© 2001 American Heart Association, Inc.
Editorials |
Cardiac Ryanodine Receptors and Accessory Proteins: Augmented Expression Does Not Necessarily Mean Big Function
From the Department of Physiology, University of Wisconsin Medical School, Madison, Wis.
Correspondence to Héctor H. Valdivia, MD, PhD, Department of Physiology, University of Wisconsin Medical School, 1300 University Ave, Madison, WI 53706. E-mail valdivia{at}physiology.wisc.edu
Key Words: FKBP12 • excitation-contraction coupling • Ca2+-induced Ca2+ release • sarcoplasmic reticulum • immunophilin
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Introduction |
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 Top Introduction References |
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In cardiac cells, depolarization of the external membrane and its infoldings, the T-tubules, opens voltage-sensitive Ca2+ channels/dihydropyridine receptors (DHPRs), allowing a small influx of extracellular Ca2+ (the inward Ca2+ current, ICa). In mature cardiomyocytes, ICa is insufficient to elevate myoplasmic [Ca2+] to fully contracting levels; however, ICa triggers the opening of Ca2+ release channels/ryanodine receptors (RyRs), which produce a rapid and massive release of Ca2+ from the sarcoplasmic reticulum (SR). This amplification process, termed Ca2+-induced Ca2+ release (CICR),1 may be graded by endogenous effectors, hormones, and neurotransmitters to regulate the intensity and duration of ventricular contractions.2
RyRs are homotetramers of more than 2 megadaltons endowed with more than a fair share of structural elements to produce a bona fide ion channel. They contain a high-conductance Ca2+-selective pore, Ca2+ activation and inactivation sites, several phosphorylation sites, and multiple binding sites for a myriad of endogenous regulators that include ATP, Mg2+, and calmodulin.3 4 Still, as if this huge structural assembly were not sufficiently complex, RyRs are also capable of protein-protein interactions that allow them to bind, in some cases steadily and in other cases in a time- and Ca2+-dependent manner, to small and independently regulated accessory proteins.5 Therefore, although the RyR homotetramer with its intrinsic regulatory domains seems to be the central processor of effector signals, its association with cytosolic (FKBP12, sorcin, and calmodulin) and lumenal (calsequestrin, junctin, and triadin) proteins seems to add another layer of versatility (and complexity) to modulation of CICR in the heart. But how extensive is the level of involvement of these accessory proteins in CICR? Are they indispensable components of Ca2+ release during excitation-contraction coupling? If not, do they play a role in other processes of the cell?
Probably for no other accessory protein of RyRs are these questions more pressing than for the immunophilin FKBP12 (FK506-binding protein 12 kDa). Originally identified in T cells as the cytosolic receptor for immunosuppressant drugs,6 FKBP12 is a cis-trans peptidyl-prolyl isomerase that may bind to RyRs with a stoichiometry of 1(RyR protomer):1(FKBP12) or 4 FKBP12s per single functional channel. The RyR-FKBP12 complex is so stable that proteolytic fragments of purified RyR often appear to be contaminated with fragments of FKBP12,7 and the immunophilin may be used in affinity columns to purify RyRs.8 Furthermore, inositol trisphosphate receptors (IP3Rs), another major type of intracellular Ca2+ release channels, also bind FKBP12 with high affinity.9 Because IP3Rs play no major role in excitation-contraction coupling of striated muscle, this would suggest that FKBP12 might also be involved in interfacing Ca2+-signal amplification pathways unrelated to CICR.
