Reconstitution of local Ca2+ signaling between cardiac L-type Ca2+ channels and ryanodine receptors: insights into regulation by FKBP12.6

Sanjeewa A. Goonasekera,1 S. R. Wayne Chen,2 and Robert T. Dirksen1

1Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York; and 2Cardiovascular Research Group, Department of Physiology and Biophysics and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada

Submitted 25 May 2005 ; accepted in final form 22 July 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Ca+-induced Ca2+ release (CICR) in the heart involves local Ca2+ signaling between sarcolemmal L-type Ca2+ channels (dihydropyridine receptors, DHPRs) and type 2 ryanodine receptors (RyR2s) in the sarcoplasmic reticulum (SR). We reconstituted cardiac-like CICR by expressing a cardiac dihydropyridine-insensitive (T1066Y/Q1070M) {alpha}1-subunit ({alpha}1CYM) and RyR2 in myotubes derived from RyR1-knockout (dyspedic) mice. Myotubes expressing {alpha}1CYM and RyR2 were vesiculated and exhibited spontaneous Ca2+ oscillations that resulted in chaotic and uncontrolled contractions. Coexpression of FKBP12.6 (but not FKBP12.0) with {alpha}1CYM and RyR2 eliminated vesiculations and reduced the percentage of myotubes exhibiting uncontrolled global Ca2+ oscillations (63% and 13% of cells exhibited oscillations in the absence and presence of FKBP12.6, respectively). {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes exhibited robust and rapid electrically evoked Ca2+ transients that required extracellular Ca2+. Depolarization-induced Ca2+ release in {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a bell-shaped voltage dependence that was fourfold larger than that of myotubes expressing {alpha}1CYM alone (maximal fluorescence change was 2.10 ± 0.39 and 0.54 ± 0.07, respectively), despite similar Ca2+ current densities. In addition, the gain of CICR in {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a nonlinear voltage dependence, being considerably larger at threshold potentials. We used this molecular model of local {alpha}1C-RyR2 signaling to assess the ability of FKBP12.6 to inhibit spontaneous Ca2+ release via a phosphomimetic mutation in RyR2 (S2808D). Electrically evoked Ca2+ release and the incidence of spontaneous Ca2+ oscillations did not differ in wild-type RyR2- and S2808D-expressing myotubes over a wide range of FKBP12.6 expression. Thus a negative charge at S2808 does not alter in situ regulation of RyR2 by FKBP12.6.

heart failure; dihydropyridine receptor; excitation-contraction coupling


DURING CARDIAC excitation-contraction (EC) coupling, membrane depolarization-induced Ca2+ entry through plasma membrane dihydropyridine receptors (DHPRs) activates nearby Ca2+ release channels in the sarcoplasmic reticulum (SR) to open and release Ca2+ via a mechanism referred to as Ca2+-induced Ca2+ release (CICR) (4, 9). In addition to the DHPR and type 2 RyR ryanodine receptor (RyR2), accessory proteins including a 12.6-kDa FK-506 binding protein (FKBP12.6), calmodulin, sorcin, calsequestrin, PKA, Ca2+/calmodulin-dependent protein kinase II, and protein phosphatases (including PP1 and PP2A) modulate the EC coupling process via targeted association with the DHPR and/or RyR2 (5, 39).

In recent years, research has focused on assessing the role of FKBP12.6 in the regulation of RyR2 function and Ca2+ release during cardiac EC coupling. These studies (both in vitro and in vivo) have shown that FKBP12.6 selectively binds to RyR2 with a 4:1 stoichiometry in a manner that alters the Ca2+ sensitivity and stabilizes the closed state of the channel during the diastolic phase of the cardiac cycle (17, 20, 37). In addition, the association of FKBP12.6 with RyR2 may facilitate coupled and coordinated gating of adjacent RyR2 channels clustered in functional arrays referred to as Ca2+ release units (23). PKA hyperphosphorylation of S2809 (residue number corresponds to rabbit RyR2 and is homologous to S2808 of mouse RyR2) during heart failure may result in structural and functional uncoupling of FKBP12.6 from the release channel (24, 38). The resulting destabilization of the channel's closed state and potential loss of coupled/coordinated RyR2 gating might then contribute to the observed decrease in EC coupling gain that occurs during heart failure (21, 24, 38, 42). Consequently, research aimed at approaches designed to reestablish a tight association of FKBP12.6 with RyR2 as a novel therapeutic approach in the treatment of heart failure has understandably stimulated considerable excitement within the field (19, 30, 40). Nevertheless, significant debate persists with regard to the precise molecular and structural determinants of the FKBP12.6-RyR2 interaction and its potential role in heart failure. Specifically, several studies have failed to confirm PKA hyperphosphorylation of S2809 (or S2808) and consequent dissociation of FKBP12.6 from the release channel during heart failure (34, 43, 44).

