kn-93 and Atrial-Fibrillation

A Pitx2c deficiency increases the risk of atrial fibrillation (AF). Atrial structural remodelling with fibrosis blocks electrical conduction and leads to arrhythmogenesis. A Pitx2c deficiency enhances profibrotic transforming growth factor (TGF)-β expression and calcium dysregulation, suggesting that Pitx2c may play a role in atrial fibrosis. The purposes of this study were to evaluate whether a Pitx2c deficiency modulates cardiac fibroblast activity and study the underlying mechanisms.. A migration assay, proliferation analysis, Western blot analysis and calcium fluorescence imaging were conducted in Pitx2c-knockdown human atrial fibroblasts (HAFs) using short hairpin (sh)RNA or small interfering (si)RNA.. Compared to control HAFs, Pitx2c-knockdown HAFs had a greater migration but a similar proliferative ability. Pitx2c-knockdown HAFs had a higher calcium influx with enhanced phosphorylation of calmodulin kinase II (CaMKII), α-smooth muscle actin and matrix metalloproteinase-2. In the presence of a CaMKII inhibitor (KN-93, 0.5 μmol/L), control and Pitx2c-knockdown HAFs exhibited similar migratory abilities.. These findings suggest that downregulation of Pitx2c may regulate atrial fibrosis through modulating calcium homeostasis, which may contribute to its role in anti-atrial fibrosis, and Pitx2c downregulation may change the atrial electrophysiology and AF occurrence through modulating fibroblast activity.

Topics: Actins; Arrhythmias, Cardiac; Atrial Fibrillation; Atrial Remodeling; Benzylamines; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Movement; Cell Proliferation; Down-Regulation; Fibroblasts; Fibrosis; Gene Knockdown Techniques; Heart Atria; Homeobox Protein PITX2; Homeodomain Proteins; Humans; In Vitro Techniques; Matrix Metalloproteinase 2; Optical Imaging; Phosphorylation; Protein Isoforms; Protein Kinase Inhibitors; RNA, Small Interfering; Sulfonamides; Transcription Factors

BACKGROUND Increased small-conductance Ca2+-activated K+ current (SK), abnormal intracellular Ca2+ handling, and enhanced expression and activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been found in clinical and/or experimental models of atrial fibrillation (AF), but the cumulative effect of these phenomena and their mechanisms in AF are still unclear. This study aimed to test the hypothesis that CaMKII increases SK current in human chronic AF. MATERIAL AND METHODS Right atrial appendage tissues from patients with either sinus rhythm (SR) or AF and neonatal rat atrial myocytes were used. Patch clamp, qRT-PCR, and Western blotting techniques were used to perform the study. RESULTS Compared to SR, the apamin-sensitive SK current (IKAS) was significantly increased, but the mRNA and protein levels of SK1, SK2, and SK3 were significantly decreased. In AF, the steady-state Ca2+ response curve of [i]IKAS[/i] was shifted leftward and the [Ca2+]i level was significantly increased. CaMKII inhibitors (KN-93 or autocamtide-2-related inhibitory peptide (AIP)) reduced the IKAS in both AF and SR. The inhibitory effect of KN-93 or AIP on [i]IKAS[/i] was greater in AF than in SR. The expression levels of calmodulin, CaMKII, and autophosphorylated CaMKII at Thr287 (but not at Thr286) were significantly increased in AF. Furthermore, KN-93 inhibited the expression of (Thr287)p-CaMKII and SK2 in neonatal rat atrial myocytes. CONCLUSIONS SK current is increased via the enhanced activation of CaMKII in patients with AF. This finding may explain the difference between SK current and channels expression in AF, and thus may provide a therapeutic target for AF.