Despite the ubiquitous presence of FKBP12 in a variety of cells and its demonstrated association to RyRs, its role as a regulator of CICR remains controversial. Although it is clear that the immunosuppressant FK506 or rapamycin removes FKBP12 from skeletal RyRs (RyR1) or FKBP12.6 (a protein isoform) from cardiac RyRs (RyR2),10 11 there is disagreement regarding the functional consequences of such removal. Several studies have found that FKBP12.6-devoid RyRs reconstituted in lipid bilayers exhibit long-lived subconductance states with high frequency and tend to dwell in the open state for longer periods than control RyRs.12 13 Both effects led to the proposal that the immunophilin serves to stabilize RyRs in the closed conformational state.11 However, other studies14 15 have found that removal of FKBP12.6 has no significant effect on the Ca2+ dependency or gating behavior of cardiac RyRs. Different interpretations have also resulted from the effect of immunosuppressants on intact ventricular myocytes. Xiao et al13 found that FK506 increased the frequency and duration of Ca2+ sparks, consistent with the role for FKBP12 postulated above, but other studies failed to detect alterations in the Ca2+ transient16 or in the SR Ca2+ content17 by similar concentrations of FK506. Instead, McCall et al17 detected predominant effects of the drug on Na+-Ca2+ exchanger activity and duBell et al16 on K+ currents of the sarcolemma.
Additional roles for FKBP12 have been proposed, and new arguments have ensued. Marx et al18 proposed that FKBP12 induces coupled gating of skeletal RyRs, whereby 2 or more individual RyRs open and close simultaneously to enhance Ca2+ release in response to a given voltage stimulus. Surprisingly, transgenic mice with disrupted expression of FKBP12 showed no functional or structural alterations in the skeletal muscle.19 Instead, major alterations were detected in the heart of the transgenic mice, which had RyRs with high incidence of long-lived subconductance states. The latter results were also puzzling, because cardiac RyRs presumably associate with FKBP12.6 only,14 15 an isoform of FKBP12 not altered in the transgenic mice. More recently, Marx et al20 postulated that phosphorylation of cardiac RyRs by protein kinase A dissociates FKBP12.6 and induces long-lived subconductance states, resulting in increased Ca2+ leak from the SR. However, previous studies21 22 did not find alterations in subconductance states of protein kinase A–phosphorylated RyRs. Although it is simplistic to directly compare results obtained with different experimental techniques, some of these apparent discrepancies remain as fundamental problems, and new studies are needed to clarify the role of the immunophilins in striated muscle in general and in CICR in particular.
In this issue of Circulation Research, Prestle et al23 describe an alternative approach to shed light on the role of the immunophilins on cardiac RyR function: overexpression of FKBP12.6. Using adenovirus-mediated gene transfer, cultured ventricular myocytes were forced to express up to 6 times the normal levels of FKBP12.6 to compare cell contraction, Ca2+ uptake rates, and their response to caffeine with the same parameters obtained in mock-infected cardiomyocytes. At variance with other approaches that promote or disrupt gene expression, adenovirus gene transfer occurs relatively fast and allows little time for changes in the expression of compensatory proteins. In this model, there were no detectable changes in FKBP12 or RyR levels,23 and the former was particularly significant for two reasons. First, experiments were conducted on cells with identical FKBP12 background. A direct comparison of control and FKBP12.6-overexpressing cells was therefore valid, because although in vitro experiments indicate that cardiac RyRs are poor substrates for FKBP12,14 15 binding of FKBP12 to cardiac RyRs in intact cells has not been ruled out. Second, some experiments involved the use of rapamycin, which blunts the effects of FKBP12 and FKBP12.6 with equal effectiveness.6 If different levels of FKBP12 had been observed, a potential indirect effect of FKBP12 on the measured parameters would have complicated the interpretation of the results. A major downside of the approach is that in an effort to reveal the function of a given protein, cells are forced to express large quantities of the same that might, in fact, generate an artificial function. Realistically, the experiments of Prestle et al23 reveal what FKBP12.6 is capable of doing more than what it actually does in a normal cell environment. Even then, interesting conclusions could be extracted.
