Massive Accumulation of Myofibroblasts in the Critical Isthmus Is Associated With Ventricular Tachycardia Inducibility in Post-Infarct Swine Heart.

OBJECTIVES: In this study the authors determined the extent of cellular infiltration and dispersion, and regional vascularization in electrophysiologically (EP) defined zones in post-myocardial infarction (MI) swine ventricle. BACKGROUND: The critical isthmus (CI) in post-MI re-entrant ventricular tachycardia (VT) is a target for catheter ablation. In vitro evidence suggests that myofibroblasts (MFB) within the scar border zone (BZ) may increase the susceptibility to slow conduction and VT, but whether this occurs in vivo remains unproven. METHODS: Six weeks after mid-left anterior descending coronary artery occlusion, EP catheter-based mapping was used to assess susceptibility to VT induction. EP data were correlated with detailed cellular profiling of ventricular zones using immunohistochemistry and spatial distribution analysis of cardiomyocytes, fibroblasts, MFB, and vascularization. RESULTS: In pigs with induced sustained monomorphic VT (mean cycle length: 353 ± 89 ms; n = 6) the area of scar that consisted of the BZ (i.e., between the normal and the low-voltage area identified by substrate mapping) was greater in VT-inducible hearts (iVT) than in noninducible hearts (non-VT) (p < 0.05). Scar in iVT hearts was characterized by MFB accumulation in the CI (>100 times that in normal myocardium and >5 times higher than that in the BZ in non-VT hearts) and by a 1.7-fold increase in blood vessel density within the dense scar region extending towards the CI. Sites of local abnormal ventricular activity potentials exhibited cellularity and vascularization that were intermediate to the CI in iVT and BZ in non-VT hearts. CONCLUSIONS: The authors reported the first cellular analysis of the VT CI following an EP-based zonal analysis of iVT and non-VT hearts in pigs post-MI. The data suggested that VT susceptibility was defined by a remarkable number of MFB in the VT CI, which appeared to bridge the few remaining dispersed clusters of cardiomyocytes. These findings define the cellular substrate for the proarrhythmic slow conduction pathway.

Unambiguous observation of blocked states reveals altered, blocker-induced, cardiac ryanodine receptor gating.

The flow of ions through membrane channels is precisely regulated by gates. The architecture and function of these elements have been studied extensively, shedding light on the mechanisms underlying gating. Recent investigations have focused on ion occupancy of the channel's selectivity filter and its ability to alter gating, with most studies involving prokaryotic K+ channels. Some studies used large quaternary ammonium blocker molecules to examine the effects of altered ionic flux on gating. However, the absence of blocking events that are visibly distinct from closing events in K+ channels makes unambiguous interpretation of data from single channel recordings difficult. In this study, the large K+ conductance of the RyR2 channel permits direct observation of blocking events as distinct subconductance states and for the first time demonstrates the differential effects of blocker molecules on channel gating. This experimental platform provides valuable insights into mechanisms of blocker-induced modulation of ion channel gating.

Calsequestrin interacts directly with the cardiac ryanodine receptor luminal domain.

Cardiac muscle contraction requires sarcoplasmic reticulum (SR) Ca2+ release mediated by the quaternary complex comprising the ryanodine receptor 2 (RyR2), calsequestrin 2 (CSQ2), junctin (encoded by ASPH) and triadin. Here, we demonstrate that a direct interaction exists between RyR2 and CSQ2. Topologically, CSQ2 binding occurs at the first luminal loop of RyR2. Co-expression of RyR2 and CSQ2 in a human cell line devoid of the other quaternary complex proteins results in altered Ca2+-release dynamics compared to cells expressing RyR2 only. These findings provide a new perspective for understanding the SR luminal Ca2+ sensor and its involvement in cardiac physiology and disease.

Structural insights into Ca(2+)-activated long-range allosteric channel gating of RyR1.

Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca(2+)-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 Å and a resolution of 4.2 Å for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca(2+) activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family.

Effect of flecainide derivatives on sarcoplasmic reticulum calcium release suggests a lack of direct action on the cardiac ryanodine receptor.

