Cardiovascular Research

Cardiovascular Research Advance Access first published online on August 21, 2007
This version [Corrected Proof] published online on October 3, 2007
Cardiovascular Research, doi:10.1093/cvr/cvm006

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 77/2/302    most recent cvm006v2 cvm006v1 E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Google Scholar Articles by George, C. H. Search for Related Content PubMed PubMed Citation Articles by George, C. H. Social Bookmarking          What's this?

Sarcoplasmic reticulum Ca²⁺ leak in heart failure: mere observation or functional relevance?

Christopher H. George

Wales Heart Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK

Corresponding author. Tel: +44 2920 744431; fax: +44 2920 743500. E-mail address: georgech{at}cf.ac.uk

Time for primary review: 26 days

	Abstract

Top Abstract 1. Introduction 2. Dissecting the role... 3. RyR phosphorylation and... 4. The arrhythmogenic... 5. Linking intracellular Ca2+... 6. Linking Ca2+ leak... 7. Therapeutic strategies to... 8. Summary Funding References

 
Heart failure (HF) is a chronic multi-factorial disease characterized by sarcoplasmic reticulum (SR) dysfunction that manifests as severely reduced contractility and increased risk of arrhythmia. Several lines of evidence have revealed the existence of defective ryanodine receptor (RyR2)-mediated Ca²⁺ leak in HF, although its relevance as a causative factor rather than a phenotypic consequence of the disease is questioned. This review will consider the relative contribution of RyR2-mediated Ca²⁺ leak to the profound cellular, transcriptional and electrical remodelling associated with HF. In particular, it will focus on our current understanding of the role of defective phosphorylation of RyR2 as a both a chronic mediator of excitation–contraction coupling (ECC) dysfunction and as a potent catalyst of RyR2-dependent arrhythmogenesis. A hypothetical concept that SR Ca²⁺ leak fundamentally underlies the increased arrhythmogenic susceptibility in HF, but that it may not directly contribute to contractile dysfunction, which may involve maladaptive perturbations in metabolism and energy utilization, is also discussed.

KEYWORDS Sarcoplasmic reticulum; Ryanodine receptor; Calcium handling; Arrhythmia

Received May 22, 2007; revised July 13, 2007; accepted July 29, 2007

	1. Introduction

Top Abstract 1. Introduction 2. Dissecting the role... 3. RyR phosphorylation and... 4. The arrhythmogenic... 5. Linking intracellular Ca2+... 6. Linking Ca2+ leak... 7. Therapeutic strategies to... 8. Summary Funding References

 
The sarcoplasmic reticulum (SR) is a key integrator of excitation–contraction coupling (ECC), transducing the intracellular milieu to synchronize the spatio-temporal dynamics of Ca²⁺ release.¹ Normal cardiac cell function critically depends on a spectrum of graded Ca²⁺ release varying from the massive Ca²⁺ efflux that underpins muscle contraction to small highly localized Ca²⁺ release events that drive cardiac gene transcription and cellular signalling.² Spatially confined, low amplitude Ca²⁺ release events, visualized as Ca²⁺ sparks,³ contribute to the phenomenon known as Ca²⁺ leak^1,4 (the ubiquitously used term Ca²⁺ ‘leak’ originates from experiments in which the increased SR Ca²⁺ load following inhibition of basal levels of Ca²⁺ efflux was consistent with the concept of a constitutive ‘leak’ in cardiomyocytes.⁴ However, the term ‘leak’ is somewhat misleading since Ca²⁺ does not simply drain away from the SR). Normal Ca²⁺ leak links sarcoplasmic events to downstream signalling processes such as gene transcription (via a process termed excitation–transcription coupling)⁵ and post-translational protein modification and is a critical determinant of cardiomyocyte viability.^6–11 Distinct from its effects on cell signalling, basal Ca²⁺ leak plays a vital role in enhancing the local regenerativity and reliability of Ca²⁺-induced Ca²⁺ release (CICR)^12,13 and may act as a protective mechanism that limits Ca²⁺ overload during higher inotropic states.¹⁴

Consequently, the crucial role of Ca²⁺ fluxes in diverse aspects of cardiac function necessitates the exquisite co-ordination of Ca²⁺ channels, pumps, and exchangers. Moreover, reciprocal feedback mechanisms exist such that Ca²⁺ fluxes modulate the cardiac Ca²⁺ handling machinery via post-translational modifications of ion channels⁷ or altered gene transcription.⁸

Heart failure (HF) is a chronic maladaptive state in which complex transcriptional, proteomic, and morphological changes result in profound perturbations in intracellular Ca²⁺ cycling that drive the progressive deterioration in cardiac function.^1,15 A hallmark feature of HF pathogenesis is impaired SR function that predominantly manifests as reduced SR Ca²⁺ load via decreased SR Ca²⁺-ATPase (SERCA) activity.^1,15,16 However, in addition to reduced SR Ca²⁺ sequestration, increased Ca²⁺ leak from the SR mediated by defective regulation of ryanodine receptors (RyR2), vast tetrameric Ca²⁺ release channels,¹⁷ has emerged as an important mechanistic paradigm in Ca²⁺ handling dysfunction in HF.^18–23 However, the pathophysiological role of Ca²⁺ leak as a primary cause of altered gene transcription, cell remodelling, and contractile dysfunction associated with HF, particularly when considered against a cellular context of the huge, rapid Ca²⁺ fluctuations that occur during every cardiac cycle, has been questioned.

This review will consider whether abnormal RyR2-dependent Ca²⁺ leak directly contributes to HF pathogenesis, or whether it merely represents a phenotypic manifestation of the disease. The link between abnormal Ca²⁺ leak and altered gene transcription in HF is rapidly developing,^6,8,24,25 but aspects of the mechanisms require further elucidation that will not be considered here. Here, the focus is on the pathophysiological relevance of Ca²⁺ leak to two facets of HF phenotype, namely increased arrhythmogenicity and contractile dysfunction. In particular, the existence of chronically remodelled ECC that occurs as a consequence of maladaptive mechanisms is reconciled with the development of a pro-arrhythmic substrate which is remarkably prone to acute perturbation. Numerous studies showing exacerbated Ca²⁺ leak following pathogenic ß-AR stimulation implicates defective channel phosphorylation as a central mechanism in both chronic and acute facets of HF phenotype. Our current understanding of the molecular consequences of aberrant cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 in ECC remodelling (chronic) and arrhythmogenesis (acute) are summarized.

