CaM or cAMP
Linking β-Adrenergic Stimulation to ‘Leaky’ RyRs
- Ca-calmodulin kinase
- sarcoplasmic reticulum
- heart failure
- ryanodine receptor
- adrenergic receptor
See related article, pages 391–398
During each heart beat a transient rise in [Ca2+]i links the electrical signal to the actual contraction. Ca2+ release from the sarcoplasmic reticulum (SR) is the major source of this Ca2+ transient and occurs through the ryanodine receptor, RyR. The RyR is a Ca2+-activated channel, responding to Ca2+ influx, commonly referred to as the trigger Ca2+, predominantly through the L-type Ca2+ channel. The Ca2+ available in the SR is the SR Ca2+ content or Ca2+ load. The SR Ca2+ content depends to a large extent on the Ca2+ uptake into the SR by SERCA, regulated by phospholamban, and also on the balance of Ca2+ fluxes across the sarcolemma. For the latter, the Na+/Ca2+ exchanger is the major efflux pathway, generating an inward current. Alterations in [Ca2+]i handling have been implicated in contractile dysfunction in heart failure, and several proteins are involved such as SERCA, phospholamban and the Na+/Ca2+ exchanger, which would all influence the available Ca2+ in the SR.
When Marks et al reported in 2000 that hyperphosphorylation of the ryanodine receptor (RyR) could underlie abnormal calcium cycling in heart failure,1 it was the start of a new area of research that has since sparked a lot of debate. Increased phosphorylation was proposed to lead to reduced binding of the stabilizing protein, FKBP12.6 or calstabin, and result in increased channel openings or ‘leaky’ RyRs. This will lead to abnormal release of Ca2+ in diastole as well as abnormal gating during excitation-contraction coupling. The abnormal release in diastole could, by depleting the sarcoplasmic reticulum, also reduce systolic Ca2+ levels. At the same time, a diastolic ‘leak’ of Ca2+ could contribute to arrhythmias by activating an inward Na+/Ca2+ exchange current. In this framework, several aspects of RyR phosphorylation and its functional consequences were investigated and (hotly) debated. At the molecular and biochemical level the question was which site of the RyR was phosphorylated during adrenergic stimulation. The debate is still ongoing as to whether there is only 1 site, Ser2808/28091 or more, such as Ser20301,2 (and see3 for commentary). Not only cAMP-activated kinase, PKA, but also CaCalmodulin-activated kinase II, CaMKII, phosphorylates RyR.4–6 In addition to specific CaMKII sites there appears to be an interaction between the kinases on the same site.7 CaMKII activity in sarcoplasmic reticulum fractions was reported to be higher in the rabbit after myocardial infarction.8
Functional Consequences of RyR Phosphorylation
Whether heart failure increases the level of RyR phosphorylation remains controversial (see eg9), but this has not deterred investigators from examining the functional consequences of RyR phosphorylation. The difficulty of these studies lies in the multiple effects of β-adrenergic stimulation that confound the interpretation of the RyR effect proper. Indeed, β-adrenergic stimulation increases the L-type Ca2+ current and thus the trigger for RyR activation, and also increases the available Ca2+ in the sarcoplasmic reticulum because of the phospholamban phosphorylation and increased SERCA activity. These effects would by themselves increase open probability of the RyR. Trafford et al bypassed the adrenergic stimulus and simulated the functional consequences of RyR phosphorylation using a low dose of caffeine to slightly increase the open probability of RyR.10 They showed that, in isolation, increased RyR opening had no lasting effect on the [Ca2+]i transient. Bers and colleagues used the PLB KO mouse, permeabilized the cells and looked at the properties of RyR release events, seen as ‘sparks’, at a fixed level of activating Ca2+.11 In that model, neither changes in trigger Ca2+ nor in SR Ca2+ content interfere with the effect of PKA on RyR. Under those conditions no effect of PKA on RyR could be detected despite significant levels of phosphorylation. In another study in intact myocytes, adrenergic stimulation had only a minor effect on Ca2+ release if the increase in sarcoplasmic reticulum Ca2+ content and in Ca2+ current were taken into account.12 In contrast, when similar studies were done for CaMKII activation, the results were quite different as both the gain of Ca2+ release and the spontaneous sparks activity were increased.13,14 These results are actually challenged by another study in the current issue by Yang et al14b which is commented on by Yamaguchi and Meissner.14c