BACKGROUND AND PURPOSE: Flecainide is a use-dependent blocker of cardiac Na(+) channels. Mechanistic analysis of this block showed that the cationic form of flecainide enters the cytosolic vestibule of the open Na(+) channel. Flecainide is also effective in the treatment of catecholaminergic polymorphic ventricular tachycardia but, in this condition, its mechanism of action is contentious. We investigated how flecainide derivatives influence Ca(2) (+) -release from the sarcoplasmic reticulum through the ryanodine receptor channel (RyR2) and whether this correlates with their effectiveness as blockers of Na(+) and/or RyR2 channels. EXPERIMENTAL APPROACH: We compared the ability of fully charged (QX-FL) and neutral (NU-FL) derivatives of flecainide to block individual recombinant human RyR2 channels incorporated into planar phospholipid bilayers, and their effects on the properties of Ca(2) (+) sparks in intact adult rat cardiac myocytes. KEY RESULTS: Both QX-FL and NU-FL were partial blockers of the non-physiological cytosolic to luminal flux of cations through RyR2 channels but were significantly less effective than flecainide. None of the compounds influenced the physiologically relevant luminal to cytosol cation flux through RyR2 channels. Intracellular flecainide or QX-FL, but not NU-FL, reduced Ca(2) (+) spark frequency. CONCLUSIONS AND IMPLICATIONS: Given its inability to block physiologically relevant cation flux through RyR2 channels, and its lack of efficacy in blocking the cytosolic-to-luminal current, the effect of QX-FL on Ca(2) (+) sparks is likely, by analogy with flecainide, to result from Na(+) channel block. Our data reveal important differences in the interaction of flecainide with sites in the cytosolic vestibules of Na(+) and RyR2 channels.

Stable expression and functional characterisation of the diamondback moth ryanodine receptor G4946E variant conferring resistance to diamide insecticides.

Diamides, such as flubendiamide and chlorantraniliprole, belong to a new chemical class of insecticides that act as conformation-sensitive activators of insect ryanodine receptors (RyRs). Both compounds are registered for use against lepidopteran species such as the diamondback moth, Plutella xylostella, a notorious global pest of cruciferous crops. Recently acquired resistance to diamide insecticides in this species is thought to be due to a target-site mutation conferring an amino acid substitution (G4946E), located within the trans-membrane domain of the RyR, though the exact role of this mutation has not yet been fully determined. To address this we have cloned a full-length cDNA encoding the P. xylostella RyR and established clonal Sf9 cell lines stably expressing either the wildtype RyR or the G4946E variant, in order to test the sensitivity to flubendiamide and chlorantraniliprole on the recombinant receptor. We report that the efficacy of both diamides was dramatically reduced in clonal Sf9 cells stably expressing the G4946E modified RyR, providing clear functional evidence that the G4946E RyR mutation impairs diamide insecticide binding.

The mechanism of flecainide action in CPVT does not involve a direct effect on RyR2.

RATIONALE: Flecainide, a class 1c antiarrhythmic, has emerged as an effective therapy in preventing arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) refractory to ß-adrenergic receptor blockade. It has been proposed that the clinical efficacy of flecainide in CPVT is because of the combined actions of direct blockade of ryanodine receptors (RyR2) and Na(+) channel inhibition. However, there is presently no direct evidence to support the notion that flecainide blocks RyR2 Ca(2+) flux in the physiologically relevant (luminal-to-cytoplasmic) direction. The mechanism of flecainide action remains controversial. OBJECTIVE: To examine, in detail, the effect of flecainide on the human RyR2 channel and to establish whether the direct blockade of physiologically relevant RyR2 ion flow by the drug contributes to its therapeutic efficacy in the clinical management of CPVT. METHODS AND RESULTS: Using single-channel analysis, we show that, even at supraphysiological concentrations, flecainide did not inhibit the physiologically relevant, luminal-to-cytosolic flux of cations through the channel. Moreover, flecainide did not alter RyR2 channel gating and had negligible effect on the mechanisms responsible for the sarcoplasmic reticulum charge-compensating counter current. Using permeabilized cardiac myocytes to eliminate any contribution of plasmalemmal Na(+) channels to the observed actions of the drug at the cellular level, flecainide did not inhibit RyR2-dependent sarcoplasmic reticulum Ca(2+) release. CONCLUSIONS: The principal action of flecainide in CPVT is not via a direct interaction with RyR2. Our data support a model of flecainide action in which Na(+)-dependent modulation of intracellular Ca(2+) handling attenuates RyR2 dysfunction in CPVT.