	2. Dissecting the role of localized RyR2 phosphorylation as the catalyst for pathological Ca2+ leak

Top Abstract 1. Introduction 2. Dissecting the role... 3. RyR phosphorylation and... 4. The arrhythmogenic... 5. Linking intracellular Ca2+... 6. Linking Ca2+ leak... 7. Therapeutic strategies to... 8. Summary Funding References

 
Normal cellular Ca²⁺ homeostasis and co-ordinated ion flux is maintained by the balanced activities of ECC machinery¹ and the stimulation of ECC, for example following ß-adrenoceptor (ß-AR) activation, results in the synchronous increase in heart rate and contractile force (the positive force–frequency relationship, or the Bowditch phenomenon) via co-ordinated post-translational modifications of L-type Ca²⁺ channel (LTCC), NCX, RyR2, and phospholamban (PLB). Phosphorylation is the most important post-translational mechanism for synchronizing the Ca²⁺ handling machinery and defective phosphorylation has long been viewed as a culprit of ECC dysfunction in cardiac disease.^1,26,27 Phosphorylation acts as a tunable ‘volume’ switch to regulate other ion channels,²⁸ and it dynamically tunes RyR2 to its ambient Ca²⁺ environment.^29,30 Exquisitely localized phosphorylation of RyR2 is enabled by a scaffold of regulatory co-proteins (termed the macromolecular complex), including PKA, CaMKII, and protein phosphatases 1 and 2a (PP1, PP2A)^31,32 that facilitates the transduction of global cellular signals at the level of individual channels. Consequently, structure-function defects in the macromolecular complex are implicated in cardiac disease,^31,32 and in particular, abnormal channel phosphorylation has emerged as a catalyst of augmented Ca²⁺ leak and is often regarded as an index of pathophysiological channel behaviour. Accordingly, much attention has focused on dissecting the precise contribution of RyR2-associated kinases to channel dysfunction.

Originally described as the site for CaMKII phosphorylation,³³ Ser2808 has since risen to prominence as the major PKA substrate in HF pathogenesis.³⁴ However, Ser2808 is a target for multiple kinases^35,36 and its constitutive high-level phosphorylation in both normal and failing hearts³⁷ suggests that the phosphorylation status of Ser2808 is not a reliable index of abnormal PKA-mediated phosphorylation of RyR2 in HF. Thus, although arguments for PKA-mediated phosphorylation in RyR2 channel dysfunction are compelling,^34,38,39 particularly when considered in the context of ß-AR stimulation (via the ‘fight-or-flight’ mechanism), the (patho)physiological consequences of PKA phosphorylation at Ser2808 remain hugely controversial.^35,40–43 Recently, an additional PKA phosphorylation epitope (Ser2031) has been proposed as a more robust marker of dynamic channel phosphorylation in HF,^35,44 although more work is needed to reconcile phosphorylation at Ser2031with channel dysfunction. Recently, CaMKII phosphorylation of RyR2 (at Ser 2814,⁴⁵ but also at Ser2808) has emerged as an important trigger of abnormal Ca²⁺ leak in HF^{20,21,23,46,47} and defects in the ability of CaMKII to decipher Ca²⁺ fluxes and modulate multiple facets of ECC has led to its candidature as an ‘arrhythmogenic kinase’.^9,10,48

The current molecular understanding of RyR2 phosphorylation from a chronological perspective is summarized in Table 1. However, despite the remarkable molecular insights into it, many aspects of RyR2 phosphorylation in vivo remain a mystery. ß-AR activation stimulates both PKA and CaMKII events in vivo⁴⁹ yet despite a common stimulatory pathway, cardiomyocytes functionally discriminate between the downstream effects of PKA and CaMKII phosphorylation, resulting in an increased rate and force of Ca²⁺ release, respectively.⁵⁰ It remains to be determined whether distinct subsets of RyR2 are phosphorylated by distinct kinases in vivo i.e. discrete populations of PKA vs. CaMKII phosphorylated channels. However, discrete RyR2 populations exhibiting distinct levels of phosphorylation may exist in vivo,⁵¹ and channels that appeared entirely normal (around 25% of total RyR2) existed against a cellular background of profoundly altered RyR2 phosphorylation in human HF.³⁴

View this table: [in this window] [in a new window]   Table 1 Ryanodine receptor phosphorylation from a chronological perspective

 
The phosphorylation status of RyR2 reflects the dynamic balance between protein kinases and phosphatases and thus disease-linked RyR2 ‘hyperphosphorylation’ may if fact arise from decreased activity of channel-associated phosphatases (PP1 and 2A) in addition to the more common perception of kinase ‘hyper-activity’. Conversely, channel hypophosphorylation has been reported to promote Ca²⁺ leak^52–55 and this apparent paradox may be explained by data showing that increased or decreased phosphorylation from the lowest point in RyR2 activation profile (that occurs at a level equivalent to phosphorylation at 75% full-stoichiometry) activated the channel via distinct mechanisms.³⁷

It is striking that the mechanistic complexity highlighted in Table 1 stems from analysis of RyR2 phosphorylation at just three experimentally identified sites (Ser2808, Ser2031, and Ser2814). However, additional phosphorylation epitopes for PKA, CaMKII, cGMP-, and Ca²⁺ dependent protein kinase (PKG and PKC respectively) are predicted (Figure 1A) but their validity as bona fide phosphorylation epitopes requires future evaluation. To date, there is experimental evidence for multiple CaMKII sites^56–59 although their molecular identity remains to be established.

View larger version (44K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1 Modulation of RyR2 by phosphorylation. (A) RyR2 phosphorylation sites at Ser2031, Ser2808, and Ser2814 have been determined experimentally (black symbols) (Table 1). Other epitopes for RyR2 phosphorylation by PKA, CaMKII, PKG, and PKC were predicted using hidden Markov models [KinasePhos, http://kinasephos.mbc.nctu.edu.tw/ (accessed July 2007)] and a cut-off score>–4.0. Grey symbols represent predictions based on 100% confidence. Nineteen additional consensuses CaMKII phosphorylation epitopes were obtained using 95% confidence levels (unfilled symbols, dotted line) and epitopes that are predicted to be substrates for both CaMKII and PKA at 95% confidence are shown (unfilled symbols, solid line). PKC is predicted to exclusively phosphorylate RyR2 at Thr residues. Ser2031 was predicted as a CaMKII phosphorylation epitope, and not a PKA substrate using this algorithm. All numbering is based on the human sequence. (B) The putative localization of functional domains within RyR2 has been mapped onto the 3D structure by cryo-electron microscopy and image analysis. CPVTI and II domains are loci for arrhythmia-linked mutations and DR represent regions of sequence diversity between RyR isoforms. The proposed site of FKBP12.6 interaction is shown (pink). The image of RyR2 is modified from116 used with permission.

