Overexpression of FK506-Binding Protein FKBP12.6 in Cardiomyocytes Reduces Ryanodine Receptor–Mediated Ca2+ Leak From the Sarcoplasmic Reticulum and Increases Contractility
Abstract—The FK506-binding protein FKBP12.6 is tightly associated with the cardiac sarcoplasmic reticulum (SR) Ca2+-release channel (ryanodine receptor type 2 [RyR2]), but the physiological function of FKBP12.6 is unclear. We used adenovirus (Ad)-mediated gene transfer to overexpress FKBP12.6 in adult rabbit cardiomyocytes. Western immunoblot and reverse transcriptase–polymerase chain reaction analysis revealed specific overexpression of FKBP12.6, with unchanged expression of endogenous FKBP12. FKBP12.6-transfected myocytes displayed a significantly higher (21%) fractional shortening (FS) at 48 hours after transfection compared with Ad-GFP–infected control cells (4.8±0.2% FS versus 4±0.2% FS, respectively; n=79 each; P=0.001). SR-Ca2+ uptake rates were monitored in β-escin–permeabilized myocytes using Fura-2. Ad-FKBP12.6–infected cells showed a statistically significant higher rate of Ca2+ uptake of 0.8±0.09 nmol/s−1/106 cells (n=8, P<0.05) compared with 0.52±0.1 nmol/s−1/106 cells in sham-infected cells (n=8) at a [Ca2+] of 1 μmol/L. In the presence of 5 μmol/L ruthenium red to block Ca2+ efflux via RyR2, SR-Ca2+ uptake rates were not significantly different between groups. From these measurements, we calculate that SR-Ca2+ leak through RyR2 is reduced by 53% in FKBP12.6-overexpressing cells. Caffeine-induced contractures were significantly larger in Ad-FKBP12.6–infected myocytes compared with Ad-GFP–infected control cells, indicating a higher SR-Ca2+ load. Taken together, these data suggest that FKBP12.6 stabilizes the closed conformation state of RyR2. This may reduce diastolic SR-Ca2+ leak and consequently increase SR-Ca2+ release and myocyte shortening.
In striated muscles, excitation-contraction (E-C) coupling involves depolarization of the plasma membrane to open voltage-gated calcium (Ca2+) channels (known as dihydropyridine receptors [DHPRs]). This event in turn triggers the release of a larger amount of Ca2+ from the sarcoplasmic reticulum (SR) to initiate muscle contraction.1 Ca2+ efflux from the SR is mediated by the SR-Ca2+ release channel (known as ryanodine receptor [RyR]), which is a tetramer comprised of 4 identical subunits. cDNA cloning revealed the existence of 3 different subtypes of RyRs: the skeletal muscle isoform RyR1, the heart muscle isoform RyR2, and the brain and smooth muscle isoform RyR3. The 3 isoforms share ≈66% sequence homology at the amino acid level and are the largest ion channels presently known.
Ca2+ release from the SR is a finely regulated process that involves not only the RyR itself but also several accessory proteins modulating RyR activity. Among these proteins are the FK506-binding protein FKBP12 and the orthologous protein FKBP12.6. The immunosuppressive drug FK506 binds to FKBP12 and FKBP12.6 and pentamerizes with calcineurin, calmodulin, and Ca2+, resulting in the inhibition of cytokine induction and the subsequent immune response.2 The physiological function of these proteins was unclear until the finding that FKBP12 associates with the RyR1 and modulates channel function.3 4 5 There is clear evidence from single-channel and lipid-bilayer studies that FKBP12 alters the kinetics of the channel activity and induces coupled gating of individual RyR1 channels in a junction.6 7 8 9 Although FKBP12 can bind to both RyR1 and RyR2, FKBP12.6 specifically associates with RyR2.10 11 12 However, the data described so far regarding the putative physiological function of FKBP12.6 are conflicting. Single-channel recordings of RyR2 activity incorporated into planar-lipid bilayers suggested that removal of FKBP12.6 from the RyR2 by FK506 or rapamycin increases the open probability of the channel and induces the appearance of long-lasting subconductance states.13 14 In comparable in vitro studies, however, removal of FKBP12.6 from the RyR2 or addition of recombinant FKBP12.6 to stripped cardiac SR preparations did not alter channel behavior.11 15 Even more puzzling, FKBP12-deficient mice, which have unchanged FKBP12.6 levels, display a severe cardiac phenotype but have normal skeletal muscle.16 The majority of the FKBP12-knockout mice died between embryonic day 14 and birth because of severe dilated cardiomyopathy. Both the skeletal and the cardiac-muscle RyRs from these mice showed similar alterations in single-channel behavior compared with RyRs from wild-type mice, ie, an increased open probability of the channel and the appearance of subconductance states.