A Systemized Approach to Investigate Ca(2+) Synchronization in Clusters of Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes.

Induced pluripotent stem cell-derived cardiomyocytes (IPS-CM) are considered by many to be the cornerstone of future approaches to repair the diseased heart. However, current methods for producing IPS-CM typically yield highly variable populations with low batch-to-batch reproducibility. The underlying reasons for this are not fully understood. Here we report on a systematized approach to investigate the effect of maturation in embryoid bodies (EB) vs. "on plate" culture on spontaneous activity and regional Ca(2+) synchronization in IPS-CM clusters. A detailed analysis of the temporal and spatial organization of Ca(2+) spikes in IPS-CM clusters revealed that the disaggregation of EBs between 0.5 and 2 weeks produced IPS-CM characterized by spontaneous beating and high levels of regional Ca(2+) synchronization. These phenomena were typically absent in IPS-CM obtained from older EBs (>2 weeks). The maintenance of all spontaneously active IPS-CM clusters under "on plate" culture conditions promoted the progressive reduction in regional Ca(2+) synchronization and the loss of spontaneous Ca(2+) spiking. Raising the extracellular [Ca(2+)] surrounding these quiescent IPS-CM clusters from ~0.4 to 1.8 mM unmasked discrete behaviors typified by either (a) long-lasting Ca(2+) elevation that returned to baseline or (b) persistent, large-amplitude Ca(2+) oscillations around an increased cytoplasmic [Ca(2+)]. The different responses of IPS-CM to elevated extracellular [Ca(2+)] could be traced back to their routes of derivation. The data point to the possibility of predictably influencing IPS-CM phenotype and response to external activation via defined interventions at early stages in their maturation.

Dantrolene rescues aberrant N-terminus intersubunit interactions in mutant pro-arrhythmic cardiac ryanodine receptors.

AIMS: The ryanodine receptor (RyR2) is an intracellular Ca(2+) release channel essential for cardiac excitation-contraction coupling. Abnormal RyR2 channel function results in the generation of arrhythmias and sudden cardiac death. The present study was undertaken to investigate the mechanistic basis of RyR2 dysfunction in inherited arrhythmogenic cardiac disease. METHODS AND RESULTS: We present several lines of complementary evidence, indicating that the arrhythmia-associated L433P mutation disrupts RyR2 N-terminus self-association. A combination of yeast two-hybrid, co-immunoprecipitation, and chemical cross-linking assays collectively demonstrate that a RyR2 N-terminal fragment carrying the L433P mutation displays substantially reduced self-interaction compared with wild type. Moreover, sucrose density gradient centrifugation reveals that the L433P mutation impairs tetramerization of the full-length channel. [(3)H]Ryanodine-binding assays demonstrate that disrupted N-terminal intersubunit interactions within RyR2(L433P) confer an altered sensitivity to Ca(2+) activation. Calcium imaging of RyR2(L433P)-expressing cells reveals substantially prolonged Ca(2+) transients and reduced Ca(2+) store content indicating defective channel closure. Importantly, dantrolene treatment reverses the L433P mutation-induced impairment and restores channel function. CONCLUSION: The N-terminus domain constitutes an important structural determinant for the functional oligomerization of RyR2. Our findings are consistent with defective N-terminus self-association as a molecular mechanism underlying RyR2 channel deregulation in inherited arrhythmogenic cardiac disease. Significantly, the therapeutic action of dantrolene may occur via the restoration of normal RyR2 N-terminal intersubunit interactions.