 
The folded 3D architecture of RyR2 is complex and many regions that are physically separated in the linear polypeptide coalesce into close proximity in the folded molecule.²⁶ Analysis of RyR domain structure (Figure 1B) indicates that phosphorylation at its N-terminus (predominantly implicating CaMKII, Figure 1A) may occur at sites that are physically separate from those in central or C-terminal regions. The functional consequences of spatially segregated phosphorylation of RyR2 remain to be investigated, but the accessibility of some phosphoepitopes may be dictated by spatial constraints and steric hinderance in vivo i.e. some sites are only accessible in the activated or inactive channels.^33,37

	3. RyR phosphorylation and irreconcilable experimental differences: time for a divorce from traditional methods?

Top Abstract 1. Introduction 2. Dissecting the role... 3. RyR phosphorylation and... 4. The arrhythmogenic... 5. Linking intracellular Ca2+... 6. Linking Ca2+ leak... 7. Therapeutic strategies to... 8. Summary Funding References

 
There has been a remarkable evolution in our understanding of RyR2 phosphorylation since the prescient speculation by Seiler and colleagues (even before the molecular characterization of RyR2) that ‘... selective phosphorylation of the SR feet proteins could modulate Ca²⁺ uptake and Ca²⁺ release at the junctional region of the SR’⁶⁰ (Table 1). Despite this, some of the fundamental issues surrounding the role of RyR2 phosphorylation remain to be unequivocally resolved. Moreover, recent conceptual developments have been obscured by persistent controversy, and despite several attempts to provide rational (and sometimes not so rational) arguments to reconcile these data,^40,43,61,62 the only consistent agreement is that phosphorylation of RyR2 does something.

Acknowledging the difficulties in mimicking all pathogenic aspects of human HF in experimental models,⁶² it is emerging that the sheer diversity of experimental strategies that are currently employed to solve the mysteries of RyR2 dysfunction may be contributing to the discrepant observations. It is difficult to reconcile all aspects of data obtained from different species and techniques including [³H]-ryanodine binding assays, microsomal Ca²⁺ leak assays, protein–protein interactions and single channel analysis, and current in vitro analyses may be incompatible with investigating channel functionality attributable to intracellular compartmentalization, or the susceptibility of the material under investigation to artefact following isolation from cells and tissues. The potential limitations of current experimental approaches have been strikingly demonstrated by the finding that the sensitivity of some widely used phosphoepitope antibodies may be influenced by the local protein environment (e.g. phosphorylation status of other residues)³⁶ and that such artefacts may have blurred crucial observations relating to RyR2 regulation by phosphorylation (Table 1).

It is therefore proposed that conventional approaches are unable to sufficiently resolve the subtleties of cardiomyocyte signalling in vivo and the following example from Yang et al.¹⁴ exemplifies this argument. Experimental evidence suggests that there are at least eight CaMKII sites per monomer, given the identification of two distinct PKA sites per RyR2 subunit and the accepted CaMKII:PKA ratio of at least four CaMKII sites to every PKA site.^56–58 This gives 2⁸ (256) patterns of CaMKII phosphorylation per monomer and an astonishing 10¹⁰ permutations for the tetramer, which when taken together with the finding that phosphorylation of specific residues within the RyR2 polypeptide may dictate its functional effects,³³ highlighting a potentially staggering functional plasticity of RyR2 modulation that could arise from phosphorylation. Consequently, attempting to resolve the precise roles of RyR2 phosphorylation using current strategies that reveal ‘all-or-nothing’ effects result in a critical undervaluing of the complexity and functional consequences of dynamic phenomena occurring in vivo. Thus, the comprehensive evaluation of RyR2 phosphorylation in the context of feedback mechanisms, localized signalling environments, and compensatory adaptation requires the development of novel research tools and strategies, e.g. non-invasive imaging tools that exquisitely report their native cellular environment.^63–66 However, some limitations of in vivo approaches should be acknowledged.^67,68 The extraordinary utility of genetic approaches to selectively modulate ECC to re-create relevant aspects of HF phenotype has been tempered by reports that the resultant cardiac phenotype in transgenic RyR2 models of HF may be dependent on the genetic background of animals used.^38,39,69,70

	4. The arrhythmogenic consequences of failing to reset the ionic equilibrium

Top Abstract 1. Introduction 2. Dissecting the role... 3. RyR phosphorylation and... 4. The arrhythmogenic... 5. Linking intracellular Ca2+... 6. Linking Ca2+ leak... 7. Therapeutic strategies to... 8. Summary Funding References

 
Normally, the synchronized interactions within ECC result in a stable, enhanced functional state in response to cardiac stimulation (the transition between A and B states in Figure 2). As discussed in Section 1, progressive deterioration of cardiac function in HF is consistent with the chronic remodelling processes that have a transcriptional basis and/or occur via persistent changes in post-translational modifications of the ECC machinery (e.g. sustained defects in RyR2 regulation as a consequence of macromolecular complex disruption)^31,32,34,71. Thus, given the functional interdependence of ECC components, the disease-linked abnormality of a single ECC component acutely perturbs the normal ionic equilibria resulting in an imbalanced state that is profoundly arrhythmogenic (Figure 2C). Figure 2 illustrates this concept from the perspective of abnormal RyR2-dependent leak, but it is implicit that perturbations in SERCA, NCX, or LTCC would also result in Ca²⁺ flux imbalance. Under most circumstances, and despite the persistence of a perturbed component, this destabilized state exists only transiently since adaptive ‘re-tuning’ of other ECC components, via a process termed auto-regulation,^72,73 normalizes disrupted Ca²⁺ fluxes and restores steady-state Ca²⁺ handling (Figure 2D). Although auto-regulation represents a powerful corrective mechanism that can negate persistent Ca²⁺ leak, it should be noted that some modes of RyR2 activation bypass auto-regulation and lead to persistent changes in contractile function (e.g. low doses of ryanodine and dantrolene). Particularly, in the context of HF progression, it has been proposed that acquired defects in the structural stability of the channel that result in a sustained Ca²⁺ leak that is not subject to auto-regulation, represents a prevalent mechanistic basis of RyR2 dysfunction.^74–76 Thus acknowledging that HF pathogenesis is linked to a multitude of long-term signalling maladaptations, it is suggested that HF may be considered, at least in part, as a disease of auto-regulation.

