Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by life threatening arrhythmias and mutations in the gene encoding the ryanodine receptor (RyR2). Disagreement exists on whether (1) RyR2 mutations induce abnormal calcium transients in the absence of adrenergic stimulation; (2) decreased affinity of mutant RyR2 for FKBP12.6 causes CPVT; (3) K201 prevent arrhythmias by normalizing the FKBP12.6-RyR2 binding. We studied ventricular myocytes isolated from wild-type (WT) and knock-in mice harboring the R4496C mutation (RyR2 ^R4496C+/− ). Pacing protocols did not elicit delayed afterdepolarizations (DADs) (n=20) in WT but induced DADs in 21 of 33 (63%) RyR2 ^R4496C+/− myocytes ( P =0.001). Superfusion with isoproterenol (30 nmol/L) induced small DADs (45%) and no triggered activity in WT myocytes, whereas it elicited DADs in 87% and triggered activity in 60% of RyR2 ^R4496C+/− myocytes ( P =0.001). DADs and triggered activity were abolished by ryanodine (10 μmol/L) but not by K201 (1 μmol/L or 10 μmol/L). In vivo administration of K201 failed to prevent induction of polymorphic ventricular tachycardia (VT) in RyR2 ^R4496C+/− mice. Measurement of the FKBP12.6/RyR2 ratio in the heavy sarcoplasmic reticulum membrane showed normal RyR2–FKBP12.6 interaction both in WT and RyR2 ^R4496C+/− either before and after treatment with caffeine and epinephrine. We suggest that (1) triggered activity is the likely arrhythmogenic mechanism of CPVT; (2) K201 fails to prevent DADs in RyR2 ^R4496C+/− myocytes and ventricular arrhythmias in RyR2 ^R4496C+/− mice; and (3) RyR2–FKBP12.6 interaction in RyR2 ^R4496C+/− is identical to that of WT both before and after epinephrine and caffeine, thus suggesting that it is unlikely that the R4496C mutation interferes with the RyR2/FKBP12.6 complex.

Background— The mineralocorticoid pathway is involved in cardiac arrhythmias associated with heart failure through mechanisms that are incompletely understood. Defective regulation of the cardiac ryanodine receptor (RyR) is an important cause of the initiation of arrhythmias. Here, we examined whether the aldosterone pathway might modulate RyR function. Methods and Results— Using the whole-cell patch clamp method, we observed an increase in the occurrence of delayed afterdepolarizations during action potential recordings in isolated adult rat ventricular myocytes exposed for 48 hours to aldosterone 100 nmol/L, in freshly isolated myocytes from transgenic mice with human mineralocorticoid receptor expression in the heart, and in wild-type littermates treated with aldosterone. Sarcoplasmic reticulum Ca ²⁺ load and RyR expression were not altered; however, RyR activity, visualized in situ by confocal microscopy, was increased in all cells, as evidenced by an increased occurrence and redistribution to long-lasting and broader populations of spontaneous Ca ²⁺ sparks. These changes were associated with downregulation of FK506-binding proteins (FKBP12 and 12.6), regulatory proteins of the RyR macromolecular complex. Conclusions— We suggest that in addition to modulation of Ca ²⁺ influx, overstimulation of the cardiac mineralocorticoid pathway in the heart might be a major upstream factor for aberrant Ca ²⁺ release during diastole, which contributes to cardiac arrhythmia in heart failure.

Background—: A hallmark of heart failure is impaired cytoplasmic Ca ²⁺ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca ²⁺ handling via altered excitation-transcription coupling contribute to the development and progression of heart failure. Methods and Results—: Using tissue and isolated cardiomyocytes from nonfailing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca ²⁺ handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment, there was also reduced expression of Ca ²⁺ pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca ²⁺ transporters. These changes were associated with altered nucleoplasmic Ca ²⁺ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca ²⁺ levels with diminished and prolonged nuclear Ca ²⁺ transients and slowed intranuclear Ca ²⁺ diffusion. Altered nucleoplasmic Ca ²⁺ levels were translated to higher activation of nuclear Ca ²⁺ /calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca ²⁺ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca ²⁺ changes that are typical of heart failure. Conclusions—: During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca ²⁺ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca ²⁺ regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling.

