kn-93 and Tachycardia--Ventricular

Calcium/calmodulin-dependent protein kinase II (CaMKII) is activated in heart failure (HF) and can contribute to arrhythmias induced by β-adrenergic receptor-mediated sarcoplasmic reticulum calcium leak.. To evaluate the effect of CaMKII inhibition on ventricular tachycardia (VT) induction in conscious HF and naive rabbits.. Nonischemic HF was induced by aortic insufficiency and constriction. Electrocardiograms were recorded in rabbits pretreated with vehicle (saline) or the CaMKII inhibitor KN-93 (300 μg/kg); VT was induced by infusion of increasing doses of norepinephrine (1.56-25 μg·kg⁻¹·min⁻¹) in naive (n = 8) and HF (n = 7) rabbits. With saline, median VT dose threshold in HF was 6.25 versus 12.5 μg·kg⁻¹·min⁻¹ norepinephrine in naive rabbits (P = 0.06). Pretreatment with KN-93 significantly increased VT threshold in HF and naive rabbits (median = 25 μg·kg⁻¹·min⁻¹, P < 0.05 vs. saline for both groups). Mean cycle length of VT initiation was shorter in HF (221 ± 20 milliseconds) than naive (296 ± 23 milliseconds, P < 0.05) rabbits with saline; this difference was not significant after treatment with KN-93.. KN-93 significantly reduced arrhythmia inducibility and slowed initiation of VT, suggesting that CaMKII inhibition may have antiarrhythmic effects in the failing human heart.

Topics: Action Potentials; Animals; Anti-Arrhythmia Agents; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinases; Disease Models, Animal; Electrocardiography; Enzyme Activation; Female; Heart Failure; Heart Rate; Male; Norepinephrine; Protein Kinase Inhibitors; Rabbits; Sulfonamides; Tachycardia, Ventricular

Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies, disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro 'patient-specific cell-based system' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited form of fatal arrhythmia. Here, we report the development of a cardiac model of CPVT through the generation of iPSC from a CPVT patient carrying a heterozygous mutation in the cardiac ryanodine receptor gene (RyR2) and their subsequent differentiation into cardiomyocytes (CMs). Whole-cell patch-clamp and intracellular electrical recordings of spontaneously beating cells revealed the presence of delayed afterdepolarizations (DADs) in CPVT-CMs, both in resting conditions and after β-adrenergic stimulation, resembling the cardiac phenotype of the patients. Furthermore, treatment with KN-93 (2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), an antiarrhythmic drug that inhibits Ca(2+)/calmodulin-dependent serine-threonine protein kinase II (CaMKII), drastically reduced the presence of DADs in CVPT-CMs, rescuing the arrhythmic phenotype induced by catecholaminergic stress. In addition, intracellular calcium transient measurements on 3D beating clusters by fast resolution optical mapping showed that CPVT clusters developed multiple calcium transients, whereas in the wild-type clusters, only single initiations were detected. Such instability is aggravated in the presence of isoproterenol and is attenuated by KN-93. As seen in our RyR2 knock-in CPVT mice, the antiarrhythmic effect of KN-93 is confirmed in these human iPSC-derived cardiac cells, supporting the role of this in vitro system for drug screening and optimization of clinical treatment strategies.

Topics: Adolescent; Adult; Animals; Arrhythmias, Cardiac; Base Sequence; Benzylamines; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Differentiation; Child; Child, Preschool; Female; HEK293 Cells; Humans; Induced Pluripotent Stem Cells; Male; Mice; Molecular Sequence Data; Myocytes, Cardiac; Pedigree; Phenotype; Protein Kinase Inhibitors; Receptors, Adrenergic, beta; Ryanodine Receptor Calcium Release Channel; Sulfonamides; Tachycardia, Ventricular