Although these studies examined the RyR properties, they did not directly measure the actual ‘leak’ that would cause the changes in SR Ca2+ content and potential arrhythmogenesis. A few years ago Shannon et al developed an elegant method to quantify the diastolic Ca2+ leak.15 In an intact cell that has been paced to steady state, caffeine is used to empty the SR and assess SR Ca2+ content; between the end of the pacing and the application of caffeine, the cell is kept at rest for 1 minute, in 0Ca2+/ 0Na+ solution to maintain SR content. This protocol is repeated but now, during the rest period before the caffeine application, tetracaine is briefly applied. This allows for 2 measurements. First, the application of tetracaine abruptly reduces [Ca2+]i, reflecting the block of SR Ca2+ leak, and this change is measured as such. Second, because of this reduced leak, the SR Ca2+ content, measured during the caffeine pulse, increases. Using this approach it has been shown that the leak through the RyR is increased in myocytes from heart failure animals16 and that this is probably because of CaMKII activation.17 This was consistent with data on increased RyR activity related to CaMKII18,19 and complemented data that CaMKII was part of the macromolecular complex of the RYR. It also fitted into a larger framework of CaMKII as an important player in heart failure and arrhythmias.20 However the missing link was the relationship between CaMKII activation and the hyperadrenergic state of heart failure; they could exist both independently or the increase in Ca2+ because of adrenergic stimulation could in itself be the stimulus for CaMKII. Venetucci et al recently showed that the propensity for diastolic Ca2+ leak strongly depends on a simultaneous increase in SR Ca2+ content.21 A combined activation of PKA and CaMKII would thus give the ideal setting of increased open probability of RyR and high Ca2+ load.
Molecular Mechanisms of Increased Leak
The present study of Curran et al22 examines the relation between increased Ca2+ load and increased RyR ‘leak’ under β-adrenergic stimulation. Using molecular and pharmacological tools, the role of PKA and CaMKII in β-adrenergic effects on load and leak were examined. In normal rabbit myocytes, the SR Ca2+ leak was quantified as described above, and this protocol was done for a range of SR Ca2+ loading conditions (obtained by varying the conditioning pacing protocol) to obtain a leak-to-load relation. The elegance of this approach is that it can examine the leak in physiological conditions, yet bypassing confounding effects of changes in trigger or uncontrolled changes in load. It was found that the relationship was shifted to the left by addition of isoproterenol (250 nmol/L); in other words, for the same SR load the leak was increased. The authors went on to investigate the underlying mechanisms. Block of CaMKII (with KN-93 or autocamtide-2 related inhibitory peptide) could suppress the leak induced by isoproterenol. In the chance that both CaMKII activation and leak proper would be consequent on the increase in cellular Ca2+ with PKA, the PKA blocker H89 was tested. Surprisingly, H-89 was unable to reverse the increase in leak despite block of ‘classic’ PKA effects such as the isoproterenol–induced increase in twitch [Ca2+]i transient amplitude and in rate of relaxation. This would suggest that it is the receptor stimulation, and not the consequent PKA activation or increase in Ca2+, which is directly responsible for the leak. Consistent with this hypothesis, forskolin which directly activates adenylate cyclase and bypasses the adrenergic receptor, could increase the [Ca2+]i transient amplitude and rate of relaxation, but not the leak.