The ryanodine receptor (RyR) calcium release channel functions as a redox sensor that is sensitive to channel modulators. The FK506-binding protein (FKBP) is an important regulator of channel activity, and disruption of the RyR2-FKBP12.6 association has been implicated in cardiac disease. In the present study, we investigated whether the RyR-FKBP association is redox-regulated. Using co-immunoprecipitation assays of solubilized native RyR2 from cardiac muscle sarcoplasmic reticulum (SR) with recombinant [³⁵S]FKBP12.6, we found that the sulfydryl-oxidizing agents, H₂O₂ and diamide, result in diminished RyR2-FKBP12.6 binding. Co-sedimentation experiments of cardiac SR vesicles with [³⁵S]FKBP12.6 also demonstrated that oxidizing reagents decreased FKBP binding. Matching results were obtained with skeletal muscle SR. Notably, H₂O₂ and diamide differentially affected the RyR2-FKBP12.6 interaction, decreasing binding to ∼75 and ∼50% of control, respectively. In addition, the effect of H₂O₂ was negligible when the channel was in its closed state or when applied after FKBP binding had occurred, whereas diamide was always effective. A cysteine-null mutant FKBP12.6 retained redox-sensitive interaction with RyR2, suggesting that the effect of the redox reagents is exclusively via sites on the ryanodine receptor. K201 (or JTV519), a drug that has been proposed to prevent FKBP12.6 dissociation from the RyR2 channel complex, did not restore normal FKBP binding under oxidizing conditions. Our results indicate that the redox state of the RyR is intimately connected with FKBP binding affinity.

The ryanodine receptor-calcium release channel complex (RyR) plays a pivotal role in excitation-contraction coupling in skeletal and cardiac muscle. RyR channel activity is modulated by interaction with FK506-binding protein (FKBP), and disruption of the RyR-FKBP association has been implicated in cardiomyopathy, cardiac hypertrophy, and heart failure. Evidence for an interaction between RyR and FKBP is well documented, both in skeletal muscle (RyR1-FKBP12) and in cardiac muscle (RyR2-FKBP12.6), however definition of the FKBP-binding site remains elusive. Early reports proposed interaction of a short RyR central domain with FKBP12/12.6, however this site has been questioned, and recently an alternative FKBP12.6 interaction site has been identified within the N-terminal half of RyR2. In this study, we report evidence for the human RyR2 C-terminal domain as a novel FKBP12.6-binding site. Using competition binding assays, we find that short C-terminal RyR2 fragments can displace bound FKBP12.6 from the native RyR2, although they are unable to exclusively support interaction with FKBP12.6. However, expression of a large RyR2 C-terminal construct in mammalian cells encompassing the pore-forming transmembrane domains exhibits rapamycin-sensitive binding specifically to FKBP12.6 but not to FKBP12. We also obtained some evidence for involvement of the RyR2 N-terminal, but not the central domain, in FKBP12.6 interaction. Our studies suggest that a novel interaction site for FKBP12.6 may be present at the RyR2 C terminus, proximal to the channel pore, a sterically appropriate location that would enable this protein to play a central role in the modulation of this critical ion channel.

Ca²⁺stores were studied in a preparation of freshly dissociated terminals from hypothalamic magnocellular neurons. Depolarization from a holding level of −80 mV in the absence of extracellular Ca²⁺elicited Ca²⁺release from intraterminal stores, a ryanodine-sensitive process designated as voltage-induced Ca²⁺release (VICaR). The release took one of two forms: an increase in the frequency but not the quantal size of Ca²⁺syntillas, which are brief, focal Ca²⁺transients, or an increase in global [Ca²⁺]. The present study provides evidence that the sensors of membrane potential for VICaR are dihydropyridine receptors (DHPRs). First, over the range of −80 to −60 mV, in which there was no detectable voltage-gated inward Ca²⁺current, syntilla frequency was increasede-fold per 8.4 mV of depolarization, a value consistent with the voltage sensitivity of DHPR-mediated VICaR in skeletal muscle. Second, VICaR was blocked by the dihydropyridine antagonist nifedipine, which immobilizes the gating charge of DHPRs but not by Cd²⁺or FPL 64176 (methyl 2,5 dimethyl-4[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate), a non-dihydropyridine agonist specific for L-type Ca²⁺channels, having no effect on gating charge movement. At 0 mV, the IC₅₀for nifedipine blockade of VICaR in the form of syntillas was 214 nmin the absence of extracellular Ca²⁺. Third, type 1 ryanodine receptors, the type to which DHPRs are coupled in skeletal muscle, were detected immunohistochemically at the plasma membrane of the terminals. VICaR may constitute a new link between neuronal activity, as signaled by depolarization, and a rise in intraterminal Ca²⁺.