Binding of FKBP12.6 to the RyR2 was shown to be regulated by phosphorylation of the channel subunits. Phosphorylation by protein kinase A, which is associated with the RyR2 via the anchoring protein mAKAP, dissociates FKBP12.6 from the channel, resulting in destabilization of the channel complex. Hyperphosphorylation of RyR2 in the failing human heart results in defective channel function and may thus account for dysregulated SR-Ca2+ handling during heart failure.17
In the present study, single-cell shortening, SR-Ca2+ uptake rates, and caffeine-evoked contractions reflecting SR-Ca2+ content were measured in rabbit cardiomyocytes overexpressing FKBP12.6 on adenovirus-mediated gene transfer. The results suggest that FKBP12.6 overexpression can modulate E-C coupling in cardiomyocytes.
Material and Methods
Recombinant Adenovirus Vector Construction
Full-length cDNA of the human FKBP12.6 gene was cloned by polymerase chain reaction (PCR) from a human heart-muscle–specific cDNA sample by the use of PCR primers that span the whole coding region of FKBP12.6 cDNA (see Table 1⇓). Recombinant adenoviruses were generated by standard procedures.
Primary Culture of Rabbit Ventricular Myocytes and Adenoviral Gene Transfer
Female Chinchilla bastard rabbits (Charles River, Sulzfeld, Germany; 2.5 to 3 kg, n=9) were heparinized and anesthetized with sodium thiopental (50 mg/kg IV). Outlines of this study were designed and carried out in accordance with institutional guidelines regarding care and use of animals. Ventricular myocytes were isolated by the enzymatic method as described.18 Myocytes were counted, and adenoviral infection with indicated multiplicity of infection (MOI) was performed during plating of the myocytes at a density of 0.5×105 rod-shaped cells/cm2 onto laminin (20 μg/mL)-coated tissue-culture dishes. After 3 hours, unattached cells were removed by 3 wash steps, and myocytes were cultured in supplemented M199 medium (Sigma).
Verification of Transgene Expression and Virus Transfection Efficiency
Reverse transcriptase–PCR (RT-PCR) analysis of transfected myocytes was performed with gene-specific primer pairs (see Table 1⇑) by the use of a hot-start Taq polymerase (Perkin Elmer) with 33 cycles each.
Western immunoblot analysis was performed with a polyclonal anti-FKBP12 (C-19) antibody (Santa Cruz Biotechnology) and an enhanced chemoluminescence detection system (Amersham) according to the manufacturer’s instructions.
Single-Cell Shortening Measurements
Myocyte shortening was measured by an edge-detection system (Crescent Electronics) at a stimulation frequency of 1 Hz and a sampling rate of 240 Hz. Online and offline analysis was performed with custom-designed Labview software (National Instruments).
Caffeine-induced contractions reflecting SR-Ca2+ load were measured after a stimulation train at 1 Hz by rapidly switching the superfusing solution to one containing 10 mmol/L caffeine for 5 to 6 seconds.
