Tachycardia Remodeling Research Articles

Abstract 13235: Enhanced Receptor-independent Nucleoside Diphosphate Kinase Mediated G s Protein Signaling Contributes to Arrhythmogenic Calcium Mishandling in Atrial Fibrillation

Background: Enhanced G-protein related cyclic AMP (cAMP) signaling and abnormal Ca 2+ handling due to dysfunctional ryanodine receptor type-2 (RyR2) are implicated in atrial fibrillation (AF). Nucleoside diphosphate kinase (NDPK) forms complexes with G s proteins, increasing cAMP signaling via receptor-independent G protein activation. Here, we studied the possible proarrhythmic role of NDPKs in clinical (chronic AF, cAF) and experimental AF. Methods: NDPK and G protein expression were assessed by immunoblotting in atria from sinus rhythm (Ctl) and cAF patients; dogs undergoing AF-mimicking tachycardia remodeling (ATR, 400 bpm, 7 days) or AF-promoting chronic atrial ischemia; and canine atrial cardiomyocytes (CMs) paced in vitro for 24-48 hrs at 1 (P1) or 3 Hz (P3). cAMP was measured by immunoassay. Immunostaining was used for protein visualization. Spontaneous sarcoplasmic reticulum (SR) Ca 2+ release events (SCaEs) in CMs overexpressing NDPKs or EGFP-control (adenoviral gene-transfer) were recorded with epifluorescence (Indo-1). Results: NDPK-B, NDPK-C, Gα s and Gβ 1 were upregulated in cAF patient atria (Fig A). ATR and chronic atrial ischemia increased NDPK-B expression in canine atria (Fig B). In vitro tachypacing reproduced AF effects on NDPK/Gα s in CMs (Fig C). Accordingly, cAMP levels were higher in cAF patients and P3-paced CMs (Fig D-E). Adenoviral NDPK-B and NDPK-C overexpression increased cAMP levels (Fig F). NDPK-B, NDPK-C and Gα s co-localized at the plasma membrane when upregulated by tachypacing canine CMs. SCaEs, a proarrhythmic consequence of diastolic SR Ca 2+ leak, were enhanced in NDPK-B or NDPK-C overexpressing CMs, and the latency from last regular SR Ca 2+ release was reduced (Fig G). Conclusions: Receptor-independent NDPK-mediated G s -protein/cAMP signaling is upregulated in clinical and experimental AF, causes arrhythmogenic SCaEs, and is likely a novel contributor to ectopy-inducing atrial triggered activity due to SR Ca 2+ leak.

Effect of irbesartan on development of atrial fibrosis and atrial fibrillation in a canine atrial tachycardia model with left ventricular dysfunction, association with p53

Effects of an angiotensin II receptor blocker, irbesartan (IRB), on the development of atrial fibrosis and atrial fibrillation (AF) were assessed in a canine model of atrial tachycardia remodeling (ATR) with left ventricular dysfunction, together with its possible association with involvement of p53. Atrial tachypacing (400bpm for 4weeks) was used to induce ATR in beagles treated with placebo (ATR-dogs, n=6) or irbesartan (IRB-dogs, n=5). Non-paced sham dogs served as control (Control-dogs, n=4). ATR- and IRB-dogs developed tachycardia-induced left ventricular dysfunction. Atrial effective refractory period (AERP) shortened (83±5ms, p<0.05), inter-atrial conduction time prolonged (72±2ms, p<0.05), and AF duration increased (29±5s, p<0.05 vs. baseline) after 4weeks in ATR-dogs. ATR-dogs also had a larger area of atrial fibrous tissue (5.2±0.5%, p<0.05 vs. Control). All these changes, except for AERP, were attenuated in IRB-dogs (92±3ms, 56±3ms, 9±5s, and 2.5±0.7%, respectively; p<0.05 vs. ATR for each). In ATR-dogs, p53 expression in the left atrium decreased by 42% compared with Control-dogs (p<0.05); however, it was highly expressed in IRB-dogs (+89% vs. ATR). Transforming growth factor (TGF)-β1 expression was enhanced in ATR-dogs (p<0.05 vs. Control) but reduced in IRB-dogs (p<0.05 vs. ATR). Irbesartan suppresses atrial fibrosis and AF development in a canine ATR model with left ventricular dysfunction in association with p53.

The ER stress-mediated mitochondrial apoptotic pathway and MAPKs modulate tachypacing-induced apoptosis in HL-1 atrial myocytes.