View larger version (66K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2 The development of unstable equilibria in HF- the mechanistic basis of increased arrhythmogenic risk. (A) A simplified schematic diagram in which Ca2+ homeostasis is maintained by the balanced activities of L-type Ca2+ channels (LTCC) and NCX, and between RyR2 and SERCA. (B) Normal cardiac activation proportionately increases LTCC, NCX, RyR2, and SERCA function to maintain synchronized Ca2+ fluxes resulting in stable, enhanced state. (C) Acute perturbation of a single ECC component relative to the others (e.g. RyR2) results in the ablation of synchronised Ca2+ fluxes and directly increases the arrhythmogenic propensity of the system. (D) Adaptation of the excitation–contraction coupling machinery rapidly abrogates this unstable state, resulting in a new steady-state equilibrium. Notably, this state, illustrated using the experimentally observed findings of increased RyR2 leak and NCX activity and diminished SERCA activity,77 is inherently more prone to destabilization and is referred to here as a ‘pseudo-stable’ state.

 
So how does a background of chronic ECC remodelling constitute the basis for the markedly pro-arrhythmic substrate that develops in HF? The answer may lie in the nature of the ‘re-tuned’ state that arises from ECC dysfunction, depicted in Figure 2D as originating from RyR2 Ca²⁺ leak and reduced SERCA-mediated sequestration offset by enhanced NCX activity (according to current mechanistic models)^1,16,77. Despite this new functional interaction within ECC appearing stable, and its capacity to preserve homeostatic ion fluxes, it is underscored by a fundamentally different mode of functional interaction between the ECC machinery relative to the basal ‘normal’ state and as such, it is profoundly unstable. Recent studies that focused on perturbing RyR2 activity provided strong support for this concept and showed that the ‘pseudo-stable’ state (Figure 2D), that occurs as a result of adaptive changes to ECC, is exquisitely susceptible to subsequent arrhythmogenic perturbation via an additional trigger (e.g. ß-AR stimulation).^78–80 In these experiments, the demonstration that ß-AR activation promoted the rapid destabilization of the ‘pseudo-stable’ state suggests a role for acute post-translational modification of ECC, and is entirely consistent with the emergence of RyR2 phosphorylation as a major arrhythmogenic mechanism.

The concept that chronic defects in single components of ECC gives rise to a chronically reset equilibrium that underpins an inherently unstable pro-arrhythmic substrate is demonstrated by the stress- or exercise-triggered nature of human arrhythmias associated with genetic mutations in calsequestrin (CSQ),⁸¹ RyR2,²⁶ or LTCC,⁸² and in animal models of cardiac dysfunction resulting from targeted genetic manipulations of single ECC components (e.g. CSQ,⁸³ triadin,⁸⁴ FKBP12.6 (calstabin 2),³⁸ and RyR2)⁸⁵. Although these are not models of HF, it is striking that in human and animal models of HF characterized by multiple chronic alterations in ECC, the function of some components of ECC are disproportionately perturbed e.g. RyR2.^{20,23,34,44,61}

	5. Linking intracellular Ca2+ leak to multi-cellular electrical dysfunction

Top Abstract 1. Introduction 2. Dissecting the role... 3. RyR phosphorylation and... 4. The arrhythmogenic... 5. Linking intracellular Ca2+... 6. Linking Ca2+ leak... 7. Therapeutic strategies to... 8. Summary Funding References

 
Arrhythmias ultimately result from disrupted myocardial electrical synchrony and so important questions are (i) how does Ca²⁺ release dysfunction manifest as an electrical abnormality, and (ii) how is cellular electrical instability propagated to a multi-cellular event? It is well established that triggered arrhythmias [e.g. delayed- or early-afterdepolarizations (DADs and EADs, respectively)], a common basis for arrhythmia in HF, are evoked as aberrant NCX-mediated inward currents in response to elevated SR Ca²⁺ leak.^{77,78,80,86,87} However, increased ß-AR stimulation that is a common feature of HF,⁸⁸ in addition to increasing the activities of intracellular Ca²⁺ handling machinery (e.g. RyR2, SERCA), would also modulate the activities of plasmalemmal ion channels thereby increasing the likelihood of their contribution to cellular electrical instability. To this end, abnormal sarcolemmal ion fluxes that sustain the resultant arrhythmia are not exclusively mediated by NCX, and it has been shown that LTCC fluxes are also involved in arrhythmogenesis.^86,89–91 Furthermore, the link between Ca²⁺ release dysfunction and inward-rectifying K⁺ channel (I_K1)-mediated alterations in surface membrane potential provides a new paradigm for altered membrane excitability in HF.⁹¹ Thus, although triggered arrhythmias originate from abnormal intracellular Ca²⁺ leak, they are ultimately generated by factors extrinsic to the SR.

Regarding how the cellular electrical instability is amplified into multi-cellular propagation through the myocardium, there is a clear role for disrupted current propagation via defective gap junction (cell-to-cell) coupling.⁹² Paradoxically, although the cellular basis of triggered arrhythmias occurs via plasmalemmal ion channels and exchangers, the amplification of cellular electrical dysfunction into multicellular arrhythmias may be crucially dependent on SR Ca²⁺ leak since gap junctional communication is exquisitely sensitive to the intracellular Ca²⁺ environment.⁹³

	6. Linking Ca2+ leak to cardiac metabolic crisis: a speculative hypothesis to explain contractile dysfunction in HF

Top Abstract 1. Introduction 2. Dissecting the role... 3. RyR phosphorylation and... 4. The arrhythmogenic... 5. Linking intracellular Ca2+... 6. Linking Ca2+ leak... 7. Therapeutic strategies to... 8. Summary Funding References

 
The concepts discussed above suggest a central role for Ca²⁺ leak in the increased arrhythmogenic risk in HF, but to what extent does Ca²⁺ leak contribute to the other facet of HF phenotype, namely reduced contractility. The magnitude of Ca²⁺ efflux from the SR governs the contractile force, and the reduced efficiency of cardiac muscle contraction in HF is widely presumed to be underpinned by a diminished SR Ca²⁺ store. However, in experimental models of Ca²⁺ leak, and despite reduced SR Ca²⁺ load, steady-state Ca²⁺ transients were preserved and contractile force remained unchanged due to increased fractional release of Ca²⁺ from the SR.^{19–21,23,94} Thus, it is plausible that the ‘pseudo-stable’ state (Figure 2D) may not directly contribute to the contractile defects observed in HF. Some important clues as to the alternative mechanisms that may underscore the reduced contractility are being uncovered from the intriguing link between systolic dysfunction and altered cellular metabolism.