Measurements of SR-Ca2+ Uptake Rates
Isolated rabbit cardiomyocytes were infected with Ad-FKBP12.6 or Ad-LacZ at an MOI of 10 and were harvested after 48-hour culture time by gentle scraping off the culture dish. An aliquot of 0.1 mL of cell suspension (1.5×106 cells/mL) was exposed to 0.1 mg/mL β-escin (Sigma) and gently stirred for 30 seconds. β-Escin was removed by centrifuging and resuspending the cells in mock intracellular solution (see the online data supplement available at http://www.circresaha.org). Cells were then placed in a cuvette, and additional solutions were added to give a final volume of 0.15 mL, containing 50 μmol/L EGTA, 5 mmol/L ATP, 10 mmol/L CrP (Sigma), the mitochondrial inhibitors carbonyl cyanide (20 μmol/L) and oligomycin (20 μmol/L) (Calbiochem), 10 mmol/L oxalate (Sigma), and 10 μmol/L Fura-2 (Molecular Probes). Cells were maintained in suspension by gentle stirring, and the Fura-2 fluorescence within the cuvette was recorded at 100 Hz using a dual-wavelength spectrophotometer (IonOptix). All experiments were done at room temperature (20°C to 22°C).
All data are presented as mean±SEM. Myocyte-shortening data were analyzed by Student’s t test for unpaired data. Ca2+ uptake rates were tested for statistically significant differences between experimental groups by 2-way repeated-measure ANOVA followed by Student-Newman-Keuls test. P<0.05 was accepted as statistically significant.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
Adenoviral Gene Transfer of FKBP12.6 in Isolated Rabbit Ventricular Myocytes
Two different recombinant adenoviruses expressing human FKBP12.6, Ad-FKBP12.6, and Ad-FKBP12.6-GFP and 2 control adenoviruses, Ad-LacZ and Ad-GFP, were used in this study. Efficiency of adenoviral gene transfer in isolated rabbit ventricular myocytes was initially verified using the green fluorescent protein (GFP) and LacZ viruses. The transfection protocol resulted in transfection efficiencies of typically more than 95% at an MOI of 10 (data not shown).
Western immunoblot analysis using an antibody that cross-reacts with both FKBP12 and FKBP12.6 was used to verify transgene protein expression in the transfected myocytes. Although both proteins consist of 108 amino acid residues, FKBP12.6 migrates slower during denaturing electrophoresis than FKBP12.10 Interestingly, as shown in Figure 1A⇓, FKBP12 is the prominent isoform in rabbit cardiomyocytes. FKBP12.6 can be detected only in the transfected cells. Similar results were obtained with the RT-PCR analysis (Figure 1B⇓). Endogenous mRNA expression of FKBP12.6 in control cells was found to be much lower than endogenous FKBP12 expression. Adenovirus-mediated overexpression of FKBP12.6 resulted in a 5-fold increase in relative FKBP12.6 mRNA levels at 24 hours after transfection and a 6-fold increase at 48 hours after transfection compared with control. FKBP12 mRNA expression was unchanged on FKBP12.6 overexpression, as was mRNA expression of RyR2.
The DNA sequence of the 172-bp PCR fragment amplified by primers. FKBP12.6 internal (see Table 1⇑) in rabbit cardiomyocytes was found to be 94.2% homologous to rat FKBP12.6 cDNA and 97.6% homologous to human FKBP12.6 cDNA.