Insights into the gating mechanism of the ryanodine-modified human cardiac Ca2+-release channel (ryanodine receptor 2).

Ryanodine receptors (RyRs) are intracellular membrane channels playing key roles in many Ca(2+) signaling pathways and, as such, are emerging novel therapeutic and insecticidal targets. RyRs are so named because they bind the plant alkaloid ryanodine with high affinity and although it is established that ryanodine produces profound changes in all aspects of function, our understanding of the mechanisms underlying altered gating is minimal. We address this issue using detailed single-channel gating analysis, mathematical modeling, and energetic evaluation of state transitions establishing that, with ryanodine bound, the RyR pore adopts an extremely stable open conformation. We demonstrate that stability of this state is influenced by interaction of divalent cations with both activating and inhibitory cytosolic sites and, in the absence of activating Ca(2+), trans-membrane voltage. Comparison of the conformational stability of ryanodine- and Imperatoxin A-modified channels identifies significant differences in the mechanisms of action of these qualitatively similar ligands.

N-terminus oligomerization regulates the function of cardiac ryanodine receptors.

The ryanodine receptor (RyR) is an ion channel composed of four identical subunits mediating calcium efflux from the endo/sarcoplasmic reticulum of excitable and non-excitable cells. We present several lines of evidence indicating that the RyR2 N-terminus is capable of self-association. A combination of yeast two-hybrid screens, co-immunoprecipitation analysis, chemical crosslinking and gel filtration assays collectively demonstrate that a RyR2 N-terminal fragment possesses the intrinsic ability to oligomerize, enabling apparent tetramer formation. Interestingly, N-terminus tetramerization mediated by endogenous disulfide bond formation occurs in native RyR2, but notably not in RyR1. Disruption of N-terminal inter-subunit interactions within RyR2 results in dysregulation of channel activation at diastolic Ca(2+) concentrations from ryanodine binding and single channel measurements. Our findings suggest that the N-terminus interactions mediating tetramer assembly are involved in RyR channel closure, identifying a crucial role for this structural association in the dynamic regulation of intracellular Ca(2+) release.

Functional characterization of the cardiac ryanodine receptor pore-forming region.

Ryanodine receptors are homotetrameric intracellular calcium release channels. The efficiency of these channels is underpinned by exceptional rates of cation translocation through the open channel and this is achieved at the expense of the high degree of selectivity characteristic of many other types of channel. Crystallization of prokaryotic potassium channels has provided insights into the structures and mechanisms responsible for ion selection and movement in these channels, however no equivalent structural detail is currently available for ryanodine receptors. Nevertheless both molecular modeling and cryo-electron microscopy have identified the probable pore-forming region (PFR) of the ryanodine receptor (RyR) and suggest that this region contains structural elements equivalent to those of the PFRs of potassium-selective channels. The aim of the current study was to establish if the isolated putative cardiac RyR (RyR2) PFR could form a functional ion channel. We have expressed and purified the RyR2 PFR and shown that function is retained following reconstitution into planar phospholipid bilayers. Our data provide the first direct experimental evidence to support the proposal that the conduction pathway of RyR2 is formed by structural elements equivalent to those of the potassium channel PFR.

Investigations of the contribution of a putative glycine hinge to ryanodine receptor channel gating.

Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling, and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately, high-resolution structural data, such as exist for K(+)-selective channels, are not available for RyR. In the absence of these data, we have used modeling to identify similarities in the structural elements of K(+) channel pore-forming regions and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G(4864)LIIDA(4869) in RyR2) analogous to the glycine hinge motif present in many K(+) channels. Gating in these K(+) channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine 4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K(+) channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast, function is altered when glycine 4864 is replaced by either valine or proline, the former preventing channel opening and the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K(+) channels. Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.

Flecainide reduces Ca(2+) spark and wave frequency via inhibition of the sarcolemmal sodium current.

