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Background: Atrial fibrillation impairs atrial contractility, inducing atrial stunning that promotes thromboembolic stroke. Action potential (AP)-prolonging drugs are reported to normalize atrial hypocontractility caused by atrial tachycardia remodeling (ATR). Here, we addressed the role of AP duration (APD) changes in ATR-induced hypocontractility., Methods and Results: ATR (7-day tachypacing) decreased APD (perforated patch recording) by ≈50%, atrial contractility (echocardiography, cardiomyocyte video edge detection), and [Ca(2+)](i) transients. ATR AP waveforms suppressed [Ca(2+)](i) transients and cell shortening of control cardiomyocytes; whereas control AP waveforms improved [Ca(2+)](i) transients and cell shortening in ATR cells. However, ATR cardiomyocytes clamped with the same control AP waveform had ≈60% smaller [Ca(2+)](i) transients and cell shortening than control cells. We therefore sought additional mechanisms of contractile impairment. Whole-cell voltage clamp revealed reduced I(CaL); I(CaL) inhibition superimposed on ATR APs further suppressed [Ca(2+)](i) transients in control cells. Confocal microscopy indicated ATR-impaired propagation of the Ca(2+) release signal to the cell center in association with loss of t-tubular structures. Myofilament function studies in skinned permeabilized cardiomyocytes showed altered Ca(2+) sensitivity and force redevelopment in ATR, possibly due to hypophosphorylation of myosin-binding protein C and myosin light-chain protein 2a (immunoblot). Hypophosphorylation was related to multiple phosphorylation system abnormalities where protein kinase A regulatory subunits were downregulated, whereas autophosphorylation and expression of Ca(2+)-calmodulin-dependent protein kinase IIδ and protein phosphatase 1 activity were enhanced. Recovery of [Ca(2+)](i) transients and cell shortening occurred in parallel after ATR cessation., Conclusions: Shortening of APD contributes to hypocontractility induced by 1-week ATR but accounts for it only partially. Additional contractility-suppressing mechanisms include I(CaL) current reduction, impaired subcellular Ca(2+) signal transmission, and altered myofilament function associated with abnormal myosin and myosin-associated protein phosphorylation. The complex mechanistic basis of the atrial hypocontractility associated with AF argues for upstream therapeutic targeting rather than interventions directed toward specific downstream pathophysiological derangements.

Background: Sustained bradycardia is associated with long-QT syndrome in human beings and causes spontaneous torsades de pointes in rabbits with chronic atrioventricular block (CAVB), at least partly by downregulating delayed-rectifier K(+)-current to cause action potential (AP) prolongation. We addressed the importance of altered Ca(2+) handling, studying underlying mechanisms and consequences., Methods and Results: We measured ventricular cardiomyocyte [Ca(2+)](i) (Indo1-AM), L-type Ca(2+)-current (I(CaL)) and APs (whole-cell perforated-patch), and Ca(2+)-handling protein expression (immunoblot). CAVB increased AP duration, cell shortening, systolic [Ca(2+)](i) transients, and caffeine-induced [Ca(2+)](i) release, and CAVB cells showed spontaneous early afterdepolarizations (EADs). I(CaL) density was unaffected by CAVB, but inactivation was shifted to more positive voltages, increasing the activation-inactivation overlap zone for I(CaL) window current. Ca(2+)-calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation was enhanced in CAVB, indicating CaMKII activation. CAVB also enhanced CaMKII-dependent phospholamban-phosphorylation and accelerated [Ca(2+)](i)-transient decay, consistent with phosphorylation-induced reductions in phospholamban inhibition of sarcoplasmic reticulum (SR) Ca(2+)-ATPase as a contributor to enhanced SR Ca(2+) loading. The CaMKII-inhibitor KN93 reversed CAVB-induced changes in caffeine-releasable [Ca(2+)](i) and I(CaL) inactivation voltage and suppressed CAVB-induced EADs. Similarly, the calmodulin inhibitor W7 suppressed CAVB-induced I(CaL) inactivation voltage shifts and EADs, and a specific CaMKII inhibitory peptide prevented I(CaL) inactivation voltage shifts. The SR Ca(2+)-uptake inhibitor thapsigargin and the SR Ca(2+) release inhibitor ryanodine also suppressed CAVB-induced EADs, consistent with an important role for SR Ca(2+) loading and release in arrhythmogenesis. AP-duration changes reached a maximum after 1 week of bradypacing, but peak alterations in CaMKII and [Ca(2+)](i) required 2 weeks, paralleling the EAD time course., Conclusions: CAVB-induced remodeling enhances [Ca(2+)](i) load and activates the Ca(2+)-calmodulin-CaMKII system, producing [Ca(2+)](i)-handling abnormalities that contribute importantly to CAVB-induced arrhythmogenic afterdepolarizations.