Contractile Parameters of Isolated Cardiomyocytes
Single-cell shortening of Ad-FKBP12.6-GFP–infected and Ad-GFP–infected myocytes was measured at 24 and 48 hours after adenoviral transfection by a video-edge detection system (Figure 2⇓). At 48 hours after transfection, myocyte fractional shortening (FS, expressed in percentage of diastolic cell length) was 21% higher in Ad-FKBP12.6-GFP–infected cells compared with Ad-GFP–infected cells (4.8±0.2% FS versus 4±0.2% FS, respectively; n=79 cells each, P=0.001). At 24 hours after transfection, at a lower level of FKBP12.6 overexpression (see Figure 1B⇑), FS was 15% higher in FKBP12.6 cells compared with GFP control cells (5.3±0.2% FS versus 4.6±0.3% FS, respectively; n=55 cells each, P=0.034). Shortening-time characteristics of both groups of myocytes are given in Table 2⇓. FKBP12.6 overexpression slightly but statistically significantly prolonged time to peak shortening after 2 days of culture time. Time to 50% relengthening was unchanged in both groups of myocytes at both time points.
SR-Ca2+ Uptake Rates in Permeabilized Cardiomyocytes
SR-Ca2+ uptake rates were monitored in β-escin–permeabilized cardiomyocytes with the use of the fluorescent dye Fura-2 (Figure 3⇓). The measurements of SR-Ca2+ uptake rates were done in the presence of 10 mmol/L oxalate. Oxalate enters the SR and acts as a Ca2+ sink, maintaining a constant [Ca2+] in the SR. A typical fluorescence signal is shown in Figure 3A⇓. Addition of aliquots of Ca2+ at the times indicated increased the [Ca2+] within the cuvette to ≈3 μmol/L. The time course of the decay of [Ca2+] represents a balance between Ca2+ uptake by the SR-Ca2+-ATPase pump and SR-Ca2+ leak. Addition of 5 μmol/L ruthenium red (RuR) caused the subsequent decay of [Ca2+] to be faster because of block of Ca2+ leak through SR-Ca2+ release channels. In a series of experiments, we observed no differences in SR-Ca2+ uptake characteristics between noninfected (control) and Ad-LacZ–infected cells (summarized in Table 3⇓). Myocytes overexpressing FKBP12.6 showed a significant higher rate of Ca2+ uptake at 1 μmol/L Ca2+ than LacZ-infected myocytes. Figures 3B⇓ (i) and 3C (i) show superimposed traces of [Ca2+] arranged to intersect at 1 μmol/L Ca2+ to illustrate the difference in the rate of Ca2+ uptake. These differences in Ca2+ uptake rates are more clearly distinguished when expressed as the rate of change of [Ca2+] (d[Ca2+]/dt) plotted against the associated [Ca2+] (Figure 3B⇓ [ii] and 3C [ii]). From these plots, it is evident that comparable rates of Ca2+ uptake were achieved at <0.5 μmol/L Ca2+ in both cell types and in the presence of RuR. However, at [Ca2+] >0.5 μmol/L, d[Ca2+]/dt values were lower in the absence of RuR, consistent with activation of a significant RyR2-mediated leak. As described in Materials and Methods, the values of d[Ca2+]/dt that were measured at 1 μmol/L Ca2+ were converted to a net Ca2+ flux using the Ca2+ buffer power, and the mean values of Ca2+ uptake are shown in Table 3⇓. In the presence of RuR, Ca2+ uptake rates in the 3 experimental groups were not significantly different. This result indicates that the 3 groups of myocytes have comparable rates of Ca2+ pump activity and background Ca2+ leak. The markedly different uptake rates in the absence of RuR (ie, in the presence of RyR2 activity) indicate that SR-Ca2+ leak through RyR2 is ≈50% lower in FKBP12.6-overexpressing cardiomyocytes at 1 μmol/L Ca2+. This difference between experimental groups is most clearly seen when the change in Ca2+ efflux rate is expressed relative to the control rate. Addition of RuR increased the rate of Ca2+ uptake ≈2-fold in noninfected and Ad-LacZ–infected cells (1.79±0.14-fold and 2.02±0.11-fold, respectively). The relative increase in Ca2+ uptake rate was significantly less in Ad-FKBP12.6–infected cells (1.3±0.16-fold, P<0.05) compared with control cells.
