were carried out at 20°C. Twenty to twenty-five traces (each representing 1000 data points) of the arsenazom signal were averaged for each experiment. The arsenazoIII signal was converted to nanomoles of Ca2+ released per mg of protein by determining the {Delta} arsenazoIII signal/{Delta}[Ca2+] coefficient from a Ca2+ calibration curve [17,21,22]. Curves were fitted by a single exponential function, y = A(1–e–kt), where y is the amount of Ca2+ released at time t, A is the final amount of Ca2+ released at an infinite time, and k is the rate constant of release.

2.5 [3H]dihydro-FK506 assay
[3H]Dihydro-FK506 binding was performed in CHAPS-solubilized SR by the LH-20-column method with slight modifications [13,24,25]. SR vesicles were first solubilized in FK506-binding buffer (20 mM NaPO4, pH 7.2, 0.5% CHAPS, 2 mM DTT, 5 mg/ml BSA and 0.02% NaN3) at a protein concentration of 0.1 mg/ml. Then, samples of the solubilized vesicles were incubated for 30 min at 37°C in the binding mixture containing 1.25–20 nM [3H]dihydro-FK506 (55 000 cpm/pmol; DuPont NEN). Following the incubation, 50 µl of the samples containing 5 µg of SR vesicles were applied to a 3-ml Sephadex LH-20 column equilibrated in LH-20 column buffer (10 mM NaPO4, 1 mM DTT, 0.25% CHAPS and 0.01% NaN3) to separate free from bound ligand. Non-specific binding was determined by the addition of 30 µM unlabelled FK506. The values for Bmax and Kd were determined using Scatchard plots.

2.6 Ca2+-ATPase activity assay
Ca2+-ATPase activity in SR vesicles was evaluated by measuring the amount of Pi released during the reaction initiated by adding ATP [9,26–28]. The assay mixture in a total assay volume of 500 µl contained 0.15 M KCl, 20 mM MES, pH 6.8 (adjusted using KOH), 0.3 mM MgCl2, 10 mM NaN3, 6 µM ionophore A-23187, 0.212 mM CaC12, 0.25 mM EGTA (free [Ca2+]=3 µM) and SR vesicles (0.1 mg). To start the reaction, 1.0 mM ATP was added to the above priming solution in the presence or absence of various concentrations of FK506. The amount of Pi released was calculated by converting nm (absorbance of 0.1% malachite green) to nmol by means of a standard linear line.

2.7 Statistics
An unpaired t-test was used to compare binding data and kinetic parameters between normal and heart failure groups. A one-way analysis of variance was employed to compare the effect of FK506 on the polylysine-induced enhancement of [3H]ryanodine binding these groups. When a significant trend was identified by the F test, Scheffe's post-hoc test was used to compare the data. A P value less than 0.05 was accepted as statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Characteristics of animal model
There were no differences between normal and heart-failure groups in terms of body weight (kg; normal, 11±1; failure 1.2±2; P = ns), LV weight (g; normal, 61±5; failure, 62±5; P = ns) or protein yield from SR vesicles (mg protein/g; normal, 1.3±0.2; failure 1.2±0.3; P = ns).

Hemodynamic data are summarized in Table 1. In the heart-failure group, LV end-diastolic pressure was significantly elevated and both the peak +dP/dt of LV pressure and the fractional shortening were decreased while the time constant of the LV pressure decay during the isovolumic relaxation period ({tau}) was increased These data indicate that both systolic and diastolic functions were impaired in the heart-failure group.


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  		Table 1 Hemodynamic dataa

 
3.2 Effects of various concentrations of FK506 on the polylysine-induced enhancement of [3H]ryanodine binding to RyR
As shown in Fig. 2, [3H]ryanodine binding in the absence of polylysine and FK506 was smaller in failing SR vesicles than in normal SR vesicles (pmol/mg; normal, 0.28±0.02; failure, 0.11±0.01; P<0.05). Polylysine (0.37 µM) significantly increased [3H]ryanodine binding in both normal and failing SR vesicles. Because ryanodine binds to RyR when it is in an open state, the increase in ryanodine binding seen upon the addition of a Ca2+-release trigger (such as polylysine) indicates activation of the receptor [20,29]. FK506 decreased the [3H]ryanodine binding induced by polylysine in a dose dependent fashion in normal SR vesicles but no significant change was detected in failing SR vesicles.


Figure 2
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  		Fig. 2 Effects of various concentrations of FK506 on the polylysine-induced enhancement of [3H]ryanodine binding in SR vesicles isolated from normal and failing canine hearts. Specific [3H]ryanodine binding was measured in the absence or presence of 0.37 µM polylysine plus the indicated concentration of FK506. Data represent means of six experiments, each performed on a different pair of normal and failing SR vesicle preparations. *, P<0.01 vs. polylysine (0 µM) plus FK506 (0 µM); #, P<0.05 vs. polylysine (0.37 µM) plus FK506 (0 µM).