At present, three approaches have been used to probe the in situ regulatory role of FKBP12.6: 1) the use of pharmacological agents to disrupt the FKBP12.6-RyR2 interaction (8, 12), 2) transgenic animal models that lack FKBP12.6 (38, 45), and 3) short-term overexpression of FKBP12.6 in cultured adult heart cells (12, 13, 22, 29). However, important limitations are associated with each approach. For example, problems associated with drug specificity and potential compensatory changes in surrogate FKBP isoforms and/or other proteins of the EC coupling machinery during FKBP12.6 deficiency confound interpretations from pharmacological and knockout approaches, respectively (8). More important, none of these approaches enables a systematic characterization of the molecular determinants of local DHPR-RyR2 Ca2+ signaling and the in situ influence of FKBP12.6 on this central component of the cardiac EC coupling machinery.

In the present study, we characterized a novel molecular model of local CICR between junctional DHPR and RyR2 Ca2+ channels by expressing a dihydropyridine-insensitive (T1066Y/Q1070M) {alpha}1C-subunit of the cardiac DHPR ({alpha}1CYM), RyR2, and FKBP12.6 in myotubes derived from RyR1-knockout (dyspedic) mice. Dyspedic myotubes were used because although they lack muscle RyR proteins, they nevertheless exhibit SR-sarcolemmal junctions (36) and express a full complement of a number of other key proteins of the triadic junction (e.g., DHPR auxiliary subunits, calsequestrin, junctin, triadin) (6). Moreover, both {alpha}1C (15) and RyR2 (28) proteins readily target to SR-sarcolemmal junctions in myotubes. These observations suggest that it should be possible to reconstitute molecularly defined local {alpha}1C-RyR2 junctional coupling in dyspedic myotubes. Our results confirm that indeed local Ca2+ signaling between {alpha}1CYM and RyR2 exhibiting many of the hallmarks cardiac CICR (e.g., requirement of extracellular Ca2+, fourfold amplification of Ca2+ influx, bell-shaped voltage dependence of Ca2+ release, and nonlinear voltage dependence on the gain of CICR) are reproduced with this approach. We used this model to probe the regulatory role of FKBP12.6 and phosphorylation of RyR2 S2808 on the fidelity of local {alpha}1C-RyR2 signaling.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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Preparation and microinjection of myotubes. Primary cultures of dyspedic myotubes were generated from skeletal muscle myoblasts obtained from neonatal dyspedic mice as previously described (2). All animal procedures were reviewed and approved by the University Committee on Animal Resources at the University of Rochester. Five to seven days after being plated, individual myotube nuclei were microinjected with cDNAs encoding a dihydropyridine-insensitive {alpha}1C (T1066Y/Q1070M, {alpha}1CYM; Ref. 25) or a combination of {alpha}1CYM and RyR2 [wild type (WT) or S2808D; Ref. 44] in the presence and absence of FKBP12.6. All constructs other than FKBP12.6 and FKBP12.0 were injected at a cDNA concentration of 0.1 µg/µl. FKBP12.6 and FKBP12.0 were injected at a cDNA concentration of 0.5 µg/µl (except for some experiments shown in Fig. 4). Experiments were carried out on the third day after microinjection. In all experiments, coinjection of CD8 cDNA (0.1 µg/µl) was used to enable identification of expressing myotubes via incubation with anti-CD8 antibody-coated beads.



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Fig. 4. An aspartic acid substitution at S2808 (S2808D) does not alter in situ regulation of RyR2 by FKBP12.6. A: representative Ca2+ response of an {alpha}1CYM/RyR2-expressing myotube exhibiting numerous spontaneous Ca2+ oscillations (left). Spontaneous Ca2+ oscillations are not observed in a {alpha}1CYM/RyR2/FKBP12.6-expressing myotube (right). WT, wild type. B: spontaneous Ca2+ oscillations in {alpha}1CYM/S2808D-expressing myotubes (left) are similarly abolished by coexpression with FKBP12.6 (right). A single exponential was used to fit the decaying phase of the final evoked Ca2+ transients (smooth solid line) in A and B (insets). The average time constant ({tau}) of Ca2+ transient decay was not different between {alpha}1CYM/RyR2/FKBP12.6- and {alpha}1CYM/S2808D/FKBP12.6-expressing myotubes. Maximal responses to 10 mM caffeine (horizontal bars in A and B) were used to confirm RyR2 expression and assess SR Ca2+ content. C: % of cells displaying spontaneous Ca2+ oscillations for different concentrations of FKBP12.6 cDNA at a fixed level of either WT RyR2 (filled bars) or S2808D (gray bars).