Background and ObjectCell apoptosis is a contributing factor in the initiation, progression and relapse of atrial fibrillation (AF), a life-threatening illness accompanied with stroke and heart failure. However, the regulatory cascade of apoptosis is intricate and remains unidentified, especially in the setting of AF. The aim of this study was to explore the roles of endoplasmic reticulum (ER) stress, mitochondrial apoptotic pathway (MAP), mitogen-activated protein kinases (MAPKs), and their cross-talking in tachypacing-induced apoptosis.Methods and ResultsHL-1 cells were cultured in the presence of tachypacing for 24 h to simulate atrial tachycardia remodeling. Results showed that tachypacing reduced cell viability measured by the cell counting kit-8, dissipated mitochondrial membrane potential detected by JC-1 staining and resulted in approximately 50% apoptosis examined by Hoechst staining and annexin V/propidium iodide staining. In addition, the proteins involved in ER stress, MAP and MAPKs were universally up-regulated or activated via phosphorylation, as confirmed by western blotting; and reversely silencing of ER stress, caspase-3 (the ultimate executor of MAP) and MAPKs with specific inhibitors prior to pacing partially alleviated apoptosis. An inhibitor of ER stress was applied to further investigate the responses of mitochondria and MAPKs to ER stress, and results indicated that suppression of ER stress comprehensively but incompletely attenuated the activation of MAP and MAPKs aroused by tachypacing, with the exception of ERK1/2, one branch of MAPKs.ConclusionsOur study suggested tachypacing-induced apoptosis is regulated by ER stress-mediated MAP and MAPKs. Thus, the above three components are all promising anti-apoptotic targets in AF patients and ER stress appears to play a dominant role due to its comprehensive effects.

Atrial Fibrillation-Associated Remodeling Does Not Promote Atrial Thrombus Formation in Canine Models

The most important complication of atrial fibrillation (AF) is thromboembolic stroke. Although AF-related remodeling is considered important in atrial thrombogenesis, its role never has been directly tested. This study assessed effects of AF-related remodeling on the atrial thrombogenic milieu by using radiofrequency ablation (RFA) to create a quantifiable prothrombotic nidus. We studied normal control dogs (control, n=16) and 3 canine AF-models: (1) atrial tachycardia remodeling (ATR; n=16) induced by atrial tachypacing (400 bpm for 1 week, with atrioventricular block and ventricular pacing at 80 bpm); (2) congestive heart failure (CHF; n=14) attributable to ventricular tachypacing (240 bpm for 2 weeks); and (3) chronic AF (CAF; n=8) induced by atrial tachypacing (35±3 days) without atrioventricular block. CAF dogs had AF for 13±1 days until euthanization. After remodeling was established, RFA lesions were created in both atria. Half the ATR and CHF dogs were subjected to atrial tachypacing during 7-day post-RFA follow-up. Electrophysiological and echocardiographic studies were performed before RFA and 7 days after RFA, and then hearts were removed and atrial thrombi were quantified by histomorphometry. Burst-pacing-induced AF duration was significantly greater in ATR, CHF, and CAF groups versus control group. The atrial effective refractory period shortened in ATR and CAF groups. Left atrial diameter was significantly larger with CHF, but not with ATR. Neither total thrombus volume nor thrombus volume per lesion differed significantly among groups. Table.Properties of Ablation Lesions and Atrial Thrombi Experimental GroupControl (n=16)ATR (n=16)CHF (n=14)CAF (n=8)N of ablation lesions per dog6.9±0.36.6±0.27.2±0.26.9±0.4Ablation lesion area, mm(2)53.1±3.558.3±4.857.7±4.944.3±3.7Ablation lesion depth, mm5.2±0.25.1±0.35.3±0.25.2±0.2Ablation lesion volume, mm(3)205.2±17.8211.6±17.6231.5±29.0176.8±22.2N of thrombi per dog5.4±0.44.7±0.35.6±0.46.5±0.4Presence of thrombus, %80±572±577±695±3Mean thrombus volume in both atria, mm(3)20.8±3.414.9±2.212.2±2.622.5±5.6Mean thrombus volume in left atria, mm(3)8.2±1.54.0±0.95.5±1.68.1±3.3Mean thrombus volume in right atria, mm(3)30.1±5.422.7±4.317.9±4.132.8±8.3Total thrombus volume in both atria, mm(3)140.5±21.399.7±16.886.1±17.5131.1±22.7Total thrombus volume in left atria, mm(3)22.8±5.311.8±3.317.0±3.723.3±6.4Total thrombus volume in right atria, mm(3)117.7±21.587.8±17.269.1±16.1107.8±23.3Thrombus volume normalized to ablation lesion area in both atria, mm(3)/mm(2)0.5±0.10.4±0.11.5±1.10.8±0.3Thrombus volume normalized to ablation lesion volume in both atria0.2±0.10.1±0.00.5±0.40.3±0.1 ATR indicates atrial tachycardia remodeling; CAF, chronic atrial fibrillation; and CHF, congestive heart failure. There were no statistically significant differences for any groups vs control group for any of these variables studied. None of the AF substrates tested, including sustained atrial tachycardia/AF itself, enhanced post-RFA atrial thrombus formation. Indices of electrical and structural remodeling did not predict post-RFA thrombogenic potential. Contrary to widely held but previously untested notions, we were unable to demonstrate prothrombotic effects of AF-related remodeling.