 
3.3 Effects of FK506 on polylysine-induced Ca2+ release
In the absence of polylysine, the Ca2+ release from normal or failing SR vesicles was negligible (because there was virtually no change in [Ca2+] after mixing Solution A with Solution B; data not shown). Hence the induced Ca2+ release was solely dependent on polylysine. Fig. 3a shows the time course of the Ca2+ release induced by 0.37 µM polylysine in the absence or presence of 1 µM thapsigargin. In confirmation of our previous report [9], the rate of Ca2+ release was found to be slower in failing SR vesicles than in normal SR vesicles. In both types of SR vesicles, the time course of the Ca2+ release was unaffected by the addition of thapsigargin indicating that there may be negligible Ca2+ reuptake during this Ca2+ release. Fig. 3b shows the time course of the Ca2+ release induced by 0.37 µM polylysine in the absence or presence of 30 µM FK506. The various parameters characterizing the kinetics of Ca2+ release in both normal and failing SR vesicles (A, amount; k, rate constant; Ak, initial rate of Ca2+ release) are summarized in Table 2. In normal SR vesicles, both the rate constant and the initial rate of Ca2+ release were decreased after the addition of 30 µM FK506, whereas they were both unchanged in the failing SR vesicles. In normal SR vesicles, the amount of Ca2+ release expressed as A was increased in the presence of FK506, suggesting the releasable fraction of sequestered Ca2+ for the total amount of Ca2+ uptake is increased like in failing SR vesicles (Fig. 3 in Ref. [9]).


Figure 3
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  		Fig. 3 Effects of thapsigargin (a) and FK506 (B) on the time course of polylysineinduced Ca2+ release in SR vesicles isolated from normal and failing canine hearts. Cardiac SR vesicles (0.5 mg/ml) were first loaded with Ca2+ by ATP-dependent uptake and then mixed with an equal volume of a solution containing 0.74 µM polylysine (final concentration, 0.37 µM after mixing) to induce Ca2+ release; this was done in the presence or absence of 1 µM thapsigargin (a) or 30 µM FK506 (b). Each trace was obtained by signal-averaging a total of 120–150 traces originating from six experiments, each performed on a different pair of normal and failing SR vesicle preparations.

 

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  		Table 2 Amount (A), rate constant (k) and initial rate of release (Ak) of polylysine (0.37 µM)-induced Ca2+ release in the presence or absence of 30 µM FK506a

 
3.4 [3H]dihydro-FK506 binding
Fig. 4 shows representative data for [3H]dihydro-FK506 binding in SR vesicles isolated from normal and failing canine hearts. The mean values for the number of [3H]dihydro-FK506 binding sites (Bmax) and the dissociation constants (Kd) are shown in Table 3. Bmax was significantly lower in failing SR vesicles than in normal SR vesicles but there was no difference in Kd.


Figure 4
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  		Fig. 4 Representative data for [3H]dihydro-FK506 binding in SR vesicles isolated from normal ({circ}) and falling (bullet) canine hearts. Data represent specific [3H]dihydro-FK506 binding. Each datum point is the average of duplicate determinations.

 

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  		Table 3 [3H]dihydro-FK506 binding in SR vesicles taken from normal and failing heartsa

 
3.5 Effect of FK506 on SR Ca2+-ATPase activity
Fig. 5 shows the effects of FK506 on Ca2+-ATPase activity. In line with our previous finding [9], the Ca2+-ATPase activity was significantly lower in failing SR vesicles than in normal SR vesicles (P<0.05). None of the concentrations of FK506 tested significantly changed the Ca2+-ATPase activity in either normal or failing SR vesicles.


Figure 5
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  		Fig. 5 Effects of various concentrations of FK506 on Ca2-ATPase activity in SR vesicles isolated from normal ({circ}) and failing (bullet) canine hearts. Ca2+-ATPase activity in SR vesicles was obtained by measuring the amount of Pi released during the reaction initiated by adding 1.0 mM ATP to the above priming solution in the presence or absence of various concentrations of FK506. The amount of Pi released was calculated by converting nm (absorbance of 0.1% malachite green) to nmol by means of a standard linear line. Data represent means±S.D. of four experiments, each performed on a different preparation.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The major findings of this study are as follows. First, the polylysine-induced enhancement of [3H]ryanodine binding was reduced after the addition of FK506 in normal SR vesicles, whereas no significant change was seen in failing SR vesicles. Second, the rate of the polylysine-induced Ca2+ release was decreased after the addition of FK506 in normal SR vesicles but unchanged in failing SR vesicles. Third, the Bmax value for [3H]dihydro-FK506 binding was significantly smaller in failing SR vesicles than in normal ones. Fourth, FK506 had no direct effect on Ca2+-ATPase activity in either normal or failing SR vesicles.