 
Measurements of resting fluorescence, electrically evoked Ca2+ transients, and caffeine-induced Ca2+ release. Myotubes grown on glass coverslips were loaded with 6 µM indo-1 AM (Molecular Probes, Eugene, OR) in rodent Ringer solution (see below) for 75 min at 37°C. Myotubes were then rinsed several times with indo-1 AM-free Ringer solution and incubated for an additional 20 min to allow for deesterification of the dye. A small rectangular region of indo-1-loaded myotubes was excited at 350 nm, and fluorescence emission at 405 and 485 nm (F405 and F485) was monitored with a x40 magnification (1.35 numerical aperture) oil-immersion objective was collected at 100 Hz with a photomultiplier detection system. Results are presented as the ratio of F405 and F485 (F405/F485). Expressing myotubes were electrically stimulated (8 V every 15 s for 50 s) with an extracellular electrode placed close to the cell of interest. The declining phase of electrically evoked Ca2+ transients was fit with a single exponential function to extract the time constant ({tau}) of Ca2+ transient decay. SR Ca2+ store content was assessed via local application of a maximal concentration of caffeine (10 mM) and subsequent washout with control Ringer solution and a rapid perfusion system (Warner Instruments, Hamden, CT) that permits local drug application/removal. Data were analyzed with FeliX (Photon Technology International, Lawrenceville, NJ) and SigmaPlot 2000 (SPSS, Chicago, IL) software packages. For the data presented in Figs. 1C and 4C, statistical significance was assessed using a {chi}2 test.



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Fig. 1. FKBP12.6 is required for tight local control of Ca2+ signaling between cardiac dihydropyridine-insensitive (T1066Y/Q1070M) {alpha}1-subunit ({alpha}1CYM) and type 2 ryanodine receptor (RyR2). A: a {alpha}1CYM/RyR2-expressing dyspedic myotube exhibiting membrane vesiculations and spontaneous contractions (left) and a representative Ca2+ response of an indo-1 AM-loaded {alpha}1CYM/RyR2-expressing dyspedic myotube displaying numerous uncontrolled, spontaneous Ca2+ oscillations (right). B: coexpression of FKBP12.6 with {alpha}1CYM and RyR2 eliminated both membrane vesiculations (left) and spontaneous Ca2+ oscillations (right). Rapid, electrically evoked global Ca2+ release events are readily observed in {alpha}1CYM/RyR2/FKBP12.6-expressing dyspedic myotubes. A single exponential (smooth solid line) was used to fit the decaying phase of the final evoked Ca2+ transient (inset). Maximal responses to 10 mM caffeine (horizontal bars in A and B) were used to confirm RyR2 expression and assess SR Ca2+ content [change in 405- to 495-nm fluorescence ratio ({Delta}F405/485) was 1.14 ± 0.04 (n = 55) and 1.15 ± 0.05 (n = 21) for {alpha}1CYM/RyR2- and {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes, respectively]. C: 63% of {alpha}1CYM/rabbit RyR2-expressing myotubes exhibited spontaneous Ca2+ oscillations (solid bar, left), whereas coexpression of FKBP12.6 significantly reduced the percentage of myotubes (13%) with spontaneous Ca2+ oscillations (open bar, left). Qualitatively similar results were obtained for mouse RyR2 (right). The incidence of spontaneous Ca2+ oscillations was not reduced when FKBP12.6 was replaced with FKBP12.0 (gray bar). *P < 0.05 relative to myotubes expressing {alpha}1CYM/RyR2/FKBP12.6.

 
Simultaneous voltage-clamp measurements of Ca2+ currents and Ca2+ transients. The whole cell patch-clamp technique was used to simultaneously measure the voltage dependence of L-type Ca2+ currents and global intracellular Ca2+ transients as previously described (2, 3). Because naive dyspedic myotubes exhibit very small L-type Ca2+ currents via the skeletal muscle isoform of the DHPR ({alpha}1S; <1 pA/pF) (2, 26), all experiments were conducted in the presence of 3 µM nifedipine. We used a dihydropyridine-insensitive variant of the {alpha}1-subunit of the cardiac L-type Ca2+ channel (T1066Y/Q1070M or {alpha}1CYM; Ref. 25) to permit Ca2+ flux through expressed cardiac L-type Ca2+ channels in the presence of nifedipine. These conditions allow definitive separation of expressed cardiac L currents from any endogenous dihydropyridine-sensitive skeletal L currents present in dyspedic myotubes. After expression in myotubes, L current kinetics and voltage dependence attributable to {alpha}1CYM (Table 1) were indistinguishable from those of WT {alpha}1C expressed in myotubes (7, 18, 31). L currents were elicited with 200-ms test pulses from –50 to +100 mV in 10-mV increments. Linear leak and capacitative currents were subtracted with a pulse amplitude/n protocol (n = 3). Peak L currents were normalized to total cell capacitance (determined from fitting a single exponential to the membrane current resulting from 10-mV hyperpolarizing pulses delivered from a holding potential of –80 mV) and plotted as a function of membrane potential (Vm) according to the following equation:

