F. Anthony Lai

Arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia: insights from a RyR2 R4496C knock-in mouse model.

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 (RyR2R4496C+/−). Pacing protocols did not elicit delayed afterdepolarizations (DADs) (n=20) in WT but induced DADs in 21 of 33 (63%) RyR2R4496C+/− 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 RyR2R4496C+/− myocytes (P=0.001). DADs and triggered activity were abolished by ryanodine (10 &mgr;mol/L) but not by K201 (1 &mgr;mol/L or 10 &mgr;mol/L). In vivo administration of K201 failed to prevent induction of polymorphic ventricular tachycardia (VT) in RyR2R4496C+/− mice. Measurement of the FKBP12.6/RyR2 ratio in the heavy sarcoplasmic reticulum membrane showed normal RyR2–FKBP12.6 interaction both in WT and RyR2R4496C+/− 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 RyR2R4496C+/− myocytes and ventricular arrhythmias in RyR2R4496C+/− mice; and (3) RyR2–FKBP12.6 interaction in RyR2R4496C+/− 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.

Ryanodine Receptor Mutations Associated With Stress-Induced Ventricular Tachycardia Mediate Increased Calcium Release in Stimulated Cardiomyocytes

Abstract— Ca2+ release from the sarcoplasmic reticulum mediated by the cardiac ryanodine receptor (RyR2) is a fundamental event in cardiac muscle contraction. RyR2 mutations suggested to cause defective Ca2+ channel function have recently been identified in catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD) affected individuals. We report expression of three CPVT-linked human RyR2 (hRyR2) mutations (S2246L, N4104K, and R4497C) in HL-1 cardiomyocytes displaying correct targeting to the endoplasmic reticulum. N4104K also localized to the Golgi apparatus. Phenotypic characteristics including intracellular Ca2+ handling, proliferation, viability, RyR2:FKBP12.6 interaction, and beat rate in resting HL-1 cells expressing mutant hRyR2 were indistinguishable from wild-type (WT) hRyR2. However, Ca2+ release was augmented in cells expressing mutant hRyR2 after RyR activation (caffeine and 4-chloro-m-cresol) or &bgr;-adrenergic stimulation (isoproterenol). RyR2:FKBP12.6 interaction remained intact after caffeine or 4-CMC activation, but was dramatically disrupted by isoproterenol or forskolin, an activator of adenylate cyclase. Isoproterenol and forskolin elevated cyclic-AMP to similar magnitudes in all cells and were associated with equivalent hyperphosphorylation of mutant and WT hRyR2. CPVT-linked mutations in hRyR2 did not alter resting cardiomyocyte phenotype but mediated augmented Ca2+ release on RyR-agonist or &bgr;-AR stimulation. Furthermore, equivalent interaction between mutant and WT hRyR2 and FKBP12.6 was demonstrated.

Redistribution of mitochondria leads to bursts of ATP production during spontaneous mouse oocyte maturation

During mammalian oocyte maturation there are marked changes in the distribution of mitochondria that supply the majority of the cellular ATP. Such redistribution of mitochondria is critical for oocyte quality, as oocytes with a poor developmental potential display aberrant mitochondrial distribution and lower ATP levels. Here we have investigated the dynamics of mitochondrial ATP production throughout spontaneous mouse oocyte maturation, using live measurements of cytosolic and mitochondrial ATP levels. We have observed three distinct increases in cytosolic ATP levels temporally associated with discrete events of oocyte maturation. These changes in cytosolic ATP levels are mirrored by changes in mitochondrial ATP levels, suggesting that mitochondrial ATP production is stimulated during oocyte maturation. Strikingly, these changes in ATP levels correlate with the distribution of mitochondria undergoing translocation to the peri‐nuclear region and aggregation into clusters. Mitochondrial clustering during oocyte maturation was concomitant with the formation of long cortical microfilaments and could be disrupted by cytochalasin B treatment. Furthermore, the ATP production bursts observed during oocyte maturation were also inhibited by cytochalasin B suggesting that mitochondrial ATP production is stimulated during oocyte maturation by microfilament‐driven, sub‐cellular targeting of mitochondria. J. Cell. Physiol. 224: 672–680, 2010.