Oxidative stress with hydrogen peroxide (H(2)O(2)) readily promotes early afterdepolarizations (EADs) and triggered activity (TA) in isolated rat and rabbit ventricular myocytes. Here we examined the effects of H(2)O(2) on arrhythmias in intact Langendorff rat and rabbit hearts using dual-membrane voltage and intracellular calcium optical mapping and glass microelectrode recordings. Young adult rat (3-5 mo, N = 25) and rabbit (3-5 mo, N = 6) hearts exhibited no arrhythmias when perfused with H(2)O(2) (0.1-2 mM) for up to 3 h. However, in 33 out of 35 (94%) aged (24-26 mo) rat hearts, 0.1 mM H(2)O(2) caused EAD-mediated TA, leading to ventricular tachycardia (VT) and fibrillation (VF). Aged rabbits (life span, 8-12 yr) were not available, but 4 of 10 middle-aged rabbits (3-5 yr) developed EADs, TA, VT, and VF. These arrhythmias were suppressed by the reducing agent N-acetylcysteine (2 mM) and CaMKII inhibitor KN-93 (1 microM) but not by its inactive form (KN-92, 1 microM). There were no significant differences between action potential duration (APD) or APD restitution slope before or after H(2)O(2) in aged or young adult rat hearts. In histological sections, however, trichrome staining revealed that aged rat hearts exhibited extensive fibrosis, ranging from 10-90%; middle-aged rabbit hearts had less fibrosis (5-35%), whereas young adult rat and rabbit hearts had <4% fibrosis. In aged rat hearts, EADs and TA arose most frequently (70%) from the left ventricular base where fibrosis was intermediate ( approximately 30%). Computer simulations in two-dimensional tissue incorporating variable degrees of fibrosis showed that intermediate (but not mild or severe) fibrosis promoted EADs and TA. We conclude that in aged ventricles exposed to oxidative stress, fibrosis facilitates the ability of cellular EADs to emerge and generate TA, VT, and VF at the tissue level.

Topics: Acetylcysteine; Action Potentials; Age Factors; Aging; Animals; Antioxidants; Benzylamines; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Communication; Computer Simulation; Disease Models, Animal; Electrocardiography; Enzyme Activation; Fibrosis; Heart Ventricles; Hydrogen Peroxide; Male; Models, Cardiovascular; Myocytes, Cardiac; Oxidants; Oxidative Stress; Perfusion; Protein Kinase Inhibitors; Rabbits; Rats; Rats, Inbred F344; Sulfonamides; Tachycardia, Ventricular; Time Factors; Ventricular Fibrillation

The recently described exchange protein directly activated by cAMP (Epac) has been implicated in distinct protein kinase A-independent cellular signalling pathways. We investigated the role of Epac activation in adrenergically mediated ventricular arrhythmogenesis. In contrast to observations in control conditions (n = 20), monophasic action potentials recorded in 2 of 10 intrinsically beating and 5 of 20 extrinsically paced Langendorff-perfused wild-type murine hearts perfused with the Epac activator 8-pCPT-2'-O-Me-cAMP (8-CPT, 1 microM) showed spontaneous triggered activity. Three of 20 such extrinsically paced hearts showed spontaneous ventricular tachycardia (VT). Programmed electrical stimulation provoked VT in 10 of 20 similarly treated hearts (P < 0.001; n = 20). However, there were no statistically significant accompanying changes (P > 0.05) in left ventricular epicardial (40.7 +/- 1.2 versus 44.0 +/- 1.7 ms; n = 10) or endocardial action potential durations (APD(90); 51.8 +/- 2.3 versus 51.9 +/- 2.2 ms; n = 10), transmural (DeltaAPD(90)) (11.1 +/- 2.6 versus 7.9 +/- 2.8 ms; n = 10) or apico-basal repolarisation gradients, ventricular effective refractory periods (29.1 +/- 1.7 versus 31.2 +/- 2.4 ms in control and 8-CPT-treated hearts, respectively; n = 10) and APD(90) restitution characteristics. Nevertheless, fluorescence imaging of cytosolic Ca(2+) levels demonstrated abnormal Ca(2+) homeostasis in paced and resting isolated ventricular myocytes. Epac activation using isoproterenol in the presence of H-89 was also arrhythmogenic and similarly altered cellular Ca(2+) homeostasis. Epac-dependent effects were reduced by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibition with 1 microM KN-93. These findings associate VT in an intact cardiac preparation with altered cellular Ca(2+) homeostasis and Epac activation for the first time, in the absence of altered repolarisation gradients previously implicated in reentrant arrhythmias through a mechanism dependent on CaMKII activity.

Topics: Action Potentials; Adrenergic beta-Agonists; Animals; Benzylamines; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cardiac Pacing, Artificial; Cyclic AMP; Female; Guanine Nucleotide Exchange Factors; Heart Ventricles; Homeostasis; In Vitro Techniques; Isoproterenol; Isoquinolines; Male; Mice; Models, Cardiovascular; Myocytes, Cardiac; Perfusion; Protein Kinase Inhibitors; Refractory Period, Electrophysiological; Sulfonamides; Tachycardia, Ventricular; Time Factors