The effects of rapamycin on RyR-mediated Ca2+ leak from the SR was studied in noninfected myocytes (control) and FKBP12.6-overexpressing cells. In the noninfected control group, rapamycin (5 μmol/L) had no significant effect on the magnitude of the RuR-sensitive leak (Table 4⇓). When normalized to control conditions, Ca2+ uptake rate was not significantly altered in the presence of rapamycin (1.11±0.07-fold increase, not significant). However, when rapamycin was applied to myocytes overexpressing FKBP12.6, there was a statistically significant slowing of the rate of Ca2+ uptake to 0.74±0.1 of the value under control conditions. This was significantly different from the effect of rapamycin on noninfected cells (P=0.021). Because comparable rates of Ca2+ uptake are achieved in the presence of RuR, these results suggest that rapamycin increased the RuR-sensitive leak in rabbit myocytes overexpressing FKBP12.6.
Caffeine-Induced Contractures in Isolated Cardiomyocytes
To investigate whether the differences in shortening amplitude and SR-Ca2+ uptake rates between FKBP12.6-overexpressing myocytes and control myocytes were associated with differences in SR-Ca2+ load, we analyzed caffeine-induced contractures in Ad-GFP–infected and Ad-FKBP12.6-GFP–infected cardiomyocytes during steady-state shortening at a stimulation frequency of 1 Hz in a separate set of experiments. The amplitude of caffeine-induced contractures provides an index of the SR-Ca2+ content.24 As shown in Figure 4⇓, caffeine-evoked contractures were statistically significantly larger in FKBP12.6-overexpressing myocytes compared with control myocytes (18.8±1.2% FS, n=23, versus 15.2±1.1% FS, n=26, respectively, P=0.037) at 48 hours after transfection. Overexpression of FKBP12.6 increased Ca2+ load of the SR by 20% compared with control cells.
Role of FKBP12.6 in Modulation of E-C Coupling in Cardiac Muscle
The present study, using adenoviral gene transfer to specifically overexpress FKBP12.6 in rabbit myocytes, indicates 3 significant effects of FKBP12.6 overexpression: (1) reduction of RyR2-mediated Ca2+ efflux from cardiac muscle SR; (2) higher SR-Ca2+ load; and (3) increased amplitude of twitch shortening in single myocytes.
The relationship between modulation of RyR2 activity and contractility is an active area of research.19 20 Contradictory evidence exists concerning the steady-state effects of changes in RyR activity. Cyclic ADP ribose is thought to increase the open probability of RyR, increase peak systolic [Ca2+]i, and increase twitch shortening in guinea pig myoctes.21 In contrast, tetracaine, a drug that decreases Ca2+ sensitivity of RyR2, had no effects on rat-myocyte shortening in the steady state.22 Furthermore, caffeine, a drug that increases the Ca2+ sensitivity of the RyR2, can decrease the peak systolic [Ca2+].22 23 Therefore, it is difficult to extrapolate directly from the effects of FKBP12.6 overexpression to the increased twitch shortening observed in this study. Yet our data strongly suggest that overexpression of FKBP12.6 reduces Ca2+ leak from the SR during diastole, thereby increasing SR-Ca2+ content, and thus increases the amount of Ca2+ available for release, which in turn increases twitch shortening amplitude. In a way analogous to that described recently for FKBP12 and RyR1, FKBP12.6 overexpression may alter not only the Ca2+ efflux through RyR2 but also the degree of cooperative activity of the RyR2 cluster.9
Effects of FKBP12.6 Ligands on Cardiac E-C Coupling
A range of effects of rapamycin and FK506 have been reported. McCall et al25 observed increased Ca2+ transient amplitude in isolated rat myocytes exposed to FK506, with no obvious change in SR-Ca2+ content. High concentrations (50 μmol/L) of FK506 caused prolonged openings of RyR2 and prolongation of the Ca2+ transient in rat myocytes.14 Yet similar concentrations of FK506 had no dramatic effect on the Ca2+ transient in voltage-clamped rat cardiac myocytes.26 To date, there seems to be no information available concerning the effect of either of these ligands on the E-C coupling of isolated rabbit ventricular myocytes. In the present study, 5 μmol/L rapamycin had no significant effect on the SR of noninfected myocytes. In contrast, rapamycin significantly decreased the rate of Ca2+ uptake at 1 μmol/L in myocytes overexpressing FKBP12.6. These results suggest that the decreased Ca2+ flux through RyR2 observed on overexpression of FKBP12.6 could be reversed by rapamycin. This is consistent with the interpretation that increased cytosolic levels of FKBP12.6 are the cause of altered RyR2 properties with reduced Ca2+ leakage through RyR2.