The first striking observation of Prestle et al23 is that the expression of FKBP12.6 assessed at the protein (Western blot) and RNA (reverse transcriptase–polymerase chain reaction) levels in control, noninfected cells is significantly lower than that of FKBP12 and perhaps insufficient to bind to RyRs with the proposed ratio of 4(FKBP12.6):1(RyR tetramer). Under these conditions, it might be possible that FKBP12 compensates for the low levels of FKBP12.6 and binds to cardiac RyRs despite the demonstrated adversity of the reaction.14 15 However, a functional assay of immunophilin effect on RyRs used in the study showed that rapamycin, which effectively blocks FKBP12 and FKBP12.6, only marginally decreased the RyR-mediated Ca2+ efflux rate in control cells but significantly increased the same in FKBP12.6-overexpressing cells. Thus, it seems that cardiac RyRs are indeed selective targets of FKBP12.6 but that their low quantity in cardiac cells draws out a barely detectable effect, at least in this aspect of RyR function. Again, with risk of oversimplification, these results tend to suggest that major roles for the immunophilins in cardiomyocytes reside in cellular aspects other than CICR.
There are results, however, in the study by Prestle et al23 that suggest that overexpression of FKBP12.6 does affect CICR if we assume that all effects of the immunophilin are attributable to regulation of RyR function. For example, the size of cell contractions was increased in FKBP12.6-overexpressing cells, suggesting that SR Ca2+ release was increased. Interestingly, the kinetics of cell contractions were not increased. Time to peak and time to 50% relengthening of cell shortenings were actually slowed or unmodified by FKBP12.6. Again, circumscribing the effects of FKBP12.6 to RyRs and barring its potential effects on other proteins of excitation-contraction coupling, the bigger but slower contractions of FKBP12.6-overexpressing cells are in agreement with the notion that a slow component of, but not the initial, RyR-mediated Ca2+ release efflux was increased by the immunophilin. However, even in such a case, the cellular effects of FKBP12.6 cannot be reconciled with those obtained with single RyR channels without invoking the involvement of additional regulatory mechanisms. In the above scheme, FKBP12.6 could be presented as a promoter of RyR openings that sustains longer than normal SR Ca2+ efflux, whereas in lipid bilayer experiments, FKBP12.6 is seen as a promoter of RyR closings, because FKBP12.6-devoid RyRs tend to dwell longer in the open state on stimulation by transient Ca2+ stimuli.13 In the cellular environment, this effect would translate as longer, sustained SR Ca2+ efflux on activation of RyRs by ICa, just as proposed to explain the outsized contractions of FKBP12.6-overexpressing cells. Prestle et al23 cleverly address this apparent discrepancy by measuring SR Ca2+ uptake rates and observing that RyRs normally leak Ca2+ in a resting cell and that overexpression of FKBP12.6 decreases the rate of Ca2+ leak. The major effect of FKBP12.6 overexpression would be, therefore, an increase in SR Ca2+ content with subsequent larger Ca2+ release. Overall, Prestle et al23 favor the idea, like others before them,11 12 13 14 that FKBP12.6 stabilizes the cardiac RyR in the closed conformational state.
Where does this leave us? The results of Prestle et
al23 with their novel
approach advance the notion that FKBP12.6 regulates cardiac RyR
function during excitation-contraction coupling, although not with the
strength predicted by experiments with isolated RyRs or fragmented
cells.11 12 13 14
Let us not lose sight of the fact that Prestle et
al23 detected only modest
effects in experiments that blocked the native population of
immunophilins in cardiac cells. It was not until cardiac cells were
forced to overexpress FKBP12.6 that the effects of the immunophilin
became more apparent. Thus, the majority of their results may be
interpreted as a gain of function for RyRs, not the unveiling of a
normal RyR function. Timerman et
al14 estimated that 
17%
of the total FKBP12.6-binding sites of dog cardiac SR were unoccupied.
If that were the case in rabbit cardiomyocytes (the cells used by
Prestle et al23 ), there
would seem to be few RyRs for new FKBP12.6 molecules to interact with.
However, initial control experiments by Prestle et
al23 showed that FKBP12.6
was hardly expressed in rabbit cardiomyocytes, and overexpression would
therefore seem justified to reveal a function. The challenge ahead is
to determine the normal levels of FKBP12.6 across species and during
different stages of cell development to determine their contribution to
CICR and other aspects of cell
function.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health and National Heart, Lung and Blood Institute (HL55438 and HL47053). H.H.V. is an Established Investigator of the American Heart Association.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionReferences |
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