With respect to myocardial contraction, Morgan [30] has nicely shown a close relationship between the Ca2+ transient and the subsequent tension development. Furthermore, in an other study, on rat papillary muscle [31], the time to peak for the Ca2+ transient was clearly shortened as the extracellular Ca2+ concentration increased, in association with an increase in the tension. On the basis of these findings, the rate of acceleration as well as of deceleration in the Ca2+ transient may greatly influence the subsequent tension development. In actual fact, there is a controversy as to the change in the peak value of the Ca2+ transient in heart failure. Using the same model of pacing-induced heart failure as us, Perreault et al. [32] demonstrated that there was no difference in the peak value of the Ca2+ transient between normal and failing hearts. In contrast, Yao et al. [33], working on rabbits, found a significant reduction in the peak of the Ca2+ transient in heart failure. However, in both studies the duration of the Ca2+ transient was significantly prolonged. The delayed fall in the descending portion of the Ca2+ transient might be caused by a decrease in the expression and/or activity of the SR Ca2+-ATPase (SERCA II) [34]. The decreased rate of acceleration in the Ca2+ transient (prolongation of time to peak) may be mainly secondary to the altered Ca2+-release function of the RyR because no other protein or receptor can induce a faster Ca2+ release than the RyR.

Recently [9], we demonstrated in a canine model of pacing-induced heart failure (i) that the rate of polylysine-induced Ca2+ release from the SR was significantly decreased and (ii) that the polylysine concentration-dependence of both the initial rate of Ca2+ release and [3H]ryanodine binding was shifted towards a lower concentration of polylysine in failing SR vesicles. This suggests that the gating function of the SR Ca2+ release channel is altered in heart failure, leading to a decrease in the acceleration of both the Ca2+ transient and the subsequent tension development.

FKBP12, which is tightly coupled with the skeletal muscle RyR, has recently been identified as a modulatory protein acting to stabilize the gating function of RyR [11,12,24]. However, the physiological significance of the cardiac FKBP12.6 in E–C coupling remains to be elucidated. Very recently, Marks et al. [35] demonstrated that PKA hyperphosphorylation of RyR in failing hearts causes a dissociation of FKBPI2.6 from RyR, resulting in the following abnormal single-channel properties: (i) increased Ca2+ sensitivity for activation and (ii) elevated channel activity associated with destabilization of the tetrameric channel complex. Also very recently, we showed (i) that FK506 caused a dose-dependent Ca2+ leak in normal SR vesicles (ii) that this leak showed a close parallelism with the conformational change in RyR and (iii) that the stoichiometry of FKBP with respect to RyR was significantly decreased in failing SR vesicles, leading to an abnormal Ca2+ leak in heart failure [16].

The present study has permitted further insight into the mechanism underlying the altered RyR function in heart failure. This greater insight is based on the following findings: (i) a confirmation of our previous finding [16] that the number of FKBP12.6 showed a tremendous decrease in failing cardiac SR vesicles, compared with normal cardiac SR vesicles, and (ii) in normal cardiac SR vesicles the addition of 30 µM FK506, which dissociates FKBP12.6 from cardiac RyR, decreased both drug-induced Ca2+ release and the drug-induced enhancement of [3H]ryanodine binding, although neither of these effects of FK506 was seen in failing SR vesicles. The decrease in the Bmax of for FK506 binding in heart failure (83% in this study and 77% in [16]) was considerably larger than that in the Bmax for RyR (66% in [9] and 50% in [16]), resulting in a decreased stoichiometry for FKRP12.6 with respect to RyR, as we reported previously [16].

Taking the present findings together with these previous reports [16,35], strongly suggests to us that when a sufficiently high concentration of FK506 (or rapamycin) is applied to cardiac myocytes, cooperation among the four RyR subunits is disrupted, thus destabilizing the channel and in turn inducing an abnormality in the channel-gating function of RyR. In heart failure, because of the partial loss of FKBP12.6 from the RyR, equivalent phenomena are presumably occurring even in the absence of FKBP dissociating agents.