Background: Congestive heart failure (CHF) is a common cause of atrial fibrillation. Focal sources of unknown mechanism have been described in CHF-related atrial fibrillation. The authors hypothesized that abnormal calcium (Ca(2+)) handling contributes to the CHF-related atrial arrhythmogenic substrate., Methods and Results: CHF was induced in dogs by ventricular tachypacing (240 bpm x2 weeks). Cellular Ca(2+)-handling properties and expression/phosphorylation status of key Ca(2+) handling and myofilament proteins were assessed in control and CHF atria. CHF decreased cell shortening but increased left atrial diastolic intracellular Ca(2+) concentration ([Ca(2+)](i)), [Ca(2+)](i) transient amplitude, and sarcoplasmic reticulum (SR) Ca(2+) load (caffeine-induced [Ca(2+)](i) release). SR Ca(2+) overload was associated with spontaneous Ca(2+) transient events and triggered ectopic activity, which was suppressed by the inhibition of SR Ca(2+) release (ryanodine) or Na(+)/Ca(2+) exchange. Mechanisms underlying abnormal SR Ca(2+) handling were then studied. CHF increased atrial action potential duration and action potential voltage clamp showed that CHF-like action potentials enhance Ca(2+)(i) loading. CHF increased calmodulin-dependent protein kinase II phosphorylation of phospholamban by 120%, potentially enhancing SR Ca(2+) uptake by reducing phospholamban inhibition of SR Ca(2+) ATPase, but it did not affect phosphorylation of SR Ca(2+)-release channels (RyR2). Total RyR2 and calsequestrin (main SR Ca(2+)-binding protein) expression were significantly reduced, by 65% and 15%, potentially contributing to SR dysfunction. CHF decreased expression of total and protein kinase A-phosphorylated myosin-binding protein C (a key contractile filament regulator) by 27% and 74%, potentially accounting for decreased contractility despite increased Ca(2+) transients. Complex phosphorylation changes were explained by enhanced calmodulin-dependent protein kinase IIdelta expression and function and type-1 protein-phosphatase activity but downregulated regulatory protein kinase A subunits., Conclusions: CHF causes profound changes in Ca(2+)-handling and -regulatory proteins that produce atrial fibrillation-promoting atrial cardiomyocyte Ca(2+)-handling abnormalities, arrhythmogenic triggered activity, and contractile dysfunction.

Cardiac tissue in the pulmonary vein sleeves plays an important role in clinical atrial fibrillation. Mechanisms leading to pulmonary vein activity in atrial fibrillation remain unclear. Indirect experimental evidence points to pulmonary vein Ca(2+) handling as a potential culprit, but there are no direct studies of pulmonary vein cardiomyocyte Ca(2+) handling in the literature. We used the Ca(2+)-sensitive dye indo-1 AM to study Ca(2+) handling in isolated canine pulmonary vein and left atrial myocytes. Results were obtained at 35 degrees C and room temperature in cells from control dogs and in cardiomyocytes from dogs subjected to 7-day rapid atrial pacing. We found that basic Ca(2+)-transient properties (amplitude: 186 +/- 28 vs. 216 +/- 25 nM; stimulus to half-decay time: 192 +/- 9 vs. 192 +/- 9 ms; atria vs. pulmonary vein, respectively, at 1 Hz), beat-to-beat regularity, propensity to alternans, beta-adrenergic response (amplitude increase at 0.4 Hz: 96 +/- 52 vs. 129 +/- 61%), number of spontaneous Ca(2+)-transient events after Ca(2+) loading (in normal Tyrode: 0.9 +/- 0.2 vs. 1.3 +/- 0.2; with 1 microM isoproterenol: 7.6 +/- 0.3 vs. 5.1 +/- 1.8 events/min), and caffeine-induced Ca(2+)-transient amplitudes were not significantly different between atrial and pulmonary vein cardiomyocytes. In an arrhythmia-promoting model (dogs subjected to 7-day atrial tachypacing), Ca(2+)-transient amplitude and kinetics were the same in cells from both pulmonary veins and atrium. In conclusion, the similar Ca(2+)-handling properties of canine pulmonary vein and left atrial cardiomyocytes that we observed do not support the hypothesis that intrinsic Ca(2+)-handling differences account for the role of pulmonary veins in atrial fibrillation.