Abstract 9723: Calcium Loading Induces a PKC Isoform Switch That Activates Constitutive Acetylcholine Regulated Potassium Current in Atrial Fibrillation-Related Remodeling

Background: AF causes atrial tachycardia remodeling (ATR), with enhanced constitutive acetylcholine regulated K + current (I KAChc ) contributing to ATR-induced APD shortening and AF promotion. The present study examined the mechanisms underlying I KAChc activation; based on preliminary evidence for an essential role of phosphorylation, we evaluated the participation of protein kinase C (PKC). Methods: Cultured dog atrial cardiomyocytes in vitro tachypaced (TP) at 3 Hz for 24 hours were compared to parallel 1 Hz paced cells and cells from ATR dogs (paced in vivo at 400 bpm x 1 week). I KAChc single channel activity was assessed in cell attached and cell free (I-O) patches. Protein expression was assessed by Western blot. Results: In vitro TP for 24 hours activated I KAChc (Fig A), mimicking effects of in vivo ATR. The classical PKC isoform PKCα inhibited, whereas the novel isoform PKCε enhanced, I KAChc when applied to the intracellular side of I-O patches. TP and ATR downregulated PKCα (by 33, 37% respectively) and caused membrane translocation of PKCε, switching PKC-predominance to the stimulatory novel isoform. TP increased [Ca 2+ ] i at 2 hours by 30% (Fig B), with subsequent return to baseline at 24 hrs. Ca 2+ activates calpain, which breaks down proteins. Decreasing calpain activity during TP with PD150606 (20 µM) prevented TP enhancement of I KAChc (Fig C). Decreasing [Ca 2+ ] i during TP with BAPTA-AM (1 µM), a cell permeable Ca 2+ chelator, also prevented TP-enhancement of I KAChc (Fig D). Both PD150606 and BAPTA-AM prevented downregulation of PKCα protein expression by TP. Exposure of TP cells to a PKCε inhibitor (Fig E) suppressed TP-induced I KAChc activation, whereas a PKCα inhibitor (Fig F) enhanced I KAChc activity in 1 Hz cells. Conclusions: Rate-related [Ca 2+ ] i loading due to ATR causes a PKC isoform switch, with downregulation of inhibitory PKCα and membrane translocation (activation) of stimulatory PKCε, which enhances I KAChc causing APD shortening and AF promotion.

HSPB1, HSPB6, HSPB7 and HSPB8 Protect against RhoA GTPase-Induced Remodeling in Tachypaced Atrial Myocytes

BackgroundWe previously demonstrated the small heat shock protein, HSPB1, to prevent tachycardia remodeling in in vitro and in vivo models for Atrial Fibrillation (AF). To gain insight into its mechanism of action, we examined the protective effect of all 10 members of the HSPB family on tachycardia remodeling. Furthermore, modulating effects of HSPB on RhoA GTPase activity and F-actin stress fiber formation were examined, as this pathway was found of prime importance in tachycardia remodeling events and the initiation of AF.Methods and ResultsTachypacing (4 Hz) of HL-1 atrial myocytes significantly and progressively reduced the amplitude of Ca2+ transients (CaT). In addition to HSPB1, also overexpression of HSPB6, HSPB7 and HSPB8 protected against tachypacing-induced CaT reduction. The protective effect was independent of HSPB1. Moreover, tachypacing induced RhoA GTPase activity and caused F-actin stress fiber formation. The ROCK inhibitor Y27632 significantly prevented tachypacing-induced F-actin formation and CaT reductions, showing that RhoA activation is required for remodeling. Although all protective HSPB members prevented the formation of F-actin stress fibers, their mode of action differs. Whilst HSPB1, HSPB6 and HSPB7 acted via direct prevention of F-actin formation, HSPB8-protection was mediated via inhibition of RhoA GTPase activity.ConclusionOverexpression of HSPB1, as well as HSPB6, HSPB7 and HSPB8 independently protect against tachycardia remodeling by attenuation of the RhoA GTPase pathway at different levels. The cardioprotective role for multiple HSPB members indicate a possible therapeutic benefit of compounds able to boost the expression of single or multiple members of the HSPB family.

