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
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ABSTRACT |
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heart failure; dihydropyridine receptor; excitation-contraction coupling
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) 1C-subunit of the cardiac DHPR (
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
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
1C-RyR2 junctional coupling in dyspedic myotubes. Our results confirm that indeed local Ca2+ signaling between
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
1C-RyR2 signaling.
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MATERIALS AND METHODS |
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RESULTS |
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In contrast to that observed for myotubes expressing only 1CYM and RyR2, coexpression of FKBP12.6 with
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
1C/RyR2- and
1CYM/RyR2/FKBP12.6-expressing myotubes, respectively) and maximum caffeine-induced Ca2+ release (peak
F405/485 was 1.14 ± 0.04 and 1.15 ± 0.05 in
1CYM/RyR2- and
1CYM/RyR2/FKBP12.6-expressing myotubes, respectively) were not different between the two populations of cells.
Extracellular field stimulation of 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
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
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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Confirmation of close functional coupling between expressed 1CYM and RyR2.
Although prior studies demonstrated efficient junctional targeting of
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
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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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 [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 1CYM/RyR2-expressing myotubes (Fig. 4C). However, the incidence of spontaneous Ca2+ oscillations in
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.
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DISCUSSION |
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Several lines of evidence support the conclusion that the voltage-gated Ca2+ transients observed with this model arise from close spatial coupling between junctional 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
1CYM/RyR2/FKBP12.6-expressing myotubes is fourfold greater than that observed in myotubes expressing
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
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 1CYM, FKBP12.6, and either WT RyR2 or S2808D. These results indicate that localized
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 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
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
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
1CYM-RyR2 signaling described herein provides a powerful tool to identify and/or test the structural determinants of these and other such mechanisms.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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