Our sisters the plants? notes from phylogenetics and botany on plant kinship blindness.

Before the upheaval brought about by phylogenetic classification, classical taxonomy separated living beings into two distinct kingdoms, animals and plants. Rooted in 'naturalist' cosmology, Western science has built its theoretical apparatus on this dichotomy mostly based on ancient Aristotelian ideas. Nowadays, despite the adoption of the Darwinian paradigm that unifies living organisms as a kinship, the concept of the "scale of beings" continues to structure our analysis and understanding of living species. Our aim is to combine developments in phylogeny, recent advances in biology, and renewed interest in plant agency to craft an interdisciplinary stance on the living realm. The lines at the origin of plant or animal have a common evolutionary history dating back to about 3.9 Ga, separating only 1.6 Ga ago. From a phylogenetic perspective of living species history, plants and animals belong to sister groups. With recent data related to the field of Plant Neurobiology, our aim is to discuss some socio-cultural obstacles, mainly in Western naturalist epistemology, that have prevented the integration of living organisms as relatives, while suggesting a few avenues inspired by practices principally from other ontologies that could help overcome these obstacles and build bridges between different ways of connecting to life.

Loss of cardiomyocyte integrin-linked kinase produces an arrhythmogenic cardiomyopathy in mice.

BACKGROUND: Integrin-linked kinase (ILK), a serine/threonine protein kinase, has roles in cell signaling and molecular scaffolding. ILK mutation/deletion causes cardiomyopathic phenotypes, but the functional and electrophysiological features have not been characterized. This study investigated the structural, functional, ion channel, and electrophysiological changes associated with cardiomyocyte-directed ILK deletion in mice. METHODS AND RESULTS: Adult mice with cardiomyocyte-directed ILK knockout were compared with littermate controls. Knockout mice showed markedly increased mortality, with sudden death beginning after 5 weeks and 100% mortality at 18 weeks. In 10-week-old knockout mice, spontaneous and inducible ventricular tachyarrhythmias were common, occurring in 60% and 86%, respectively, and absent in controls (P<0.001, P<0.05 versus knockout mice). Ventricular refractoriness was prolonged, along with both QRS and QT interval. Action potentials were prolonged and displayed triggered activity. A wide range of ion currents were downregulated, including total, fast and slow components of transient outward K(+) current and inward rectifier K(+) current, along with corresponding ion channel subunit genes, providing a plausible explanation of action potential prolongation. At 5 weeks, only voltage-dependent K(+) currents were reduced, possibly related to direct ILK-Kv4.2 subunit interactions. Action potentials were prolonged, but no arrhythmias or cardiac dysfunction were noted. Structural remodeling was prominent at 10 weeks: connexin-43 was downregulated and redistributed to lateral cell margins, and left ventricular fibrosis occurred, with a strong regional distribution (predominating in the basal left ventricle). Conduction was slowed. High-throughput quantitative polymerase reaction gene-expression studies in 10-week-old ILK knockout showed upregulation of structural, remodeling and fibrosis-related genes, and downregulation of a wide range of ion channel and transporter subunits. CONCLUSIONS: Cardiomyocyte ILK deletion produces a lethal arrhythmogenic cardiomyopathy associated with important ion channel and structural remodeling.

Differential effectiveness of pharmacological strategies to reveal dormant pulmonary vein conduction: a clinical-experimental correlation.

BACKGROUND: Atrial fibrillation recurs in ∼30%-40% of patients after pulmonary vein (PV) isolation (PVI) procedures, often because of restored PV-left atrial (LA) conduction. Adenosine or isoproterenol are used clinically to reveal dormant PV conduction and guide additional ablation. OBJECTIVE: The purpose of this study was to assess the differential efficacy of adenosine and/or isoproterenol in revealing dormant PV conduction. METHODS: In 25 patients undergoing PVI, dormant conduction was assessed sequentially in response to intravenous adenosine, isoproterenol, and adenosine plus isoproterenol in 100 PVs. To study mechanisms, PVs were isolated by radiofrequency ablation in coronary-perfused canine LA-PV preparations. After PVI, resting membrane potential from PV cells was recorded before and after 1 mM adenosine, 1 µM isoproterenol, 1 µM isoproterenol plus 1 mM adenosine, or no drug (controls). RESULTS: Clinical PVI was successful in all 100 PVs, with dormant conduction in 31. Sensitivity for dormant conduction was isoproterenol 10%; adenosine 87% (P <.001 vs. isoproterenol); and isoproterenol + adenosine 100% (P = .13 vs. adenosine). Dormant PV conduction in vitro was revealed with adenosine (53%) and adenosine + isoproterenol (60%) but not with isoproterenol alone or in controls (P <.01). Radiofrequency lesions producing PVI depolarized resting membrane potential, causing inexcitability. Postablation, resting membrane potential hyperpolarized after both adenosine and isoproterenol, but adenosine-induced changes were greater (9.1 ± 0.6 mV, vs. 3.8 ± 0.6 mV; P <0.001), with no significant additional effect when isoproterenol was added to adenosine. CONCLUSION: Adenosine is superior to isoproterenol in revealing dormant PVs clinically and experimentally because of more effective adenosine-induced hyperpolarization. Adding isoproterenol to adenosine had no significant additional value.