Effects of different small HSPB members on contractile dysfunction and structural changes in a Drosophila melanogaster model for Atrial Fibrillation

The most common clinical tachycardia, Atrial Fibrillation (AF), is a progressive disease, caused by cardiomyocyte remodeling, which finally results in contractile dysfunction and AF persistence. Recently, we identified a protective role of heat shock proteins (HSPs), especially the small HSPB1 member, against tachycardia remodeling in experimental AF models. Our understanding of tachycardia remodeling and anti-remodeling drugs is currently hampered by the lack of suitable (genetic) manipulatable in vivo models for rapid screening of key targets in remodeling. We hypothesized that Drosophila melanogaster can be exploited to study tachycardia remodeling and protective effects of HSPs by drug treatments or by utilizing genetically manipulated small HSP-overexpressing strains. Tachypacing of Drosophila pupae resulted in gradual and significant cardiomyocyte remodeling, demonstrated by reduced contraction rate, increase in arrhythmic episodes and reduction in heart wall shortening, compared to normal paced pupae. Heat shock, or pre-treatment with HSP-inducers GGA and BGP-15, resulted in endogenous HSP overexpression and protection against tachycardia remodeling. DmHSP23 overexpressing Drosophilas were protected against tachycardia remodeling, in contrast to overexpression of other small HSPs (DmHSP27, DmHSP67Bc, DmCG4461, DmCG7409, and DmCG14207). (Ultra)structural evaluation of the tachypaced heart wall revealed loss of sarcomeres and mitochondrial damage which were absent in tachypaced DmHSP23 overexpressing Drosophila. In addition, tachypacing induced a significant increase in calpain activity, which was prevented in tachypaced Drosophila overexpressing DmHSP23. Tachypacing of Drosophila resulted in cardiomyocyte remodeling, which was prevented by general HSP-inducing treatments and overexpression of a single small HSP, DmHSP23. Thus, tachypaced D. melanogaster can be used as an in vivo model system for rapid identification of novel targets to combat AF associated cardiomyocyte remodeling.

The role of pulmonary veins vs. autonomic ganglia in different experimental substrates of canine atrial fibrillation

Pulmonary vein (PV)-encircling ablation, which is effective in suppressing atrial fibrillation (AF), damages autonomic ganglia near the PV ostia. This study examined the effects of PV isolation (PVI) vs. peri-PV ganglionic plexus ablation (GPA) in two discrete canine AF models: ventricular tachypacing (240 bpm, 2 weeks)-induced congestive heart failure (CHF), and atrial tachypacing (400 bpm, 1 week)-induced atrial tachycardia remodeling (ATR). All PVs were isolated with an epicardial radiofrequency clamp in nine CHF and eight ATR dogs. Peri-PV ganglionic plexi (identified by bradycardic responses to high-frequency stimulation) were ablated in six CHF and five ATR dogs with an epicardial radiofrequency-ablation pen. Electrophysiologic measurements, including 240-electrode AF mapping, were obtained and dominant frequencies (DFs) determined. Atrial growth associated protein-43 (GAP-43) and neurofilament-M (NF-M) expression were determined immunohistologically. In CHF, neither PVI nor GPA affected AF duration, DF or the already low AF vulnerability. In ATR, PVI reduced AF vulnerability (75 ± 6% to 55 ± 11%, P< 0.05) but did not alter AF duration or DF. In contrast, GPA prolonged atrial refractory period and decreased AF vulnerability (75 ± 8 to 30 ± 10%, P< 0.05), AF duration (617 ± 246 to 39 ± 23 s, **P< 0.01), and DF (11.4 ± 0.6 to 8.6 ± 0.3** Hz, left atrium) in ATR dogs. Both GAP-43 and NF-M expression were decreased in CHF (by 63.1** and 60.0%**) and increased in ATR (by 65.5** and 92.1%, P< 0.001) compared with control. PVs play a minor role in experimental AF due to CHF or ATR, but autonomic ganglia are important in AF related to ATR. Differential neural remodelling may contribute to varying effects of GPA in discrete AF substrates.

Multiple Potential Molecular Contributors to Atrial Hypocontractility Caused by Atrial Tachycardia Remodeling in Dogs

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. 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. 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.

The involvement of ZnT‐1, a new modulator of cardiac L‐type calcium channels, in remodeling atrial tachycardia

Atrial fibrillation (AF), the highest occurring cardiac arrhythmia in the Western world, is associated with substantial morbidity and increased mortality. In spite of extensive research, the cause of atrial electrical remodeling, a major factor in the self‐perpetuating nature of AF, is still unknown. Downregulation of L‐type Ca2+ channel (LTCC) activity is the hallmark of atrial electrical remodeling. ZnT‐1 is a ubiquitous membrane protein that was recently suggested to inhibit the LTCC. We have studied and shown that ZnT‐1 expression inhibits LTCC function in an oocyte expression system as well as in isolated cardiomyocytes. Our data also show that rapid electrical pacing can augment ZnT‐1 expression in culture as well as in the atria of rats in vivo. Finally, in a pilot study, ZnT‐1 expression was found to be augmented in the atria of AF patients. These findings position ZnT‐1 as a probable missing link in the mechanism underlying atrial tachycardia remodeling.