Arrhythmogenic mutation-linked defects in ryanodine receptor autoregulation reveal a novel mechanism of Ca2+ release channel dysfunction

Arrhythmogenic cardiac ryanodine receptor (RyR2) mutations are associated with stress-induced malignant tachycardia, frequently leading to sudden cardiac death (SCD). The causative mechanisms of RyR2 Ca2+ release dysregulation are complex and remain controversial. We investigated the functional impact of clinically-severe RyR2 mutations occurring in the central domain, and the C-terminal I domain, a key locus of RyR2 autoregulation, on interdomain interactions and Ca2+ release in living cells. Using high-resolution confocal microscopy and fluorescence resonance energy transfer (FRET) analysis of interaction between fusion proteins corresponding to amino- (N-) and carboxyl- (C-) terminal RyR2 domains, we determined that in resting cells, RyR2 interdomain interaction remained unaltered after introduction of SCD-linked mutations and normal Ca2+ regulation was maintained. In contrast, after channel activation, the abnormal Ca2+ release via mutant RyR2 was intrinsically linked to altered interdomain interaction that was equivalent with all mutations and exhibited threshold characteristics (caffeine >2.5 mmol/L; Ca2+ >150 nmol/L). Noise analysis revealed that I domain mutations introduced a distinct pattern of conformational instability in Ca2+ handling and interdomain interaction after channel activation that was absent in signals obtained from the central domain mutation. I domain–linked channel instability also occurred in intact RyR2 expressed in CHO cells and in HL-1 cardiomyocytes. These new insights highlight a critical role for mutation-linked defects in channel autoregulation, and may contribute to a molecular explanation for the augmented Ca2+ release following RyR2 channel activation. Our findings also suggest that the mutational locus may be an important mechanistic determinant of Ca2+ release channel dysfunction in arrhythmia and SCD.

Mineralocorticoid Modulation of Cardiac Ryanodine Receptor Activity Is Associated With Downregulation of FK506-Binding Proteins

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 Ca2+ 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 Ca2+ 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 Ca2+ influx, overstimulation of the cardiac mineralocorticoid pathway in the heart might be a major upstream factor for aberrant Ca2+ release during diastole, which contributes to cardiac arrhythmia in heart failure.

Na+-dependent SR Ca2+ overload induces arrhythmogenic events in mouse cardiomyocytes with a human CPVT mutation

AIMS Mutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR Ca(2+) content--in the absence of protein kinase A activation--affects RyR2 function and arrhythmogenesis in CPVT remains elusive. METHODS AND RESULTS Isolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100 micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+) load. Changes in membrane potential and intracellular [Ca(2+)] were monitored with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+) release, and SR Ca(2+) load were comparable in wild-type (WT) and RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic [Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release, and SR Ca(2+) content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2(R4496C+/-) when compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events. CONCLUSION The elevation of SR Ca(2+) load--in the absence of beta-adrenergic stimulation--is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.

Binding of Phosphoinositide-specific Phospholipase C-ζ (PLC-ζ) to Phospholipid Membranes POTENTIAL ROLE OF AN UNSTRUCTURED CLUSTER OF BASIC RESIDUES

Phospholipase C-ζ (PLC-ζ) is a sperm-specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-δ1, but lacks the PH domain that anchors PLC-δ1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-ζ interacts with membranes. The linker region between the X and Y catalytic domains of PLC-ζ, however, contains a cluster of basic residues not present in PLC-δ1. Application of electrostatic theory to a homology model of PLC-ζ suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-ζ to phospholipid vesicles: for 2:1 phosphatidylcholine/phosphatidylserine (PC/PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP2 into the 2:1 PC/PS vesicles increases K about 10-fold, to 5 × 103 m-1, a biologically relevant value. Expressed fragments corresponding to the PLC-ζ X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge =+7) within the linker region, PLC-ζ-(374-385), binds to PC/PS vesicles with higher affinity than PLC-ζ, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-ζ may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP2 adjacent to the catalytic domain.

Ca2+ Syntillas, Miniature Ca2+ Release Events in Terminals of Hypothalamic Neurons, Are Increased in Frequency by Depolarization in the Absence of Ca2+ Influx

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of ∼250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of –80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.

Ryanodine receptor-mediated arrhythmias and sudden cardiac death.

The cardiac ryanodine receptor-Ca2+ release channel (RyR2) is an essential sarcoplasmic reticulum (SR) transmembrane protein that plays a central role in excitation–contraction coupling (ECC) in cardiomyocytes. Aberrant spontaneous, diastolic Ca2+ leak from the SR due to dysfunctional RyR2 contributes to the formation of delayed after-depolarisations, which are thought to underlie the fatal arrhythmia that occurs in both heart failure (HF) and in catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is an inherited disorder associated with mutations in either the RyR2 or a SR luminal protein, calsequestrin. RyR2 shows normal function at rest in CPVT but the RyR2 dysfunction is unmasked by physical exercise or emotional stress, suggesting abnormal RyR2 activation as an underlying mechanism. Several potential mechanisms have been advanced to explain the dysfunctional RyR2 observed in HF and CPVT, including enhanced RyR2 phosphorylation status, altered RyR2 regulation at luminal/cytoplasmic sites and perturbed RyR2 intra/inter-molecular interactions. This review considers RyR2 dysfunction in the context of the structural and functional modulation of the channel, and potential therapeutic strategies to stabilise RyR2 function in cardiac pathology.

Redox Sensitivity of the Ryanodine Receptor Interaction with FK506-binding Protein

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.