(1)
where Gmax is the maximal L channel conductance, VG1/2 is the voltage required for half-maximal activation of Gmax, Vrev is the extrapolated reversal potential, and kG is a slope factor. Relative changes in intracellular Ca2+ in patch-clamp experiments (depolarization-induced Ca2+ transients) were measured with fluo-4 introduced through the patch pipette. Relative changes in the magnitude of depolarization-induced Ca2+ release were calculated by taking the difference between the baseline fluorescence immediately before the voltage step (Fbase) and the peak fluorescence (Fpeak) at the end of the 200-ms voltage step and then normalizing to Fbase [{Delta}F/F = (Fpeak – Fbase)/Fbase]. {Delta}F/F was then plotted as a function of Vm and fitted according to the following equation:

(2)
where ({Delta}F/F)max is the maximal fluorescence change, VF1/2 is the half-maximal voltage, and kF is a slope factor. The additional variable k' is a scaling factor that varies with ({Delta}F/F)max (33). Pooled current-voltage (I-V) and fluorescence-voltage ({Delta}F/F-V) data in Table 1 are expressed as means ± SE. The ratio between the maximal rate of Ca2+ release ({Delta}F/{Delta}t) and the integral of the triggering Ca2+ current () was used as a quantitative index of the relative gain of CICR . The rate of Ca2+ release was obtained by taking the maximum slope of a linear fit of the fluorescence increase following each depolarization. The rate of change in fluorescence represents an accurate approximation of the rate of release in myotubes because these cells have a negligible calcium removal flux (10). Statistically significant differences between {alpha}1CYM- and {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes were determined using an unpaired Student's t-test. For all statistical analyses, differences were considered significant at P < 0.05.


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Table 1. Parameters of fitted I-V and {Delta}F/F-V curves

 
Solutions. Myoplasmic Ca2+ levels in intact myotubes were determined in the presence of a normal rodent Ringer solution consisting of (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.4 with NaOH). Equimolar substitution of MgCl2 for CaCl2 was used in Ca2+-free experiments. Whole cell patch-clamp experiments were recorded with an internal pipette solution containing (in mM) 145 Cs-aspartate, 0.1 Cs2-EGTA, 1.2 MgCl2, 5 MgATP, 0.2 K5-fluo-4, and 10 HEPES (pH 7.4 with CsOH). The external recording solution contained (in mM) 145 tetraethylammonium (TEA)-Cl, 10 CaCl2, and 10 HEPES, with 3 µM nifedipine (pH 7.4 with TEA-OH).


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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{alpha}1CYM/RyR2-expressing dyspedic myotubes exhibit spontaneous Ca2+ oscillations/contractions that are eliminated by coexpression of FKBP12.6. Consistent with the results of Nakai et al. (28), myotubes expressing RyR2 alone or RyR2 plus {alpha}1CYM (T1066Y/Q1070M; Ref. 25) displayed regions of spontaneous contractions, and within these regions, the myotube membrane morphology appeared rounded and vesiculated [Fig. 1A, left, and supplemental Fig. 1A (supplemental figures available at http://ajpcell.physiology.org/cgi/content/full/00250.2005/DC1)]. No such contractions or vesiculations were observed in either noninjected dyspedic myotubes or myotubes expressing {alpha}1CYM alone. Furthermore, the occurrence of vesiculations in the presence of RyR2 alone and their absence in the presence of {alpha}1CYM alone (data not shown) indicate that uncontrolled Ca2+ release, rather than Ca2+ influx, underlies the formation of membrane vesiculations. To confirm the source of the spontaneous contractions, changes in intracellular Ca2+ levels were assessed in indo-1 AM-loaded myotubes. Myotubes expressing both {alpha}1CYM and RyR2 exhibited numerous chaotic and uncontrolled global Ca2+ oscillations in the presence of either control (Fig. 1A, right) or Ca2+-free Ringer (data not shown) solutions. Confirmation of functional Ca2+ release channel expression and determination of total releasable SR Ca2+ content in these cells were obtained by local application of a maximal activating concentration (10 mM) of caffeine (Fig. 1A, right).