Differences in atrial fibrillation properties under vagal nerve stimulation versus atrial tachycardia remodeling

There are many similarities between atrial effects of atrial tachycardia remodeling (ATR) and vagal nerve stimulation (VS): both promote atrial fibrillation (AF), reduce atrial effective refractory period (AERP) and AERP rate accommodation, enhance AERP heterogeneity, and increase inward-rectifier K+ current. This study sought to compare the consequences of ATR and VS at similar levels of AERP abbreviation in dogs. ATR dogs (n = 6) were subjected to 7-day atrial tachypacing at 400 beats/min, with radiofrequency-induced atrioventricular block and ventricular demand pacing (80 beats/min) to control ventricular response. VS was applied in 6 matched dogs with stimulation parameters selected to produce similar mean AERP values to ATR dogs. ATR and VS produced similarly short AERPs (79 +/- 12 and 80 +/- 12 ms, respectively), AERP rate-adaptation loss, and AERP heterogeneity increases. Although both ATR and VS increased AF duration, VS was significantly more effective in AF promotion, with mean AF duration of 992 +/- 134 seconds, versus 440 +/- 240 seconds (P <.05) under ATR. The greater AF-promoting effect of VS was associated with greater mean dominant frequency values during AF (11.7 +/- 1.8 versus 10.0 +/- 1.3 Hz ATR, P <.05). VS greatly enhanced the spatial dominant frequency variability, increasing the coefficient of variation to 15.2 +/- 1.9 Hz, versus 8.9 +/- 1.5 Hz for ATR (P <.05), primarily by increasing the per-dog maximum dominant frequency (15.4 +/- 0.6 Hz versus 12.5 +/- 0.6 for ATR, P <.01). For matched AERP values, VS promotes AF more strongly than ATR. Despite similar AERP changes, VS produces considerably greater increases in dominant frequencies, particularly maximum values, consistent with previous suggestions that inward-rectifier K+ current enhancement is particularly effective at accelerating and stabilizing spiral wave rotors that maintain AF.

Abstract 1516: Role of Oxidative Stress in Atrial Tachycardia Remodelling

Atrial tachycardia remodeling (ATR) promotes atrial fibrillation (AF). Oxidant stress (OxS) occurs in atria of AF patients and antioxidants may be beneficial in AF. This study used a previously validated in vitro paced canine cardiomyocyte model to assess the potential role of OxS in ATR. Cultured canine atrial cardiomyocytes were paced at 1 or 3 Hz (P1, P3) for 24 hrs. I CaL was recorded with whole cell voltage clamp. Single cell superoxide production was assessed by dihydroethidium fluorescence (DHEF) imaging. ATR (3 Hz pacing x 24 hrs) decreased I CaL (Fig. A ) and induced OxS (Fig. B ). Short term induction of OxS (H 2 O 2 100 μM x 10 mins) increased I CaL (Fig. C ) and enhanced Ca 2+ loading (Indo-1 AM). 24-Hr H 2 O 2 100 μ M increased DHEF in P1 cells by 250%* (*P&lt;0.05) and mimicked ATR, decreasing I CaL by 51%* (Fig. C ). H 2 O 2 -mediated DHEF changes were suppressed by inhibiting calmodulin (W7) or CaMKII (KN93). H 2 O 2 -induced I CaL suppression at 1 Hz was prevented by: decreasing Ca 2+ i loading by I CaL blockade (nimodipine) or Ca 2+ chelation (BAPTA-AM); W7 or KN93; antioxidants (2-MPG or N-acetylcysteine, NAC); or suppression of free radical generation via NADPH-oxidase (apocynin). 2-MPG, NAC, and apocynin prevented I CaL downregulation by 3-Hz pacing and W7, KN93 and apocynin abolished ATR-induced DHEF increases. H 2 O 2 mimics ATR by causing Ca 2+ i loading and CaMKII activation coupled to NADPH-oxidase stimulation. ATR-induced I CaL -downregulation is mediated in part by OxS generation. These findings provide insights into the pathways by which OxS contributes to ATR and present a mechanistic framework for understanding the effects of antioxidant interventions in AF.