Several limitations of this study need to be addressed

  • First, since FK506 binds to FKBP12 as well as to FKBP12.6, the Bmax for [3H]dihydro-FK506 might be thought to indicate mixed binding of FK506, i.e. involving FKBP12.6 and FKBP12. In actual fact, the type of FKBP bound to RyR is solely FKBP12.6 in cardiac muscle [13,14], while FKBP12 exists in a soluble form in the cytoplasm [36]. In addition, in our study the binding isotherm of [3H]dihydro-FK506 to cardiac SR vesicles was a simple hyperbola, yielding a straight line on Scatchard analysis, which indicates a single class of FK506-binding site. Taken together, the above findings strongly suggest that the [3H]dihydro-FK506 binding in cardiac SR vesicles represents specific [3H]dihydro-FK506–FKBP12.6 binding.
  • Second, in both normal and failing SR vesicles no Ca2+ release was present in the absence of polylysine, although there was significant ryanodine binding. However, the experimental conditions used for the ryanodine binding study (0.1 M NaCl, pH 7.2, incubation time 120 min, etc.) were quite different from those used for our examination of Ca2+ release (0.15 M KCl, pH 6.8, incubation time 5 min), although the extra-vesicular Ca2+ concentration was kept at 3 µM in both cases. Therefore, the ryanodine binding present in SR vesicles in the absence of polylysine may not necessarily indicate a significant Ca2+ release through the ryanodine receptor.
  • Third, with regard to the inhibition by FK506 of polylysine-induced ryanodine binding, it is unlikely that FK506 has a direct inhibitory effect on the site of ryanodine binding because the FK506 and ryanodine have different binding sites within the ryanodine-FKBP12.6 complex [11,12,29]. Rather, the inhibition by FK506 of ryanodine binding and Ca2+ release may result from a conformational change in RyR that follows a dissociation of FKBP12.6 from RyR. Since the FK50.6-induced decrease in ryanodine binding appeared to be smaller than that in the rate of Ca2+ release, the FK50.6-induced conformational change in RyR may modulate ryanodine binding and Ca2+ release in different ways.
  • Fourth, we need to address the question as to whether or not Ca2+ release is influenced by a possible direct effect of FK506 on Ca2+ reuptake, which may occur simultaneously during Ca2+ release. As mentioned above, there was no direct effect of FK506 on Ca2+- ATPase activity in either normal or failing SR vesicles. Furthermore, to eliminate Ca2+ uptake, polylysine-induced Ca2+ release was evaluated in the presence of 1 µM thapsigargin (SR Ca2+-ATPase inhibitor). However, both in normal and failing SR vesicles, the time course of Ca2+ release was not affected at all by the presence of thapsigargin, indicating that Ca2+ reuptake during Ca2+ release is probably negligible in both normal and failing SR vesicles.
  • Fifth, although in normal SR vesicles treated with polylysine, FK506 reduced the rate of Ca2+ release and inhibited the enhancement of [3H]ryanodine binding, these effects might not fully explain the tremendous impairment of the Ca2+-release function of the cardiac RyR seen in heart failure. Probably, a decreased density of cardiac RyR [9,37] is also a factor. Moreover, other proteins found in RyR (e.g. triadin) may modulate the SR Ca2+-release function although their role remains to be elucidated. It also remains to be explained why, after the addition of FK506 to normal SR vesicles, the rate constant for Ca2+ release was decreased to a level similar to that seen in failing SR vesicles (see Table 2) even though the ryanodine binding was still higher in normal SR vesicles than in failing SR vesicles. This may be partly explained by our observation that in the presence of FK506, the amount of Ca2+ released was less in failing SR vesicles than in normal SR vesicles (see Table 2), presumably leading to a faster saturation of Ca2+ release in the former than in the latter.
  • Sixth, it might be questioned why the concentration of FK506 required to induce a Ca2+ leak and protein conformational change in RyR was considerably higher than that needed for FK506-FKBP12.6 binding. Ahern et al. [38] showed that the concentration of FK506 required for an increase in the open probability of RyR is 3–20 µmol/l. while Timerman et al. [24] demonstrated that the EC50 for the dissociation of FKBP from RyR was as high as 0.12–0.5 µmol/l Possibly, the concentration of FK506 required for its binding to FKBP12.6 may be higher under physiological conditions than under the solubilized conditions used for [3H]dihydro-FK506 binding assays.
  • Seventh, although the present model of tachycardia-induced heart failure involves a well-defined, predictable and progressive LV dilatation as well as a contractile dysfunction and neurohumoral activation [39], it differs from chronic models of heart failure in two ways: it lacks a hypertrophic compensatory phase [40] and the heart failure disappears if pacing is discontinued after 3 weeks [41]. Clearly more work using other models of heart failure are needed to clarify whether there really is an abnormality in the kinetics of RyR in chronic heart failure.

In conclusion, in heart failure an altered interaction between FKBP12.6 and the ryanodine receptor may induce an instability of RyR-channel properties and a resulting impairment of the Ca2+-release function of RyR.

Time for primary review 25 days.


   Acknowledgements
 
FK506 was kindly provided by the Fujisawa Pharmaceutical Co. (Osaka, Japan). This work was supported by a grant-in-aid for scientific research from The Ministry of Education in Japan (Grant C 11670684) and by a Health Sciences Research Grant for Comprehensive Research on Aging and Health from the Ministry of Health and Welfare, Japan.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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