In contrast to that observed for myotubes expressing only {alpha}1CYM and RyR2, coexpression of FKBP12.6 with {alpha}1CYM and RyR2 resulted in myotubes that lacked vesiculations, displayed an appearance similar to that of noninjected myotubes (Fig. 1B, left, and supplemental Fig. 1B), and exhibited a drastically reduced incidence of spontaneous Ca2+ oscillations/contractions (Fig. 1B, right). However, basal indo-1 fluorescence (F405/485 was 0.60 ± 0.01 and 0.56 ± 0.02 in {alpha}1C/RyR2- and {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes, respectively) and maximum caffeine-induced Ca2+ release (peak {Delta}F405/485 was 1.14 ± 0.04 and 1.15 ± 0.05 in {alpha}1CYM/RyR2- and {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes, respectively) were not different between the two populations of cells.

Extracellular field stimulation of {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes elicited large, rapidly activating (time to peak ~100 ms) evoked Ca2+ transients (Fig. 1B, right) with an average decay {tau} of 320 ± 70 ms (Fig. 1B, right, inset). These evoked transients were completely abolished on removal of extracellular Ca2+, consistent with their being linked to a Ca2+ influx-dependent trigger (supplemental Fig. 2). Figure 1C depicts the percentage of myotubes displaying one or more spontaneous Ca2+ oscillations within the first 100 s of recording in both the presence and absence of FKBP12.6. For rabbit RyR2 (Fig. 1C, left), 63% of the myotubes displayed one or more Ca2+ oscillations in the absence of FKBP12.6, whereas only 13% of myotubes displayed Ca2+ oscillations in the presence of FKBP12.6. Similar results were obtained for mouse RyR2 (Fig. 1C, right), indicating that the effects of FKBP12.6 on spontaneous SR Ca2+ release are species independent. To assess the specificity of FKBP12.6 inhibition of spontaneous Ca2+ release, we determined whether FKBP12.0 expression could also reduce the incidence of spontaneous Ca2+ oscillations (Fig. 1C, right). FKBP12.0 expression was confirmed by immunocytochemistry (data not shown). Eighty-seven percent of {alpha}1CYM/RyR2/FKBP12.0-expressing myotubes exhibited spontaneous Ca2+ oscillations, demonstrating that FKBP12.0 cannot functionally replace FKBP12.6. However, it is possible that inhibition might be achieved at higher levels of FKBP12 expression.



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Fig. 2. Whole cell patch-clamp experiments demonstrate functional coupling between {alpha}1CYM and RyR2. A and B: representative L-type Ca2+ currents (bottom) and intracellular Ca2+ transients (top) recorded in response to 200-ms depolarizations to the indicated potentials in myotubes expressing {alpha}1CYM alone (A) or {alpha}1CYM + RyR2 + FKBP12.6 (B). C and D: voltage dependence of average (±SE) peak Ca2+ currents (ICa, C) and Ca2+ transients (D) in {alpha}1CYM-expressing and {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes. Peak Ca2+ transients were 4-fold larger in {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes [({Delta}F/F)max = 2.10 ± 0.39; n = 15] compared with those in {alpha}1CYM-expressing myotubes [({Delta}F/F)max = 0.54 ± 0.07; n = 7]. Ca2+-induced-Ca2+ release (CICR) refers to the difference between the magnitude of Ca2+ transients observed in the presence of {alpha}1CYM, RyR2, and FKBP12.6 from that observed for {alpha}1CYM alone (this difference is plotted in Fig. 3A). Vm, membrane potential.

 
Voltage dependence of SR Ca2+ release mirrors that of Ca2+ influx. Whole cell patch-clamp experiments were used to assess the voltage dependence of CICR between junctional {alpha}1CYM-RyR2 channels. Figure 2, A and B, depicts representative macroscopic L-type Ca2+ currents (bottom) and global intracellular Ca2+ transients (top) elicited at the indicated potentials in dyspedic myotubes expressing either {alpha}1CYM alone (Fig. 2A) or {alpha}1CYM + RyR2 + FKBP12.6 (Fig. 2B). The magnitude and voltage dependence of peak L-type Ca2+ current density were not significantly different in {alpha}1CYM- and {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes (Fig. 2C and Table 1). In the absence of RyR2, global intracellular Ca2+ responses were minimal (Fig. 2D and Table 1). However, despite similar Ca2+ current magnitudes, peak intracellular Ca2+ transients [({Delta}F/F)max] were fourfold larger in {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes (Fig. 2D and Table 1). Measurements of voltage-gated L-type Ca2+ currents and Ca2+ transients could not be obtained in myotubes expressing {alpha}1CYM and RyR2 in the absence of FKBP12.6, because these cells exhibited high-frequency spontaneous contractions that disrupted the formation and maintenance of high-resistance gigaohm seals required for whole cell patch-clamp experiments.