Cellular Signaling Underlying Atrial Tachycardia Remodeling of L-type Calcium Current

Atrial tachycardia (AT) downregulates L-type Ca(2+) current (I(CaL)) and causes atrial fibrillation-promoting electric remodeling. This study assessed potential underlying signal transduction. Cultured adult canine atrial cardiomyocytes were paced at 0, 1, or 3 Hz (P0, P1, P3) for up to 24 hours. Cellular tachypacing (P3) mimicked effects of in vivo AT: decreased I(CaL) and transient outward current (I(to)), unchanged I(CaT), I(Kr), and I(Ks), and reduced action potential duration (APD). I(CaL) was unchanged in P3 at 2 and 8 hours but decreased by 55+/-6% at 24 hours. Tachypacing caused Ca(2+)(i) accumulation in P3 cells at 2 to 8 hours, but, by 24 hours, Ca(2+)i returned to baseline. Ca(v)1.2 mRNA expression was not altered at 2 hours but decreased significantly at 8 and 24 hours (32+/-4% and 48+/-4%, respectively) and protein expression was decreased (47+/-8%) at 24 hours only. Suppressing Ca(2+)(i) increases during tachypacing with the I(CaL) blocker nimodipine or the Ca(2+) chelator BAPTA-AM prevented I(CaL) downregulation. Calcineurin activity increased in P3 at 2 and 8 hours, respectively, returning to baseline at 24 hours. Nuclear factor of activated T cells (NFAT) nuclear translocation was enhanced in P3 cells. Ca(2+)-dependent signaling was probed with inhibitors of Ca(2+)/calmodulin (W-7), calcineurin (FK-506), and NFAT (INCA6): each prevented I(CaL) downregulation. Significant APD reductions ( approximately 30%) at 24 hours in P3 cells were prevented by nimodipine, BAPTA-AM, W-7, or FK-506. Thus, rapid atrial cardiomyocyte activation causes Ca(2+) loading, which activates the Ca(2+)-dependent calmodulin-calcineurin-NFAT system to cause transcriptional downregulation of I(CaL), restoring Ca(2+)i to normal at the cost of APD reduction. These studies elucidate for the first time the molecular feedback mechanisms underlying arrhythmogenic AT remodeling.

Atrial Remodeling and Atrial Fibrillation

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. It can occur at any age but is very rare in children and becomes extremely common in the elderly, with a prevalence approaching 20% in patients >85 years of age.1 AF is associated with a wide range of potential complications and contributes significantly to population morbidity and mortality. Present therapeutic approaches to AF have major limitations, including limited efficacy and significant adverse effect liability. These limitations have inspired substantial efforts to improve our understanding of the mechanisms underlying AF, with the premise that improved mechanistic insights will lead to innovative and improved therapeutic approaches.2 Our understanding of AF pathophysiology has advanced significantly over the past 10 to 15 years through an increased awareness of the role of “atrial remodeling.” Any persistent change in atrial structure or function constitutes atrial remodeling. Many forms of atrial remodeling promote the occurrence or maintenance of AF by acting on the fundamental arrhythmia mechanisms illustrated in Figure 1. Both rapid ectopic firing and reentry can maintain AF. Reentry requires a suitable vulnerable substrate, as well as a trigger that acts on the substrate to initiate reentry. Ectopic firing contributes to reentry by providing triggers for reentry induction. Atrial remodeling has the potential to increase the likelihood of ectopic or reentrant activity through a multitude of potential mechanisms. This article reviews the types of atrial remodeling, their underlying pathophysiology, the molecular basis of their occurrence, and finally, their potential therapeutic significance. Figure 1. General schema representing AF mechanisms and the role of remodeling. The mechanisms underlying AF are portrayed schematically in Figure 2. AF can be maintained by rapid focal firing, which may itself be regular but result in fibrillatory activity because of wave breakup in portions of the atrium that …

Cardiomyocyte Ca2+ overload in atrial tachycardia: is blockade of L-type Ca2+ channels a promising approach to prevent electrical remodeling and arrhythmogenesis?

Electrical remodeling paradigm has important implications for the understanding of atrial fibrillation (AF) and improvement of current treatment. Cardiomyocyte Ca(2+) overload is generally accepted as the initiating signal for the tachycardia-induced changes in atrial electrical properties (electrical remodeling). The precise role of cardiomyocyte Ca(2+) overload in AF-related ion channel alterations that contribute to AF maintenance is not fully understood. Clinically, patients with AF are often treated with Ca(2+) channel blockers such as verapamil to control their ventricular rate and to improve the success rate of cardioversion procedures. However, verapamil may produce an increased L-type Ca(2+) channel current (I(Ca,L)) that may reinforce Ca(2+) overload thereby promoting AF in the atrium. Ca(2+) channel blockers which target T-type Ca(2+) channels in addition to I(Ca,L) (for instance, efonidipine) may be more efficient at preventing Ca(2+) overload and arrhythmogenic electrical remodeling, but the potential benefits of these drugs have usually been tested in experimental models where drug administration preceded the initiation of electrical remodeling. Studies in animal models with established atrial tachycardia remodeling and in patients with AF are clearly warranted to prove the efficacy of Ca(2+) channel blockers that additionally target T-type Ca(2+) channels.