The pseudo-stable state (Figure 2D) places a significantly increased energy demand on the cell, predominantly via the futile attempts of SERCA to reload the ‘leaky’ Ca²⁺ SR store. The glycogenolytic pathway links ECC and intermediary metabolism and it is established that disrupted Ca²⁺-fluxes are associated with altered metabolism^95–100 and electrical perturbations.^89,101 Consequently, the chronic persistence of a Ca²⁺ flux imbalance and energy-intensive state (Figure 2D) may contribute to a gradual metabolic rundown that eventually results in an energy crisis (described as the ‘engine out of fuel’)¹⁰⁰. We showed that in cells chronically over-expressing human recombinant RyR2, increased RyR2-dependent Ca²⁺ leak was offset by increased SERCA activity such that steady-state cytoplasmic Ca²⁺ levels were preserved (Figure 3). However, the augmented Ca²⁺ fluxes and elevated SERCA activity were associated with impaired cell growth and a remarkable loss in cell viability (Figure 3).¹⁰² Thus, it is tentatively proposed that the relationship between increased RyR2-dependent Ca²⁺ leak, metabolic perturbation, and cell toxicity may partly explain the increased levels of cell death in HF.¹⁰³

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 3 The pathological implications of ‘re-tuned’ Ca2+ fluxes. The expression of recombinant human RyR2 in RyR2-null Chinese Hamster ovary (CHO) cell lines induced profound changes in steady-state Ca2+ handling (A). Unaltered cytoplasmic Ca2+ levels (B) masked a remarkable increase in intracellular Ca2+ fluxes that was quantified using noise analysis of cytoplasmic Ca2+ signals (C). Steady-state Ca2+ handling in response to RyR2 over-expression was maintained by concordant upregulation of SERCA activity (D), but this new equilibrium was associated with pronounced cytotoxicity (E). Data are modified from ref. 102,117.

 
A surprising finding is that ATP bioavailability sufficient to maintain contractile function is preserved even in severe HF,¹⁰⁰ and it has been proposed that contractile defects probably arise from alterations in substrate utilization, high-energy phosphate metabolism (creatine kinase energy shuttle), and oxidative phosphorylation.¹⁰⁰ Mitochondria match cardiac energy supply and demand and thus Ca²⁺- linked defects in mitochondrial function^104,105 and gene expression^96,106 may be critical mediators of metabolic dysfunction in HF. However, the notion that Ca²⁺ release dysfunction is a cause rather than a consequence of metabolic crisis is contradicted by the development of cardiac phenotypes that bear striking resemblances to human HF, including reduced contractile reserve and hypertrophy, in mice lacking key regulators of metabolism.^100,107

	7. Therapeutic strategies to normalize Ca2+ leak- not all Ca2+ ‘leak’ is bad

Top Abstract 1. Introduction 2. Dissecting the role... 3. RyR phosphorylation and... 4. The arrhythmogenic... 5. Linking intracellular Ca2+... 6. Linking Ca2+ leak... 7. Therapeutic strategies to... 8. Summary Funding References

 
Novel strategies aimed at normalizing Ca²⁺ leak in HF have been reviewed recently^67,71,108 and will not be extensively considered here. However, a key consideration is that the functional synergism of ECC components predicts that the complete elimination of Ca²⁺ fluxes would be catastrophic and as a result, emergent strategies are focused on modulating rather than blocking Ca²⁺ leak. Furthermore, considering the negative outcomes from strategies centred on discrete ion channel targets,¹⁰⁹ it is not surprising that some of the more promising strategies are based on targeting multiple components of ECC. A notable example that fulfils the above criteria is K201 (JTV519), a broad-spectrum ion channel modulator that suppresses arrhythmias and restores normal cardiac function.^26,110,111 We have previously speculated that honing the molecular specificity of K201 for a specific target (e.g. RyR2) may reduce its therapeutic potential.¹¹² Consistent with the predicted efficacy of drugs that modulate diverse facets of ECC, there has been a resurgent interest in adapting pleiotropic pharmacology to prevent Ca²⁺ leak that has extended to the use of ß-AR blockers,¹¹³ anti-oxidants,¹¹⁴ and angiotensin receptor antagonists,¹¹⁵ although the molecular basis of their actions needs to be evaluated in robust cell-based experimental models.

	8. Summary

Top Abstract 1. Introduction 2. Dissecting the role... 3. RyR phosphorylation and... 4. The arrhythmogenic... 5. Linking intracellular Ca2+... 6. Linking Ca2+ leak... 7. Therapeutic strategies to... 8. Summary Funding References

 
SR Ca²⁺ leak is an important biological phenomenon that fundamentally contributes to cardiomyocyte function and under normal circumstances is strictly regulated by the co-ordinated activities of Ca²⁺ pumps, channels, and exchangers. This review has considered mechanisms by which abnormal regulation of RyR2 (predominantly by phosphorylation) underscores the aberrant SR Ca²⁺ leak which directly contributes to ECC dysfunction in both chronic and acute facets of HF phenotype. The progressive deterioration of cardiac function in HF occurs, in part, as a consequence of abnormal Ca²⁺ leak that is initially compensated for by powerful regulatory mechanisms, but over the course of many years and against a background of transcriptional and other cellular alterations these innate regulatory mechanisms fail. Here, a hypothesis is proposed in which the pathological retuning of ECC in HF provides a plausible mechanistic basis for the development of a remarkably pro-arrhythmogenic substrate.

However, Ca²⁺ leak may not directly contribute to all phenotypic manifestations of HF. It is speculated that increased Ca²⁺ leak may indirectly cause reduced cardiac muscle contractility as a consequence of chronically increased energy demands that ultimately result in an energy crisis. Although there is robust evidence to support this concept, the precise mechanisms linking Ca²⁺ leak to metabolic dysfunction requires further experimental testing. Strategies that concentrate on modulating rather than blocking RyR2-dependent Ca²⁺ leak are emerging as attractive therapeutic approaches to tackle HF, but it is crucial that the rational design of new approaches are based on a full understanding of the molecular basis and consequences of RyR2-dependent Ca²⁺ leak in vivo.

	Funding

Top Abstract 1. Introduction 2. Dissecting the role... 3. RyR phosphorylation and... 4. The arrhythmogenic... 5. Linking intracellular Ca2+... 6. Linking Ca2+ leak... 7. Therapeutic strategies to... 8. Summary Funding References

 
CHG gratefully acknowledges the financial support of the British Heart Foundation (BS/04/02,FS/04/088,FS/06/082), the Royal Society (2004/R2) and Cardiff University.

Conflict of interest: none declared.