AIMS: Ca(2+) waves are thought to be important in the aetiology of ventricular tachyarrhythmias. There have been conflicting results regarding whether flecainide reduces Ca(2+) waves in isolated cardiomyocytes. We sought to confirm whether flecainide inhibits waves in the intact cardiomyocyte and to elucidate the mechanism. METHODS AND RESULTS: We imaged spontaneous sarcoplasmic reticulum (SR) Ca(2+) release events in healthy adult rat cardiomyocytes. Variation in stimulation frequency was used to produce Ca(2+) sparks or waves. Spark frequency, wave frequency, and wave velocity were reduced by flecainide in the absence of a reduction of SR Ca(2+) content. Inhibition of I(Na) via alternative pharmacological agents (tetrodotoxin, propafenone, or lidocaine) produced similar changes. To assess the contribution of I(Na) to spark and wave production, voltage clamping was used to activate contraction from holding potentials of -80 or -40 mV. This confirmed that reducing Na(+) influx during myocyte stimulation is sufficient to reduce waves and that flecainide only causes Ca(2+) wave reduction when I(Na) is active. It was found that Na(+)/Ca(2+)-exchanger (NCX)-mediated Ca(2+) efflux was significantly enhanced by flecainide and that the effects of flecainide on wave frequency could be reversed by reducing [Na(+)](o), suggesting an important downstream role for NCX function. CONCLUSION: Flecainide reduces spark and wave frequency in the intact rat cardiomyocyte at therapeutically relevant concentrations but the mechanism involves I(Na) reduction rather than direct ryanodine receptor (RyR2) inhibition. Reduced I(Na) results in increased Ca(2+) efflux via NCX across the sarcolemma, reducing Ca(2+) concentration in the vicinity of the RyR2.

The contribution of hydrophobic residues in the pore-forming region of the ryanodine receptor channel to block by large tetraalkylammonium cations and Shaker B inactivation peptides.

Although no high-resolution structural information is available for the ryanodine receptor (RyR) channel pore-forming region (PFR), molecular modeling has revealed broad structural similarities between this region and the equivalent region of K(+) channels. This study predicts that, as is the case in K(+) channels, RyR has a cytosolic vestibule lined with predominantly hydrophobic residues of transmembrane helices (TM10). In K(+) channels, this vestibule is the binding site for blocking tetraalkylammonium (TAA) cations and Shaker B inactivation peptides (ShBPs), which are stabilized by hydrophobic interactions involving specific residues of the lining helices. We have tested the hypothesis that the cytosolic vestibule of RyR fulfils a similar role and that TAAs and ShBPs are stabilized by hydrophobic interactions with residues of TM10. Both TAAs and ShBPs block RyR from the cytosolic side of the channel. By varying the composition of TAAs and ShBPs, we demonstrate that the affinity of both species is determined by their hydrophobicity, with variations reflecting alterations in the dissociation rate of the bound blockers. We investigated the role of TM10 residues of RyR by monitoring block by TAAs and ShBPs in channels in which the hydrophobicity of individual TM10 residues was lowered by alanine substitution. Although substitutions changed the kinetics of TAA interaction, they produced no significant changes in ShBP kinetics, indicating the absence of specific hydrophobic sites of interactions between RyR and these peptides. Our investigations (a) provide significant new information on both the mechanisms and structural components of the RyR PFR involved in block by TAAs and ShBPs, (b) highlight important differences in the mechanisms and structures determining TAA and ShBP block in RyR and K(+) channels, and (c) demonstrate that although the PFRs of these channels contain analogous structural components, significant differences in structure determine the distinct ion-handling properties of the two species of channel.

A mechanistic description of gating of the human cardiac ryanodine receptor in a regulated minimal environment.