The authors relate their finding to the earlier observations of a role for CaMKII in mediating β-adrenergic stimulation during a 24 hour exposure to noradrenaline.23 That study found that the long-term adrenergic effects on Ca2+ handling were very similar to the acute effects but that they were mediated not through PKA but through CaMKII. However, there is a caveat in this comparison. The time course of CaMKII activation was slow, with a time constant of 10 minutes, reaching its plateau only after an hour. In the present study22 the activation of CaMKII appears to be immediate. The link of the receptor to CaMKII also remains elusive. There seems to be no relation to the increase in Ca2+ mediated by cAMP, and this is consistent with the observations of Wang et al where activation of CaMKII occurred in unstimulated myocytes. Identifying this link will be an important topic for future study.
Relation Between Leak and Gain During Excitation-Contraction Coupling
Because the leak reflects an increased probability of opening of RyR, in a further analysis of Ca2+ fluxes during a single twitch, the authors examine the ‘gain’ of excitation-contraction coupling. Gain here is defined as the ratio of calculated SR Ca2+ release versus trigger Ca2+ influx via the L-type Ca2+ current. Consistent with earlier findings, the increase in this gain is mostly related to the increase in the available Ca2+ and larger SR Ca2+ content, with a small contribution of the increased sensitivity.
Quantification Equals Knowledge, and More Is Needed
The beauty of the current study is the precise quantification of the actual leak. Several studies have looked at the leak as an increase in spontaneous release events or sparks in diastole, but such an approach allows a comparative evaluation, not absolute quantification. Yet, there are some limitations to the present study as well. The protocol requires a long period of rest of ≈1 minute. After such a long rest period, the effects on changes in SR content, with or without leak are clear. However, in most conditions, the diastolic time intervals are short. This is particularly the case during adrenergic stimulation. In vivo, as we sometimes forget during cellular experiments in vitro, adrenergic stimulation increases heart rate and shortens dramatically the diastolic period, even to the extent of limiting ventricular filling. It would be of interest to know whether in fact there is an increase in loss of Ca2+ from the SR under those conditions.
The other issue that awaits quantification is the relation between this leak and arrhythmogenesis. It is assumed that the arrhythmogenic effect of increased leak is similar to what is known for waves of spontaneous Ca2+ release, which activate Na+/Ca2+ exchange current. The quantitative relation between leak, probability of occurrence of waves and amplitude of the Na+/Ca2+ exchange current is, however, unknown. Experiments performed earlier during caffeine-induced Ca2+ release defined a threshold for the size of Ca2+ release needed to depolarize the membrane for triggering an action potential.24 This approach may still underestimate the Ca2+ load and release needed for an arrhythmogenic release. During caffeine-induced Ca2+ release, the release is more or less synchronous, certainly in comparison to spontaneous release, which travels in a wave across the cell. In the latter case the mean exchanger current per time unit will be smaller. The exchange current associated with a leak, though still to be quantified, will be even less and is likely to provide a background current, rather than a triggering current. The impact of this current will need to be evaluated and quantified, in the light of the other changes in membrane currents, eg, IK1.24 It is interesting that reduction of IK1 may actually be the result of a diastolic Ca2+ leak.25
The Interplay Between SR Ca2+ Load and Leak
Another important aspect that comes of the quantification in this study is the steep relation between leak and increase in SR Ca2+ load. To have a significant leak, a substantial increase in SR load is required. Very recently, Venetucci et al, using a very different approach of increasing RyR gating with varying SR Ca2+ load, also clearly demonstrated the need for a substantial increase in SR load to have diastolic release.21 On the other hand, the leak itself reduces the SR Ca2+ load. As suggested in the present article, this would help prevent SR Ca2+ overload. Suppressing the leak may then not be unequivocally beneficial. Much will depend on the magnitude of the increase in SR content, and potential benefits of synchronizing systolic release.26 The initial enthusiasm of studies with drugs like JTV519 to reduce arrhythmias27 have been tempered,28 but with careful quantitative studies such as the present, we can expect to define better ways to target a RyR leak in treating heart failure or arrhythmias.
Sources of Funding
K.R.S. receives support from the FWO, the Fund for Scientific Research Flanders (G.0384.07), the Belgian Science Policy Fund (IAP07–36), and the 6th Framework Program of the European Union (LSHM-CT-2005–018833, EUGeneHeart).
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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