	Acknowledgements

 
I am indebted to Donald M. Bers and Sabine Huke for invaluable discussion and sharing data.

	References

Top Abstract 1. Introduction 2. Dissecting the role... 3. RyR phosphorylation and... 4. The arrhythmogenic... 5. Linking intracellular Ca2+... 6. Linking Ca2+ leak... 7. Therapeutic strategies to... 8. Summary Funding References

 

1. Bers DM. Excitation-contraction coupling and contractile force (2001) 2nd ed. Kluwer Academic Publishers.
2. Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol (2000) 1:11–21.[CrossRef][ISI][Medline]
3. Cheng H, Lederer WJ, Cannell MB. Calcium sparks—elementary events underlying excitation-contraction coupling in heart-muscle. Science (1993) 262:740–744.[Abstract/Free Full Text]
4. Shannon TR, Ginsburg KS, Bers DM. Quantitative assessment of the SR Ca²⁺ leak-load relationship. Circ Res (2002) 91:594–600.[Abstract/Free Full Text]
5. Atar D, Backx PH, Appel MM, Gao WD, Marban E. Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels. J Biol Chem (1995) 270:2473–2477.[Abstract/Free Full Text]
6. Frey N, McKinsey TA, Olson EN. Decoding calcium signals involved in cardiac growth and function. Nat Med (2000) 6:1221–1227.[CrossRef][ISI][Medline]
7. Zucchi R, Ghelardoni S, Carnicelli V, Frascarelli S, Ronca F, Ronca-Testoni S. Ca²⁺ channel remodeling in perfused heart: effects of mechanical work and interventions affecting Ca²⁺ cycling on sarcolemmal and sarcoplasmic reticulum Ca²⁺ channels. FASEB J 2002. 16:1976–1978.
8. Wu X, Zhang T, Bossuyt J, Li X, McKinsey TA, Dedman JR, et al. Local InsP3-dependent perinuclear Ca²⁺ signaling in cardiac myocyte excitation-transcription coupling. J Clin Invest (2006) 116:675–682.[CrossRef][ISI][Medline]
9. McKinsey TA. Derepression of pathological cardiac genes by members of the CaM kinase superfamily. Cardiovasc Res (2007) 73:667–677.[Abstract/Free Full Text]
10. Anderson ME. Multiple downstream proarrhythmic targets for calmodulin kinase II: moving beyond an ion channel-centric focus. Cardiovasc Res (2007) 73:657–666.[Abstract/Free Full Text]
11. George CH, Rogers SA, Bertrand BMA, Tunwell REA, Thomas NL, Steele DS, et al. Alternative splicing of ryanodine receptors modulates cardiomyocyte Ca²⁺ signaling and susceptibility to apoptosis. Circ Res (2007) 100:874–883.[Abstract/Free Full Text]
12. Keller M, Kao JPY, Egger M, Niggli E. Calcium waves driven by sensitization wave-fronts. Cardiovasc Res (2007) 74:39–45.[Abstract/Free Full Text]
13. Lindegger N, Niggli E. Paradoxical SR Ca²⁺ release in guinea-pig cardiac myocytes after ß-adrenergic stimulation revealed by two-photon photolysis of caged Ca²⁺. J Physiol (2005) 565:801–813.[Abstract/Free Full Text]
14. Yang D, Zhu WZ, Xiao B, Brochet DXP, Chen SRW, Lakatta EG, et al. Ca²⁺/Calmodulin kinase II-dependent phosphorylation of ryanodine receptors suppresses Ca²⁺ sparks and Ca²⁺ waves in cardiac myocytes. Circ Res (2007) 100:399–407.[Abstract/Free Full Text]
15. Houser SR, Piacentino V, Weisser J. Abnormalities of calcium cycling in the hypertrophied and failing heart. J Mol Cell Cardiol (2000) 32:1595–1607.[CrossRef][ISI][Medline]
16. Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhythmogenesis and contractile dysfunction in heart failure: roles of sodium-calcium exchange, inward rectifier potassium current, and residual beta-adrenergic responsiveness. Circ Res (2001) 88:1159–1167.[Abstract/Free Full Text]
17. George CH, Yin CC, Lai FA. Toward a molecular understanding of the structure: function of ryanodine receptor Ca²⁺ release channels: perspectives from recombinant expression systems. Cell Biochem Biophys (2005) 42:197–222.[CrossRef][ISI][Medline]
18. Shannon TR, Pogwizd SM, Bers DM. Elevated sarcoplasmic reticulum Ca²⁺ leak in intact ventricular myocytes from rabbits in heart failure. Circ Res (2003) 93:592–594.[Abstract/Free Full Text]
19. Maier LS, Zhang T, Chen L, DeSantiago J, Brown JH, Bers DM. Transgenic CaMKIIdeltaC overexpression uniquely alters cardiac myocyte Ca²⁺ handling. Circ Res (2003) 92:904–911.[Abstract/Free Full Text]
20. Ai X, Curran JW, Shannon TR, Bers DM, Pogwizd SM. Ca²⁺/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation sarcoplasmic reticulum Ca²⁺ leak in heart failure. Circ Res (2005) 97:1314–1322.[Abstract/Free Full Text]
21. Kohlhaas M, Zhang T, Seidler T, Zibrova D, Dybkova N, Steen A, et al. Increased sarcoplasmic reticulum calcium leak but unaltered contractility by acute CaMKII overexpression in isolated rabbit cardiac myocytes. Circ Res (2006) 98:235–244.[Abstract/Free Full Text]
22. Lehnart SE, Terrenoire C, Reiken S, Wehrens XHT, Song LS, Tillman EJ, et al. Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias. Proc Natl Acad Sci USA (2006) 103:7906–7910.[Abstract/Free Full Text]
23. Curran J, Hinton MJ, Rios E, Bers DM, Shannon TR. B-adrenergic enhancement of sarcoplasmic reticulum calcium leak in cardiac myocytes is mediated by calcium/calmodulin-dependent protein kinase. Circ Res (2007) 100:391–398.[Abstract/Free Full Text]
24. Molkentin JD. Dichotomy of Ca²⁺ in the heart: contraction versus intracellular signalling. J Clin Invest (2006) 116:623–626.[CrossRef][ISI][Medline]
25. McKinsey TA, Zhang CL, Olson EN. MEF2: a calcium dependent regulator of cell division, differentiation and death. Trends Biochem Sci (2002) 27:40–47.[CrossRef][ISI][Medline]
26. George CH, Jundi H, Thomas NL, Fry DL, Lai FA. Ryanodine receptors and ventricular arrhythmias: emerging trends in mutations, mechanisms and therapies. J Mol Cell Cardiol (2007) 42:34–50.[CrossRef][ISI][Medline]