Topics: Animals; Atrial Fibrillation; Benzylamines; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Membrane Permeability; Chronic Disease; Coronary Sinus; Cytosol; Down-Regulation; Female; Heart Atria; Humans; Ion Channel Gating; Male; Middle Aged; Patch-Clamp Techniques; Peptides; Phosphorylation; Rats, Sprague-Dawley; RNA, Messenger; Small-Conductance Calcium-Activated Potassium Channels; Sulfonamides; Up-Regulation

Increases in late Na(+) current (late INa) and activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) are associated with atrial arrhythmias. CaMKII also phosphorylates Nav1.5, further increasing late INa. The combination of a CaMKII inhibitor with a late INa inhibitor may be superior to each compound alone to suppress atrial arrhythmias. Therefore, we investigated the effect of a CaMKII inhibitor in combination with a late INa inhibitor on anemone toxin II (ATX-II, a late INa enhancer)-induced atrial arrhythmias.. Rat right atrial tissue was isolated and preincubated with either the CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP), the late INa inhibitor GS458967, or both, and then exposed to ATX-II. ATX-II increased diastolic tension and caused fibrillation of isolated right atrial tissue. AIP (0.3μmol/L) and 0.1μmol/L GS458967 alone inhibited ATX-II-induced arrhythmias by 20±3% (mean±SEM, n=14) and 34±5% (n=13), respectively, whereas the two compounds in combination inhibited arrhythmias by 81±4% (n=10, p<0.05, vs either AIP or GS458967 alone or the calculated sum of individual effects of both compounds). AIP and GS458967 also attenuated the ATX-induced increase of diastolic tension. Consistent with the mechanical and electrical data, 0.3μmol/L AIP and 0.1μmol/L GS458967 each inhibited ATX-II-induced CaMKII phosphorylation by 23±3% and 32±4%, whereas the combination of both compounds inhibited CaMKII phosphorylation completely.. The effects of an enhanced late INa to induce arrhythmic activity and activation of CaMKII in atria are attenuated synergistically by inhibitors of late INa and CaMKII.

Topics: Action Potentials; Animals; Ataxin-2; Atrial Fibrillation; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Heart Atria; Male; Myocardial Contraction; Rats; Sodium; Sulfonamides

Delayed afterdepolarizations (DADs) carried by Na(+)-Ca(2+)-exchange current (I(NCX)) in response to sarcoplasmic reticulum (SR) Ca(2+) leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined.. Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca(2+)](i) (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca(2+)](i) and SR Ca(2+) content (integrated I(NCX) during caffeine-induced Ca(2+) transient) were unchanged, whereas diastolic SR Ca(2+) leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was ≈50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca(2+) leak. Calmodulin expression (60%), Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca(2+) leak, the frequency of spontaneous Ca(2+) release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca(2+) sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca(2+)](i) and I(NCX) density revealed I(NCX) upregulation in cAF. Spontaneous Ca(2+) release events accompanied by inward I(NCX) currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca(2+)](i) (diastolic [Ca(2+)](i)-voltage coupling gain) was higher in cAF compared with control.. Enhanced SR Ca(2+) leak through CaMKII-hyperphosphorylated RyR2, in combination with larger I(NCX) for a given SR Ca(2+) release and increased diastolic [Ca(2+)](i)-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

Topics: Action Potentials; Aged; Animals; Atrial Fibrillation; Benzylamines; Biological Transport, Active; Caffeine; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calmodulin; Chronic Disease; Female; Gene Knock-In Techniques; Humans; Lipid Bilayers; Male; Membrane Potentials; Mice; Myocytes, Cardiac; Patch-Clamp Techniques; Phosphorylation; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium-Calcium Exchanger; Sulfonamides