Omega-3 Polyunsaturated Fatty Acids Prevent Atrial Fibrillation Associated With Heart Failure but Not Atrial Tachycardia Remodeling

There is epidemiological evidence that omega-3 polyunsaturated fatty acids (PUFAs) reduce the risk of atrial fibrillation (AF), but clinical data are conflicting. The present study assessed the effects of PUFA on AF in experimental models. We studied the effects of oral PUFA supplements in 2 experimental AF paradigms: electrical remodeling induced by atrial tachypacing (400 bpm for 1 week) and congestive heart failure-associated structural remodeling induced by ventricular tachypacing (240 bpm for 2 weeks). PUFA pretreatment did not directly change atrial effective refractory period (128+/-6 [mean+/-SEM] versus 127+/-2 ms; all effective refractory periods at 300-ms cycle lengths) or burst pacing-induced AF duration (5+/-4 versus 34+/-18 seconds). Atrial tachypacing dogs had shorter refractory periods (73+/-6 ms) and greater AF duration (1185+/-300 seconds) than shams (119+/-5 ms and 20+/-11 seconds; P<0.01 for each). PUFAs did not significantly alter atrial tachypacing effects on refractory periods (77+/-8 ms) or AF duration (1128+/-412 seconds). PUFAs suppressed ventricular tachypacing-induced increases in AF duration (952+/-221 versus 318+/-249 seconds; P<0.05) and attenuated congestive heart failure-related atrial fibrosis (from 19.2+/-1.1% to 5.8+/-1.0%; P<0.001) and conduction abnormalities. PUFAs also attenuated ventricular tachypacing-induced hemodynamic dysfunction (eg, left ventricular end-diastolic and left atrial pressure from 12.2+/-0.5 and 11.4+/-0.6 mm Hg, respectively, to 6.4+/-0.5 and 7.0+/-0.8 mm Hg; P<0.01) and phosphorylation of mitogen-activated protein kinases (extracellular-signal related and P38 kinase). PUFAs suppress congestive heart failure-induced atrial structural remodeling and AF promotion but do not affect atrial tachycardia-induced electrical remodeling. The beneficial effects of PUFAs on structural remodeling, possibly related to prevention of mitogen-activated protein kinase activation, may contribute to their clinical anti-AF potential.

Abstract 354: ZnT-1 Expression in the Human Atria and New Insights on its Activity as an Inhibitor of L-type Calcium Channels

BACKGROUND : Recent findings indicate that the membranous protein ZnT-1 acts as an inhibitor of L-type calcium channels (LTCC). However, no direct interaction between ZnT-1 and the LTCC α 1 C subunit was demonstrated. Recently, we found that atrial tachypacing in rats augments the expression of ZnT-1 in conjunction with AERP reduction. Therefore, we postulated that ZnT-1 may play a role in atrial tachycardia remodeling of patients with AF. In this study, we assessed the atrial expression of ZnT-1 in humans with AF or sinus rhythm (SR). In addition, in order to explore the mechanism of action of ZnT-1 we used heterologous expression system, and tested the possibility that ZnT-1 interacts with the β-subunit of the LTCC. METHODS AND RESULTS : From 39 patients (27 in SR and 12 in AF) undergoing open heart surgery, we collected right atrial appendage tissue. The expression of ZnT-1 was analyzed. β-actin served as an internal loading control and data was compared to a rat standard (STD). Overall, AF patients (n = 12) had median ZnT-1/β-actin of 1.80 STD (inter-quartile range 1.26 to 2.85) vs. 0.73 STD (0.24 to 1.64) in the SR group (p = 0.002). Multivariate analysis determined that AF and increased BMI are the only independent variables associated with higher levels of ZnT-1. In Xenopus oocytes, the absence of the β-subunit prevented the inhibition of LTCC activity by ZnT-1 (127 ± 43nA and 153 ± 39nA for control and ZnT-1 containing oocytes, respectively, p&gt;0.5, n = 8). In addition, ZnT-1 failed to inhibit the current when the α-subunit was truncated in the part essential for the interaction with the β subunit (2037 ± 300nA and 1938 ± 341nA for control and ZnT-1 containing oocytes, respectively, p&gt;0.5, n = 3). Direct interaction between ZnT-1 and the LTCC β-subunit was demonstrated by co-immunoprecipitation of ZnT-1-myc and the β-subunit, using anti-myc antibodies (n = 3) CONCLUSIONS : This work provides first evidence for an increased ZnT-1 expression in patients with AF and marks it as a putative missing link in the development of atrial electrical remodeling. An interaction of the LTCC β-subunit and the ZnT-1 plays a role in the molecular mechanism by which ZnT-1 inhibits the LTCC.