27. Marks AR, Reiken SR, Marx SO. Progression of heart failure: is protein kinase A hyperphosphorylation of the ryanodine receptor a contributing factor. Circulation (2002) 105:272–275.[Free Full Text]
28. Park K-S, Mohaptra DP, Misonou H, Trimmer JS. Graded regulation of the Kv2.1 potassium channel by variable phosphorylation. Science (2006) 313:976–979.[Abstract/Free Full Text]
29. Valdivia HH, Kaplan JH, Ellis-Davies GCR, Lederer WJ. Rapid adaptation of cardiac ryanodine receptors: modulation by Mg²⁺ and phosphorylation. Science (1995) 267:1997–2000.[Abstract/Free Full Text]
30. Sobie EA, Guatimosim S, Gomez-Viquez L, Song LS, Hartmann H, Jafri MS, et al. The Ca²⁺ leak paradox and ‘rogue ryanodine receptor’: SR Ca²⁺ efflux theory and practice. Prog Biophys Mol Biol (2006) 90:172–185.[CrossRef][ISI][Medline]
31. Marks AR, Marx SO, Reiken S. Regulation of ryanodine receptors via macromolecular complexes: a novel role for leucine/isoleucine zippers. Trends Cardiovasc Med (2002) 12:166–170.[CrossRef][ISI][Medline]
32. Bers DM. Macromolecular complex regulating cardiac ryanodine receptor function. J Mol Cell Cardiol (2004) 37:417–429.[CrossRef][ISI][Medline]
33. Witcher DR, Kovacs RJ, Schulman H, Cefali DC, Jones LR. Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity. J Biol Chem (1991) 266:11144–11152.[Abstract/Free Full Text]
34. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell (2000) 101:365–376.[CrossRef][ISI][Medline]
35. Xiao B, Zhong G, Obayashi M, Yang D, Chen K, Walsh MP, et al. Ser-2030 but not Ser-2808, is the major phosphorylation site in cardiac ryanodine receptors responding to protein kinase A activation upon B-adrenergic stimulation in normal and failing hearts. Biochem J (2006) 396:7–16.[CrossRef][ISI][Medline]
36. Huke S, Bers DM. Ryanodine receptor phosphorylation at Serine 2030, 2808 and 2814 in intact rat cardiomyocytes. Biophys J (2007) (Suppl. S):132a.
37. Carter S, Colyer J, Sitsapesan R. Maximum phosphorylation of the cardiac ryanodine receptor at Ser-2809 by protein kinase A produces unique modifications to channel gating and conductance not observed at lower levels of phosphorylation. Circ Res (2006) 98:1506–1513.[Abstract/Free Full Text]
38. Wehrens XHT, Lehnart SE, Huang F, Vest JA, Reiken SR, Mohler PJ, et al. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell (2003) 113:829–840.[CrossRef][ISI][Medline]
39. Wehrens XHT, Lehnart SE, Reiken S, Vest JA, Wronska A, Marks AR. Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression. Proc Natl Acad Sci USA (2006) 103:511–518.[Abstract/Free Full Text]
40. Bers DM, Eisner DA, Valdivia HH. Sarcoplasmic reticulum Ca²⁺ and heart failure. Roles of diastolic leak and Ca²⁺ transport. Circ Res (2003) 93:487–490.[Free Full Text]
41. Jiang MT, Lokuta AJ, Farrell EF, Wolff MR, Haworth RA, Valdivia HH. Abnormal Ca²⁺ release, but normal ryanodine receptors, in canine and human heart failure. Circ Res (2002) 91:1015–1022.[Abstract/Free Full Text]
42. Li Y, Kranias EG, Mignery GA, Bers DM. Protein kinase A phosphorylation of the ryanodine receptor does not affect Ca²⁺ sparks in mouse ventricular myocytes. Circ Res (2002) 90:309–316.[Abstract/Free Full Text]
43. Bers DM. Cardiac ryanodine receptor phosphorylation: target sites and functional consequences. Biochem J (2006) 396:e1–e3.[CrossRef][Medline]
44. Xiao B, Jiang MT, Zhao M, Yang D, Sutherland C, Lai FA, et al. Characterization of a novel PKA phosphorylation site, serine-2030, reveals no PKA hyperphosphorylation of the cardiac ryanodine receptor in canine heart failure. Circ Res (2005) 96:847–855.[Abstract/Free Full Text]
45. Wehrens XHT, Lehnart SE, Reiken SR, Marks AR. Ca²⁺/calmodulin-dependent protein kinase II phosphorylation regulates the cardiac ryanodine receptor. Circ Res (2004) 94:e61–e70.[Abstract/Free Full Text]
46. Guo T, Zhang T, Mestril R, Bers DM. Ca/Calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes. Circ Res (2006) 99:398–406.[Abstract/Free Full Text]
47. Maier LS, Bers DM. Role of Ca²⁺/calmodulin-dependent protein kinase (CaMKII) in excitation-contraction coupling in the heart. Cardiovasc Res (2007) 73:631–640.[Abstract/Free Full Text]
48. Anderson ME. Calmodulin kinase signaling in the heart: an intriguing candidate target for therapy of myocardial dysfunction and arrhythmias. Pharm Ther (2005) 106:39–55.[CrossRef][ISI][Medline]
49. Zheng M, Zhu W, Han Q, Xiao RP. Emerging concepts and therapeutic implications of B-adrenergic receptor subtype signaling. Pharm Ther (2005) 108:257–268.[CrossRef][ISI][Medline]
50. Ginsburg KS, Bers DM. Modulation of excitation-contraction coupling by isoproterenol in cardiomyocytes with controlled SR Ca²⁺ load and Ca²⁺ current trigger. J Physiol (2004) 556:463–480.[Abstract/Free Full Text]
51. Hain J, Onoue H, Mayrleitner M, Fleischer S, Schindler H. Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from cardiac muscle. J Biol Chem (1995) 270:2074–2081.[Abstract/Free Full Text]
52. Terentyev D, Viatchenko-Karpinski S, Gyorke I, Terentyeva R, Gyorke I. Protein phosphatases decreases sarcoplasmic reticulum calcium content by stimulating calcium release in cardiac myocytes. J Physiol (2003) 552:109–118.[Abstract/Free Full Text]
53. Yamada M, Ikeda Y, Yano M, Yoshimura K, Nishino S, Aoyama H, et al. Inhibition of protein phosphatase 1 by inhibitor-2 gene delivery ameliorates heart failure progression in genetic cardiomyopathy. FASEB J (2006) 20:E346–E356.