Although research suggests that diastolic Ca(2+) levels might be increased in atrial fibrillation (AF), this hypothesis has never been tested. Diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR) might increase diastolic Ca(2+) levels and play a role in triggering or maintaining AF by transient inward currents through Na(+)/Ca(2+) exchange. In ventricular myocardium, ryanodine receptor type 2 (RyR2) phosphorylation by Ca(2+)/calmodulin-dependent protein kinase (CaMK)II is emerging as an important mechanism for SR Ca(2+) leak.. We tested the hypothesis that CaMKII-dependent diastolic SR Ca(2+) leak and elevated diastolic Ca(2+) levels occurs in atrial myocardium of patients with AF.. We used isolated human right atrial myocytes from patients with AF versus sinus rhythm and found CaMKII expression to be increased by 40+/-14% (P<0.05), as well as CaMKII phosphorylation by 33+/-12% (P<0.05). This was accompanied by a significantly increased RyR2 phosphorylation at the CaMKII site (Ser2814) by 110+/-53%. Furthermore, cytosolic Ca(2+) levels were elevated during diastole (229+/-20 versus 164+/-8 nmol/L, P<0.05). Most likely, this resulted from an increased SR Ca(2+) leak in AF (P<0.05), which was not attributable to higher SR Ca(2+) load. Tetracaine experiments confirmed that SR Ca(2+) leak through RyR2 leads to the elevated diastolic Ca(2+) level. CaMKII inhibition normalized SR Ca(2+) leak and cytosolic Ca(2+) levels without changes in L-type Ca(2+) current.. Increased CaMKII-dependent phosphorylation of RyR2 leads to increased SR Ca(2+) leak in human AF, causing elevated cytosolic Ca(2+) levels, thereby providing a potential arrhythmogenic substrate that could trigger or maintain AF.

Topics: Action Potentials; Anesthetics, Local; Atrial Fibrillation; Benzylamines; Calcium Channels, L-Type; Calcium Signaling; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Case-Control Studies; Cell Size; Diastole; Heart Atria; Humans; Microscopy, Confocal; Myocardium; Patch-Clamp Techniques; Phosphorylation; Protein Kinase Inhibitors; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium-Calcium Exchanger; Sulfonamides; Systole; Tetracaine; Time Factors; Up-Regulation

The autonomic nervous system and calcium regulation play important roles in the pathophysiology of atrial fibrillation. Calmodulin regulates the calcium homeostasis and may mediate the proarrhythmic effects of autonomic nervous agents. The purpose of this study was to compare the effects of beta- and alpha-adrenoceptor agonists on the pulmonary vein electrical activity and evaluate whether calmodulin kinase II inhibitors may change the effects of the adrenoceptor agonists on the pulmonary vein arrhythmogenesis. Conventional microelectrodes were used to record the action potentials in isolated rabbit pulmonary vein tissue specimens before and after the administration of isoproterenol, phenylephrine and KN-93 (a calmodulin kinase II inhibitor). In the tissue preparation, isoproterenol (0, 0.1, 3 microM) increased the beating rates (1.5+/-0.2, 1.6+/-0.2, 2.3+/-0.3 Hz, n=10, P<0.001) with the genesis of early afterdepolarizations (EADs, 0%, 40%, 50%, P<0.05) and increased the amplitude of the delayed afterdepolarizations (DADs, 0.6+/-0.3, 1.7+/-0.4, 3.9+/-1.0 mV, P<0.05). Phenylephrine (0, 1, 10 microM) also increased the beating rates (1.4+/-0.2, 1.6+/-0.2, 1.9+/-0.2 Hz, n=12, P<0.001), incidence of EADs (0%, 8%, 50%, P<0.05) and amplitude of the DADs (0.4+/-0.2, 1.2+/-0.4, 2.6+/-0.8 mV, P<0.05). KN-93 did not change the pulmonary vein beating rates or action potential duration. However, in the presence of KN-93 (1 microM), isoproterenol (3 microM) and phenylephrine (10 microM) did not induce any EADs or DADs in the pulmonary veins. In conclusion, calmodulin kinase II inhibition may prevent adrenergic induced pulmonary vein arrhythmogenesis.

Topics: Action Potentials; Adrenergic alpha-Agonists; Adrenergic beta-Agonists; Animals; Anti-Arrhythmia Agents; Atrial Fibrillation; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Dose-Response Relationship, Drug; In Vitro Techniques; Isoproterenol; Kinetics; Phenylephrine; Protein Kinase Inhibitors; Pulmonary Veins; Rabbits; Sulfonamides