Confirmation of close functional coupling between expressed {alpha}1CYM and RyR2. Although prior studies demonstrated efficient junctional targeting of {alpha}1C (15) and RyR2 (28) proteins after transient expression in myotubes, functional coupling between these proteins after coexpression has not been assessed previously. Two key observations confirmed tight, local functional coupling between expressed {alpha}1CYM and RyR2 in our experiments. First, we defined CICR in our model as the difference between the magnitude of global Ca2+ transients measured in the presence and absence of RyR2 (as shown in Fig. 2D). CICR defined in this manner exhibits a bell-shaped voltage dependence that precisely mirrors that of the simultaneously recorded L-type Ca2+ current (Fig. 3A).



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Fig. 3. Confirmation of local voltage-dependent functional coupling between {alpha}1CYM and RyR2. A: voltage dependence of CICR (difference in Ca2+ transient magnitude obtained in presence and absence of RyR2/FKBP12.6 as shown in Fig. 2D) precisely mirrors that of the triggering L-type Ca2+ current. B: normalized gain of CICR , calculated as the ratio of the maximal rate of Ca2+ release ({Delta}F/{Delta}t) and the integral of triggering Ca2+ current (), displays a steep voltage dependence at threshold potentials. *P < 0.05 relative to the gain of CICR at –10 mV.

 
A second hallmark of local {alpha}1C-RyR2 coupling is that the gain of CICR is not constant with voltage but rather is greater at threshold potentials (1, 14). Therefore, we determined whether a similar nonlinear gain of CICR was also reproduced in our model. We calculated the relative gain of CICR (Ca2+ release per unit of Ca2+ influx) in our experiments by taking the ratio between the maximal rate of Ca2+ release and the integral of the triggering Ca2+ current and plotted the quotient as a function of voltage (Fig. 3B). Similar to that shown in adult cardiomyocytes (1, 14), the gain of CICR assessed in this manner exhibits a steep voltage dependence, with a greater CICR gain observed at threshold potentials.

Negative charge at S2808 does not alter in situ regulation of RyR2 by FKBP12.6. Marx and colleagues (24) proposed that PKA-mediated hyperphosphorylation of RyR2 during heart failure results in the dissociation of FKBP12.6 and the subsequent loss of coordinated and coupled release channel gating. Specifically, they proposed that phosphorylation of S2809 disrupts FKBP12.6 binding and an aspartic acid substitution of S2809 (S2809D) behaves as a constitutively phosphorylated RyR2 channel that does not readily bind FKBP12.6 (38). However, controversy persists with regard to the exact RyR2 PKA phosphorylation site and the relevance of S2809 phosphorylation in regulating FKBP12.6 binding to and regulation of RyR2 (34, 44).

In light of this controversy, we determined whether a negative charge at residue S2808 (the analogous residue in mouse RyR2) modifies the ability of FKBP12.6 to regulate spontaneous in situ RyR2 activity. Similar to WT RyR2 (Fig. 4A, left), in the absence of FKBP12.6 coexpression S2808D-expressing myotubes were also vesiculated and displayed numerous uncontrolled spontaneous Ca2+ oscillations/contractions (Fig. 4B, left). However, vesiculations and spontaneous Ca2+ oscillations/contractions were largely abolished on coexpression of FKBP12.6, suggesting that FKBP regulation was not disrupted by the S2808D mutation (Fig. 4, A and B, right). In addition, electrically evoked Ca2+ transients exhibited similar peak magnitudes [{Delta}ratio was 0.21 ± 0.03 (n = 14) and 0.15 ± 0.02 (n = 11) in WT RyR2- and S2808D-expressing myotubes, respectively] and kinetics (compare time to peak and decay rates in Fig. 4, A and B, right) in WT RyR2- and S2808D-expressing myotubes.

Because FKBP12.6 overexpression could potentially drive binding to RyR2 under conditions in which FKBP12.6 affinity is only moderately lowered (e.g., S2809D) (41), we conducted functional FKBP12.6 dose-response curves in WT RyR2- and S2808D-expressing myotubes (Fig. 4C). We reasoned that if the S2808D mutation significantly reduced the in situ affinity of RyR2 for binding FKBP12.6, then an increased incidence of spontaneous Ca2+ oscillations should be observed at threshold levels of FKBP12.6 expression. A clear correlation was found between the concentration of injected FKBP12.6 cDNA and the incidence of cells exhibiting spontaneous Ca2+ oscillations in {alpha}1CYM/RyR2-expressing myotubes (Fig. 4C). However, the incidence of spontaneous Ca2+ oscillations in {alpha}1CYM/S2808D-expressing myotubes was not increased at any concentration of FKBP12.6 studied (Fig. 4C). These data indicate that the S2808D mutation does not produce a measurable change in the inhibition of spontaneous RyR2 activity by FKBP12.6.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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Although Ca2+ influx through L-type Ca2+ channels triggers junctional SR Ca2+ release via CICR during cardiac EC coupling, the exact molecular details governing and/or regulating this process remain elusive. Our results indicate that coexpression of {alpha}1CYM and RyR2 in dyspedic myotubes provides a novel molecular model for probing the structural determinants of localized Ca2+ signaling between junctional cardiac L-type and SR Ca2+ release channels that is analogous to that which occurs during EC coupling in the heart (9). Differences in the complement of proteins present in dyspedic myotubes from that found in genuine cardiac myocytes limit the application of this model to more integrated questions regarding the cardiac EC coupling process. For example, whereas dyspedic myotubes possess a full complement of muscle EC coupling proteins (e.g., SERCA, L channel auxiliary subunits, calsequestrin, junction, triadin), a limitation of our model is that these cells lack the cardiac isoforms of these proteins (except for the expressed {alpha}1CYM, RyR2, and FKBP12.6 proteins). Thus the model described herein is best applied to questions regarding the molecular determinants and/or regulation of localized {alpha}1C-RyR2 Ca2+ signaling.