Induction of Heat Shock Response Protects the Heart Against Atrial Fibrillation

There is evidence suggesting that heat shock proteins (HSPs) may protect against clinical atrial fibrillation (AF). We evaluated the effect of HSP induction in an in vitro atrial cell line (HL-1) model of tachycardia remodeling and in tachypaced isolated canine atrial cardiomyocytes. We also evaluated the effect of HSP induction on in vivo AF promotion by atrial tachycardia-induced remodeling in dogs. Tachypacing (3 Hz) significantly and progressively reduced Ca(2+) transients and cell shortening of HL-1 myocytes over 4 hours. These reductions were prevented by HSP-inducing pretreatments: mild heat shock, geranylgeranylacetone (GGA), and transfection with human HSP27 or the phosphorylation-mimicking HSP27-DDD. However, treatment with HSP70 or the phosphorylation-deficient mutant HSP27-AAA failed to alter tachycardia-induced Ca(2+) transient and cell-shortening reductions, and downregulation (short interfering RNA) of HSP27 prevented GGA-mediated protection. Tachypacing (3 Hz) for 24 hours in vitro significantly reduced L-type Ca(2+) current and action potential duration in canine atrial cardiomyocytes; these effects were prevented when tachypacing was performed in cells exposed to GGA. In vivo treatment with GGA increased HSP expression and suppressed refractoriness abbreviation and AF promotion in dogs subjected to 1-week atrial tachycardia-induced remodeling. In conclusion, our findings indicate that (1) HSP induction protects against atrial tachycardia-induced remodeling, (2) the protective effect in HL-1 myocytes requires HSP27 induction and phosphorylation, and (3) the orally administered HSP inducer GGA protects against AF in a clinically relevant animal model. These findings advance our understanding of the biochemical determinants of AF and suggest the possibility that HSP induction may be an interesting novel approach to preventing clinical AF.

Abstract 190: HSP27 is Sufficient and Required for Prevention of Tachycardia Induced Remodeling in a Cell Model for Tachypacing

We previously found evidence that heat shock protein (HSP) induction protects against the promotion of atrial fibrillation (AF) by atrial tachypacing-remodeling in dogs. In this study, we evaluated the effect of HSP induction in a tachypaced atrial cell-line (HL-1) model of tachycardia-remodeling. Methods: The effects of tachypacing HL-1 myocytes (3 Hz, 0 – 4 hrs) on Ca 2+ transients (CaT) and cell shortening (CS), were studied with and without HSP-inducing pre-treatments like mild heat shock (HS), or the non-toxic inducer geranylgeranylacetone (GGA), transient transfection of human HSP70, HSP27, a phosphorylation deficient mutant HSP27-AAA or the phospho-mimicking mutant HSP27-DDD. Knockdown of HSP27 was performed with short-hairpin forming siRNA. Results: In HL-1 myocytes, tachypacing significantly and progressively reduced CaT and CS after 4 hours (Fig A ). HSP induction by HS or GGA protected HL-1 myocytes against suppression of CaT and CS by 4 hours of tachypacing. Protective effects were also seen upon transfection with HSP27 and a phosphorylation-mimicking HSP27 mutant, but not by HSP70 or a non-phosphorylatable HSP27-mutant construct (Fig B ). Knockdown of HSP27 with shRNA prevented GGA-mediated protection (Fig C ). Conclusions: Our findings indicate that HSP27 induction protects against AF-promoting remodeling, that the protective effect requires HSP27 phosphorylation, and HSP27 is sufficient for protection. These findings advance our understanding of the biochemical determinants of the protective effects of HSP induction against atrial tachycardia remodeling.

P3-17: Electrophysiological and atrial antiarrhythmic effects of a novel IKUR/Kv1.5 blocker in dogs with atrial tachycardia remodeling

Presently-available antiarrhythmic agents for atrial fibrillation (AF) have a potentially serious risk of proarrhythmia due to collateral ventricular actions. The ultrarapid delayed rectifier current IKur has atrial-specific expression in man and is a promising new antiarrhythmic target. In this study, we evaluated the effects of a novel selective IKur/Kv1.5 blocker, XEN-D0101, on electrophysiology and AF indexes in atrial tachycardia remodeled dogs.