Objectives: Indirect evidence suggests a role for Ca(2+)-overload in electrical and mechanical alterations caused by atrial tachycardia. The present study assessed the alterations in cellular [Ca(2+)] and contractile function caused by rapid atrial cellular activation., Methods: Intracellular Ca(2+) transients (CaT) and cell shortening (CS) were measured by microfluorometry (Indo-1 AM) and video edge-detection in isolated, field-stimulated canine atrial myocytes (37 degrees C)., Results: Abrupt increases in frequency (0.3-3 Hz) caused rapid increases in diastolic [Ca(2+)]i (DCa) that were maintained during rapid-pacing for up to 50 min. When short-term (3-min) rapid-pacing was imposed, CaT and CS increased initially upon returning to 0.3 Hz, but then declined rapidly to 64+/-5 and 49+/-7%, respectively, of pre-tachycardia values, returning to control after approximately 15 min. Post-tachycardia CaT and CS reductions were prevented by decreasing [Ca(2+)]o during tachycardia to prevent Ca(2+)-overload. CS reductions correlated with indices of Ca(2+) loading during tachycardia. Restoration of CaT to normal during post-tachycardia contractile dysfunction (by increasing [Ca(2+)]o) returned CS to normal, indicating that reduced Ca(2+) release, not reduced myofilament Ca(2+)-sensitivity, caused post-tachycardia contractile failure. Estimation of sarcoplasmic-reticulum Ca(2+)-stores (caffeine-induced Ca(2+)-release) confirmed tachycardia-induced Ca(2+)-loading and suggested that reduced Ca(2+)-stores decreased Ca(2+)-release post-tachycardia., Conclusions: Atrial tachycardia increases cellular Ca(2+)-loading, leading to post-tachycardia abnormalities in Ca(2+)-handling that produce contractile dysfunction. These findings are the first direct evidence for the frequently-postulated role of Ca(2+)-overload in tachycardia-induced abnormalities of atrial function.

This study was undertaken to test the hypothesis that, in the absence of extracellular Na+, Ca2+ can permeate tetrodotoxin (TTX)-sensitive Na+ channels in Cs(+)-loaded whole cell voltage-clamped guinea pig ventricular myocytes (22-24 degrees C). With 10 mM extracellular Ca2+, 50-ms step depolarizations (-50 to +25 mV) from holding potentials of -100 or -80 mV elicited fast and slow types of inward current: 1) a small (< 400 pA) dihydropyridine-insensitive inward current that exhibited similar voltage dependence to that of Na+ channels, with an activation threshold and peak near -45 mV and -30 mV, respectively; and 2) a larger and slower L-type Ca2+ current that activated and peaked at more positive potentials. Extracellular replacement of Ca2+ by Mg2+ abolished both currents. The lack of sensitivity of the low-threshold Ca(2+)-current amplitude to 50 or 200 microM Ni2+ suggests that this current is not produced by T-type Ca2+ current. In contrast, TTX dose dependently inhibited the low-threshold Ca2+ current, with a half-maximal inhibition concentration of 2.4 microM. Veratridine (10-50 microM), a plant alkaloid that alters the gating and permeability properties of Na+ current, induced an outward shift of time-dependent current during steps to -25 mV and typical slowly decaying inward tail currents after repolarization to -80 mV. Cell exposure to 10 and 50 microM extracellular Na+ inhibited the inward current by 21.2 +/- 3.9% (n = 23) and 14.0 +/- 3.0% (n = 14), respectively, whereas 1 microM Na+ (n = 14) was without effect. The application of 200 microM Na+ produced a small enhancement of the current (+6.2 +/- 4.1%; n = 14) which was just at the limit of significance. Our data support the notion that Ca2+ can permeate cardiac Na+ channels in the absence of an agonist and external Na+.