Several lines of evidence support the conclusion that the voltage-gated Ca2+ transients observed with this model arise from close spatial coupling between junctional {alpha}1CYM and RyR2 Ca2+ channels. First, electrically evoked Ca2+ release transients are activated rapidly (time to peak ~100 ms; Figs. 1B and 4, A and B) and require extracellular Ca2+ (supplemental Fig. 2). Second, the magnitude of voltage-gated Ca2+ transients in {alpha}1CYM/RyR2/FKBP12.6-expressing myotubes is fourfold greater than that observed in myotubes expressing {alpha}1CYM alone, although they exhibit similar Ca2+ current densities (Fig. 2, C and D). Thus Ca2+ release through expressed RyR2 release channels amplifies the Ca2+ influx signal to a degree comparable to that reported for adult cardiomyocytes (4). Third, voltage-activated myoplasmic Ca2+ transients are graded in nature and exhibit a bell-shaped voltage dependence that precisely mirrors that of Ca2+ influx through expressed cardiac L-type Ca2+ channels (Fig. 3A). Finally, our observation that the gain of CICR is increased at negative/threshold Vm provides a strong functional argument for local {alpha}1CYM activation of RyR2 in this model (Fig. 3B).

We used this molecular model of cardiac CICR to probe the influence of FKBP12.6 on junctional RyR2 function and localized cardiac DHPR-RyR2 Ca2+ signaling. Our results demonstrate that in the absence of FKBP12.6 junctional RyR2 release channels exhibit a high incidence of uncontrolled spontaneous SR Ca2+ release. This increased propensity for spontaneous Ca2+ release was not due to differences in SR Ca2+ content, because Ca2+ release in response to application of a maximal concentration of caffeine was similar in the presence and absence of FKBP12.6 (Fig. 1, A and B). Rather, the most parsimonious explanation for the FKBP12.6-mediated reduction in spontaneous Ca2+ release is that FKBP12.6 acts as a negative allosteric regulator of the RyR2 Ca2+ release channel by stabilizing a closed state of the channel under basal conditions. This action is consistent with postulated effects of FKBP12.6 on release channel gating observed in both biochemical and lipid bilayer studies (24, 37, 38, 40). Our results are also consistent with the findings of Ji et al. (16), in which the frequency of nontriggered Ca2+ sparks and spontaneous transient outward currents is significantly increased in smooth muscle cells from FKBP12.6-knockout mice. Together, these results indicate that the FKBP12.6-RyR2 interaction stabilizes the resting closed state of the release channel. However, others have failed to observe direct effects of FKBP12.6 on RyR2 function (37). Thus our results do not exclude a possible role of more indirect effects of FKBP12.6 expression on spontaneous Ca2+ release.

Normally, sufficient levels of FKBP12.6 are present in native cardiomyocytes to ensure adequate stabilization of basal RyR2 activity (38, 45). However, pathophysiological conditions that lead to either decreased FKBP12.6 levels or reduced RyR2 affinity for FKBP12.6 could result in an increased propensity for spontaneous Ca2+ release during diastole, activation of a Ca2+ dependent inward current, and generation of an arrhythmogenic focus (38, 40). Interestingly, although cardiomyocytes of FKBP12.6–/– mice exhibit dysregulated Ca2+ release, these mice display normal growth rates and fertility (38, 45). The relatively benign basal cardiac phenotype of FKBP12.6–/– mice suggests that compensatory changes or factors in addition to FKBP12.6 may serve to limit the incidence of spontaneous, nontriggered Ca2+ oscillations in cardiomyocytes (45) (but not in smooth muscle; Ref. 16).