54. Carr AN, Schmidt AG, Suzuki Y, del Monte F, Sato Y, Lanner C, et al. Type 1 phosphatase, a negative regulator of cardiac function. Mol Cell Biol (2002) 22:4124–4135.[Abstract/Free Full Text]
55. Neumann J, Eschenhagen T, Jones LR, Linck B, Schmitz W, Scholz H, et al. Increased expression of cardiac phosphatases in patients with end-stage heart failure. J Mol Cell Cardiol (1997) 29:265–272.[CrossRef][ISI][Medline]
56. Takasago T, Imagawa T, Furukawa K-I, Ogurusu T, Shigekawa M. Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation. J Biochem (1991) 109:163–170.[Abstract/Free Full Text]
57. Rodriguez P, Bhogal MS, Colyer J. Stoichiometric phosphorylation of cardiac ryanodine receptor on serine 2809 by calmodulin-dependent kinase II and protein kinase A. J Biol Chem (2003) 278:38593–38600.[Abstract/Free Full Text]
58. Stange M, Xu L, Balshaw D, Yamaguchi N, Meissner G. Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants. J Biol Chem (2003) 278:51693–51702.[Abstract/Free Full Text]
59. Zhong G-F, Xiao B-L, Chen SRW. Molecular basis and functional significance of CaMKII phosphorylation of the cardiac calcium release channel (ryanodine receptor, RyR2). Biophys J (2006) (Suppl. S):390a.
60. Seiler S, Wegener AD, Whang DD, Hathaway DR, Jones LR. High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phosphorylated, and are degraded by Ca²⁺ activated protease. J Biol Chem (1984) 259:8550–8557.[Abstract/Free Full Text]
61. Reiken S, Gaburjakova M, Guatimosim S, Gomez AM, D'Armiento J, Burkhoff D, et al. Protein kinase A phosphorylation of the cardiac calcium release channel (ryanodine receptor) in normal and failing hearts: role of phosphatases and response to isoproterenol. J Biol Chem (2003) 278:444–453.[Abstract/Free Full Text]
62. Marks AR. A guide for the perplexed: towards an understanding of the molecular basis of heart failure. Circulation (2003) 107:1456–1459.[Free Full Text]
63. Violin JD, Zhang J, Tsien RY, Newton AC. A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol (2003) 161:899–909.[Abstract/Free Full Text]
64. Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, et al. Fluorescent indicators for Ca²⁺ based on green fluorescent proteins and calmodulin. Nature (1997) 388:882–887.[CrossRef][Medline]
65. Tallini Y, Ohkura M, Choi BR, Ji G, Imoto K, Doran R, et al. Imaging cellular signals in the heart in vivo: cardiac expression of the high-signal Ca²⁺ indicator GCaMP2. Proc Natl Acad Sci USA (2006) 103:4753–4758.[Abstract/Free Full Text]
66. George CH, Yeung WY, Walters N, Thomas NL, Claycomb WC, Lai FA. A novel cardiomyocyte FRET-based bioassay for investigating the molecular basis of RyR2 dysfunction in arrhythmogenesis. Biophys J (2006) (Suppl. S):264a.
67. Hoshijima M. Gene therapy targeted at calcium handling as an approach to the treatment of heart failure. Pharmacol Ther (2005) 105:211–218.[CrossRef][ISI][Medline]
68. Yutzey KE, Robbins J. Principles of genetic murine models for cardiac disease. Circulation (2007) 115:792–799.[Abstract/Free Full Text]
69. Xin HB, Senbonmatsu T, Cheng DS, Wang YX, Copello JA, Ji GJ, et al. Oestrogen protects FKBP12.6 null mice from cardiac hypertrophy. Nature (2002) 416:334–337.[CrossRef][Medline]
70. Benkusky NA, Weber CS, Scherman JA, Farrell EF, Hacker TA, John MC, et al. Intact ß-adrenergic response and unmodified progression toward heart failure in mice with genetic ablation of a major protein kinase A phosphorylation site in the cardiac ryanodine receptor. Circ Res (2007) (doi:10.1161/CIRCRESAHA.107.153007).
71. Wehrens XHT, Marks AR. Novel therapeutic approaches for heart failure by normalizing calcium cycling. Nat Rev Drug Disc (2004) 3:1–9.[CrossRef][ISI]
72. Trafford AW, Diaz ME, Sibbring GC, Eisner DA. Modulation of CICR has no maintained effect on systolic Ca²⁺: simultaneous measurements of sarcoplasmic reticulum and sarcolemmal Ca²⁺ fluxes in rat ventricular myocytes. J Physiol (2000) 522:259–270.[Abstract/Free Full Text]
73. Eisner DA, Choi HS, Diaz ME, O'Neill SC, Trafford AW. Integrative analysis of calcium cycling in cardiac muscle. Circ Res (2000) 87:1087–1094.[Abstract/Free Full Text]
74. Yang Z, Ikemoto N, Lamb G, Steele DS. The RyR2 central domain peptide DPc10 lowers the threshold for spontaneous Ca²⁺ release in permeabilized cardiomyocytes. Cardiovasc Res (2006) 70:475–485.[Abstract/Free Full Text]
75. Oda T, Yano M, Yamamoto T, Tokuhisa T, Okuda S, Doi M, et al. Defective regulation of interdomain interactions within the ryanodine receptor plays a key role in the pathogenesis of heart failure. Circulation (2005) 111:3400–3410.[Abstract/Free Full Text]
76. George CH, Jundi H, Thomas NL, Walters N, West RR, Lai FA. Arrhythmogenic mutation-linked defects in ryanodine receptor autoregulation reveal a novel mechanism of Ca²⁺ release channel dysfunction. Circ Res (2006) 98:88–97.[Abstract/Free Full Text]
77. Pogwizd SM, Bers DM. Cellular basis of triggered arrhythmias in heart failure. Trends Cardiovasc Med (2004) 14:61–66.[CrossRef][ISI][Medline]
78. Nam G-B, Burashnikov A, Antzelevitch C. Cellular mechanisms underlying the development of catecholaminergic ventricular tachycardia. Circulation (2005) 111:2727–2733.[Abstract/Free Full Text]
79. Venetucci LA, Trafford AW, Eisner DA. Increasing ryanodine receptor open probability alone does not produce arrhythmogenic calcium waves- threshold sarcoplasmic reticulum calcium content is required. Circ Res (2007) 100:105–111.[Abstract/Free Full Text]
80. Katra RP, Oya T, Hoek