Cardiac muscle contraction, triggered by the action potential, is mediated by the release of Ca(2+) from the sarcoplasmic reticulum through ryanodine receptor (RyR)2 channels. In situ, RyR2 gating is modulated by numerous physiological and pharmacological agents, and altered RyR2 function underlies the occurrence of arrhythmias in both inherited and acquired diseases. To understand fully the mechanisms underpinning the regulation of RyR2 in the normal heart and how these systems are altered in pathological conditions, we must first gain a detailed knowledge of the fundamental processes of RyR2 gating. In this investigation, we provide key novel mechanistic insights into the physical reality of RyR2 gating revealed by new experimental and analytical approaches. We have examined in detail the single-channel gating kinetics of the purified human RyR2 when activated by cytosolic Ca(2+) in a stringently regulated environment where the modulatory influence of factors external to the channel were minimized. The resulting gating schemes are based on an accurate description of single-channel kinetics using hidden Markov model analysis and reveal several novel aspects of RyR2 gating behavior: (a) constitutive gating is observed as unliganded opening events; (b) binding of Ca(2+) to the channel stabilizes it in different open states; (c) RyR2 exists in two preopening closed conformations in equilibrium, one of which binds Ca(2+) more readily than the other; (d) the gating of RyR2 when bound to Ca(2+) can be described by a kinetic scheme incorporating bursts; and (e) analysis of flicker closing events within bursts reveals gating activity that is not influenced by ligand binding. The gating schemes generated in this investigation provide a framework for future studies in which the mechanisms of action of key physiological regulatory factors, disease-linked mutations, and potential therapeutic compounds can be described precisely.

Techniques and methodologies to study the ryanodine receptor at the molecular, subcellular and cellular level.

In excitable tissues, the ryanodine receptor Ca(2+) release channel (RyR) protein complex regulates excitation-contraction coupling, exocytosis, gene expression and apoptosis. Defects in RyR function, in genetic or acquired pathologies, lead to massive disruptions of Ca(2+) release that can be lethal. Therefore, RyR has emerged as a putative therapeutic target and an increasing number of RyR-targeting drugs are currently being tested.Nonetheless this large-size channel is still a mystery in terms of structure, which hinders full characterization of the properties of this central protein. This chapter is dedicated to the methods available to examine RyR structure and function. The aim of the article is to concentrate on contemporary methodologies rather than focusing overtly on the progress that has been achieved using these techniques. Here we review a series of reliable approaches that are routinely employed to investigate this channel. Technical limitations are discussed, and technological developments are presented. This work is not a handbook, but it can be used as a resource and a starting point for the investigation of RyR at different levels of resolution.

Disparities in the association of the ryanodine receptor and the FK506-binding proteins in mammalian heart.

The FK506-binding proteins (FKBP12 and FKBP12.6; also known as FKBP1A and FKBP1B, respectively) are accessory subunits of the ryanodine receptor (RyR) Ca(2+) release channel. Aberrant RyR2-FKBP12.6 interactions have been proposed to be the underlying cause of channel dysfunction in acquired and inherited cardiac disease. However, the stoichiometry of the RyR2 association with FKBP12 or FKBP12.6 in mammalian heart is currently unknown. Here, we describe detailed quantitative analysis of cardiac stoichiometry between RyR2 and FKBP12 or FKBP12.6 using immunoblotting and [(3)H]ryanodine-binding assays, revealing striking disparities between four mammalian species. In mouse and pig heart, RyR2 is found complexed with both FKBP12 and FKBP12.6, although the former is the most abundant isoform. In rat heart, RyR2 is predominantly associated with FKBP12.6, whereas in rabbit it is associated with FKBP12 only. Co-immunoprecipitation experiments demonstrate RyR2-specific interaction with both FKBP isoforms in native cardiac tissue. Assuming four FKBP-binding sites per RyR2 tetramer, only a small proportion of available sites are occupied by endogenous FKBP12.6. FKBP interactions with RyR2 are very strong and resistant to drug (FK506, rapamycin and cyclic ADPribose) and redox (H(2)O(2) and diamide) treatment. By contrast, the RyR1-FKBP12 association in skeletal muscle is readily disrupted under oxidative conditions. This is the first study to directly assess association of endogenous FKBP12 and FKBP12.6 with RyR2 in native cardiac tissue. Our results challenge the widespread perception that RyR2 associates exclusively with FKBP12.6 to near saturation, with important implications for the role of the FK506-binding proteins in RyR2 pathophysiology and cardiac disease.