Controversy persists with regard to the effects of phosphorylation of S2808 (or S2809) on the affinity of RyR2 for FKBP12.6. Marx et al. (24) were the first to show that PKA hyperphosphorylation of S2809 occurs during heart failure. Wehrens et al. (38) subsequently reported that a PKA phosphomimetic mutant of RyR2 (S2809D) similarly disrupted FKBP12.6 binding/regulation. However, other studies have failed to correlate changes in FKBP12.6 binding/regulation of RyR2 after phosphorylation of this residue (34, 44). As a common thread, these studies relied on the S2809D (or S2808D) mutation acting as a constitutively phosphorylated RyR2 release channel. Herein we have demonstrated for the first time that FKBP12.6 regulation of junctional RyR2 activity in intact muscle cells is unaffected by the S2808D mutation in RyR2. Specifically, the results presented in Fig. 4, A and B, indicate that FKBP12.6 regulates basal RyR2 and S2808D activity to a similar degree. In addition, we found that the magnitude and kinetics (time to peak and decay rate) of electrically evoked Ca2+ release were similar in myotubes expressing {alpha}1CYM, FKBP12.6, and either WT RyR2 or S2808D. These results indicate that localized {alpha}1CYM-RyR2 signaling during EC coupling is also unaltered by the PKA phosphomimetic S2808D mutation in RyR2.

FKBP12.6 binding to S2808D could occur under conditions in which FKBP12.6 is overexpressed to a level that overwhelms a shift in Kd induced by the mutation (41). We investigated this possibility by carefully titrating the levels of FKBP12.6 cDNA required to regulate a fixed level of WT RyR2 or S2808D. In these experiments, if the S2808D mutation significantly reduced the affinity of RyR2 for FKBP12.6, then an increased incidence of spontaneous Ca2+ oscillations at threshold levels of FKBP12.6 expression should have been observed. However, although the data in Fig. 4C document a clear and strong correlation between the concentration of FKBP12.6 cDNA and the incidence of Ca2+ oscillations, no statistical difference was observed between the incidence of spontaneous Ca2+ oscillations in WT RyR2- and S2808D-expressing myotubes at any concentration of FKBP12.6. Thus, although PKA hyperphosphorylation of RyR2 may indeed lead to FKBP12.6 dissociation during heart failure, our data strongly argue against any role of phosphorylation of S2808 in this process. Interestingly, although PKA and Ca2+/calmodulin-dependent protein kinase (CaMK)II phosphorylate two other serine residues in RyR2 (S2030 and S2815, respectively), phosphorylation of these residues does not significantly alter FKBP12.6 binding to RyR2 (41, 43).

The model described herein provides a novel means for probing fundamental questions of localized {alpha}1C-RyR2 signaling. For example, experiments could characterize the influence of other proteins thought to regulate cardiac DHPR and RyR2 function, including calmodulin (46), sorcin (32), DHPR {beta}2-subunits (35), and CaMKII (41). Alternatively, the Ca2+ activation site(s) in RyR2 stimulated by Ca2+ influx through the DHPR could also be examined. In this regard, it is interesting to note that the magnitude of Ca2+ influx-induced Ca2+ release in our study is three- to fourfold larger than that reported for {alpha}1C-mediated Ca2+ release through RyR1 (11, 18). This observation suggests that the in situ Ca2+ sensitivity of RyR2 is better suited to supporting CICR than RyR1. Consistent with this idea, biochemical studies have shown that the Ca2+ dependence of RyR2 activation/inactivation is broader than that of RyR1 (47). Conceivably, chimeric RyR1-RyR2 constructs could reveal potential in situ Ca2+ activation/inactivation sites that differentially tune RyR1 and RyR2 to activation by rapid elevations in junctional Ca2+ (27). The molecular model of local {alpha}1CYM-RyR2 signaling described herein provides a powerful tool to identify and/or test the structural determinants of these and other such mechanisms.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-44657 (to R. T. Dirksen), and research grants from the American Heart Association (to R. T. Dirksen), the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada (to S. R. W. Chen, who is a Senior Scholar of the Alberta Heritage Foundation for Medical Research), and a Predoctoral Fellowship (to S. Goonasekera) from the New York State Affiliate of the American Heart Association.


    ACKNOWLEDGMENTS
 
We thank Dr. Paul D. Allen for providing access to the dyspedic mice used in this study and Linda Groom for excellent technical assistance. We also thank Drs. Kurt G. Beam and Manfred Grabner for providing the dihydropyridine-insensitive {alpha}1C (T1066Y/Q1070M) construct and Dr. David C. Sheridan for assistance with curve fitting the voltage dependence of global intracellular Ca2+ transients.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. T. Dirksen, Dept. of Pharmacology and Physiology, Univ. of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642 (e-mail: robert_dirksen{at}urmc.rochester.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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