Molecular Studies of RyR2 and FKBP12.6
Each RyR subunit contains only one FKBP-binding site, resulting in the structural formulas (RyR1 protomer)4-(FKBP12)4 and (RyR2 protomer)4(FKBP12.6)4, respectively.4 10 However, from in vitro binding studies using 35S-labeled FKBP12.6 and purified SR vesicles, Timerman et al11 calculated that ≈17% of the total FKBP12.6-binding sites in dog SR vesicles seem to be unoccupied. Marx et al17 recently showed that association of FKBP12.6 to RyR2 depends on phosphorylation state of RyR2 subunits.
Previous studies regarding the functional relevance of FKBPs (either FKBP12 or FKBP12.6) for RyR2 function are inconsistent. Single-channel analyses of RyR2 activity in planar-lipid bilayers are conflicting. In some studies, neither FKBP12 nor FKBP12.6 affects channel behavior,11 15 whereas other studies show marked changes in channel activity on dissociation of FKBP.13 14 In contrast, our data indicate that FKBP12.6 plays an important role in cardiac SR-Ca2+ release, which in turn depends on RyR2 function. This discrepancy may be explained by differences in the complexity of E-C–coupling mechanisms between cardiac and skeletal muscle in vivo. In cardiac myocytes, SR-Ca2+ release via RyR2 is triggered by depolarization-dependent Ca2+-influx through DHPRs, a phenomenon referred to as Ca2+-induced Ca2+ release.1 In skeletal muscle cells, a voltage-induced change in DHPR conformation directly initiates RyR1 receptor opening without a requirement for Ca2+ influx.27 Moreover, there is a functional diversity among the RyR subtypes despite their structural homology of ≈66%. RyR2 is not capable of supporting skeletal muscle–type E-C coupling in RyR1 knockout mice.28 Furthermore, keeping in mind that several other accessory proteins besides FKBP12 or FKBP12.6, such as sorcin, calmodulin, and calsequestrin, and phosphorylation of the RyR itself may be involved in proper RyR function, it is reasonable to speculate that the complex nature of the SR-Ca2+ release process in cardiac and skeletal muscle cells is difficult to reconstitute in vitro.
It is still an open question as to whether FKBP12 and FKBP12.6 are exchangeable with respect to function. Although a physical and functional association of FKBP12 and RyR1 has been demonstrated for animal species from all 5 classes of vertebrates,29 RyR2 can bind both FKBP12.6 and FKBP12, and both are expressed in myocardial tissue from different species, including humans (unpublished data, May 2000). Our data support the hypothesis that FKBP12.6 can modulate rabbit RyR2 in a similar manner as FKBP12 modulates RyR1 activity.
This study was supported in part by the Deutsche Stiftung Volkswagenwerk. G.L.S. was a recipient of a traveling grant from the Physiological Society. L.B. is funded by the Medical Research Council (UK). The authors thank Dr S. Dieterich for help with the cloning of human FKBP12.6 and S. Ott-Gebauer and M. Kothe for excellent technical assistance.
Original received July 27, 2000; revision received November 20, 2000; accepted November 22, 2000.
- © 2001 American Heart Association, Inc.
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