k201-compound and Tachycardia--Ventricular

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.. 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.. 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.

Topics: Action Potentials; Animals; Calcium; Calcium Signaling; Catecholamines; Enzyme Inhibitors; Female; Gene Knock-In Techniques; Humans; Male; Mice; Mice, Transgenic; Microscopy, Confocal; Mutation; Myocytes, Cardiac; Ouabain; Patch-Clamp Techniques; Phosphorylation; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sodium; Sodium-Potassium-Exchanging ATPase; Tachycardia, Ventricular; Thiazepines; Time Factors

Topics: Action Potentials; Animals; Calcium; Calcium Signaling; Catecholamines; Enzyme Inhibitors; Gene Knock-In Techniques; Humans; Mice; Mice, Transgenic; Mutation; Myocytes, Cardiac; Ouabain; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sodium; Sodium-Potassium-Exchanging ATPase; Tachycardia, Ventricular; Terminology as Topic; Thiazepines

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 micromol/L) but not by K201 (1 micromol/L or 10 micromol/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.

Topics: Animals; Arrhythmias, Cardiac; Caffeine; Cells, Cultured; Epinephrine; Membrane Potentials; Mice; Mice, Mutant Strains; Mutation, Missense; Myocytes, Cardiac; Protein Binding; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tachycardia, Ventricular; Tacrolimus Binding Proteins; Thiazepines

Ventricular arrhythmias can cause sudden cardiac death (SCD) in patients with normal hearts and in those with underlying disease such as heart failure. In animals with heart failure and in patients with inherited forms of exercise-induced SCD, depletion of the channel-stabilizing protein calstabin2 (FKBP12.6) from the ryanodine receptor-calcium release channel (RyR2) complex causes an intracellular Ca2+ leak that can trigger fatal cardiac arrhythmias. A derivative of 1,4-benzothiazepine (JTV519) increased the affinity of calstabin2 for RyR2, which stabilized the closed state of RyR2 and prevented the Ca2+ leak that triggers arrhythmias. Thus, enhancing the binding of calstabin2 to RyR2 may be a therapeutic strategy for common ventricular arrhythmias.

Topics: Animals; Anti-Arrhythmia Agents; Calcium; Calcium-Transporting ATPases; Cell Line; Cyclic AMP-Dependent Protein Kinases; Death, Sudden, Cardiac; Electric Stimulation; Electrocardiography; Heart; Humans; Isoproterenol; Mice; Myocardial Contraction; Phosphorylation; Physical Exertion; Protein Binding; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Tachycardia, Ventricular; Tacrolimus Binding Proteins; Thiazepines

Familial polymorphic ventricular tachycardia (FPVT) is characterized by exercise-induced arrhythmias and sudden cardiac death due to missense mutations in the cardiac ryanodine receptor (RyR2), an intracellular Ca2+ release channel required for excitation-contraction coupling in the heart.. Three RyR2 missense mutations, P2328S, Q4201R, and V4653F, which occur in Finnish families, result in similar mortality rates of approximately 33% by age 35 years and a threshold heart rate of 130 bpm, above which exercise induces ventricular arrhythmias. Exercise activates the sympathetic nervous system, increasing cardiac performance as part of the fight-or-flight stress response. We simulated the effects of exercise on mutant RyR2 channels using protein kinase A (PKA) phosphorylation. All 3 RyR2 mutations exhibited decreased binding of calstabin2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. After PKA phosphorylation, FPVT-mutant RyR2 channels showed a significant gain-of-function defect consistent with leaky Ca2+ release channels and a significant rightward shift in the half-maximal inhibitory Mg2+ concentration (IC50). Treatment with the experimental drug JTV519 enhanced binding of calstabin2 to RyR2 and normalized channel function.. Sympathetic activation during exercise induces ventricular arrhythmias above a threshold heart rate in RyR2 mutation carriers. Simulating the downstream effects of the sympathetic activation by PKA phosphorylation of RyR2 channels containing these FPVT missense mutations produced a consistent gain-of-function defect. RyR2 function and calstabin2 depletion were rescued by JTV519, suggesting stabilization of the RyR2 channel complex may represent a molecular target for the treatment and prevention of exercise-induced arrhythmias and sudden death in these patients.

Topics: Adult; Amino Acid Substitution; Calcium; Cyclic AMP-Dependent Protein Kinases; Death, Sudden, Cardiac; Finland; Genes, Dominant; Humans; Inhibitory Concentration 50; Ion Transport; Magnesium; Middle Aged; Mutation, Missense; Patch-Clamp Techniques; Phosphorylation; Physical Exertion; Point Mutation; Protein Binding; Protein Processing, Post-Translational; Protein Subunits; Recombinant Fusion Proteins; Ryanodine Receptor Calcium Release Channel; Structure-Activity Relationship; Tachycardia, Ventricular; Tacrolimus Binding Proteins; Thiazepines

Topics: Animals; Anti-Arrhythmia Agents; Calcium; Heart Failure; Humans; Mice; Myocardium; Ryanodine Receptor Calcium Release Channel; Tachycardia, Ventricular; Tacrolimus Binding Proteins; Thiazepines