Arrhythmogenic left atrial cellular electrophysiology in a murine genetic long QT syndrome model.

AIMS: Increasing evidence indicates that congenital long QT syndromes (LQTSs) promote atrial fibrillation. The atrial action potential (AP) has a short plateau, and whether LQTS atrial cardiomyocytes generate triggered activity via early afterdepolarizations (EADs) is unclear. Atrial cellular arrhythmia mechanisms have not been defined in congenital LQTS. Therefore, we studied atrial cardiomyocyte electrophysiology in mice with an LQTS3 SCN5A inactivation-impairing mutation (ΔKPQ heterozygotes). METHODS AND RESULTS: Peak and late Na(+) current (I(NaP) and I(NaL)) were measured with whole-cell patch clamp in left atrial (LA) cardiomyocytes. APs were recorded in multicellular LA preparations with floating microelectrodes. I(NaL) was increased by 110% in LA cardiomyocytes of ΔKPQ mice, whereas I(NaP) was unchanged. AP duration (APD) was prolonged over all frequencies in ΔKPQ mice, but particularly at lower frequencies [e.g. APD(90) at 0.5 Hz: 197 ± 8 ms vs. wild-type (WT) 82 ± 2 ms, P< 0.001]. EADs occurred at 0.5 Hz in 10/18 ΔKPQ (56%) vs. 1/10 WT (10%) atria (P< 0.05). EADs immediately preceded premature APs in other LA regions, suggesting triggered activity. Ranolazine preferentially inhibited I(NaL) (50% inhibitory concentration: 12.5 vs. 151.8 µM for I(NaP)) in ΔKPQ myocytes. At 10 µM, ranolazine shortened APD (e.g. APD(90) at 0.5 Hz to 122 ± 4 ms, P= 0.01) without changing APD in WT and suppressed EAD occurrence and triggered activity (from 10/18 to 1/9 preparations, 11%, P< 0.05). CONCLUSION: This study implicates increased I(NaL) in excessive atrial APD prolongation and arrhythmic EAD occurrence in a congenital LQTS3 mouse model. Our observations provide the first direct demonstration of atrial EADs and triggered activity in a genetically defined animal model of human LQTS and have potential clinically-relevant mechanistic and therapeutic implications.

Mechanisms of atrial tachyarrhythmias associated with coronary artery occlusion in a chronic canine model.

BACKGROUND: Coronary artery disease predisposes to atrial fibrillation (AF), but the effects of chronic atrial ischemia/infarction on AF-related substrates are unknown. METHODS AND RESULTS: Regional right atrial myocardial infarction (MI) was created in 40 dogs by ligating an artery that supplies the right atrial free wall and not the ventricles; 35 sham dogs with the same artery isolated but not ligated were controls. Dogs were observed 8 days after MI and subjected to open-chest study, in vitro optical mapping, and/or cell isolation for patch-clamp and Ca(2+) imaging on day 8. Holter ECGs showed more spontaneous atrial ectopy in MI dogs (eg, 662±281 on day 7 versus 34±25 ectopic complexes per day at baseline; 52±21 versus 1±1 atrial tachycardia episodes per day). Triggered activity was increased in MI border zone cells, which had faster decay of caffeine-evoked Ca(2+) transients and enhanced (by ≈73%) Na(+)-Ca(2+) exchange current. Spontaneous Ca(2+) sparks (confocal microscopy) occurred under ß-adrenergic stimulation in more MI dog cells (66±9%) than in control cells (29±4%; P<0.01). Burst pacing induced long-lasting AF in MI dogs (1146±259 versus 30±14 seconds in shams). Increased border zone conduction heterogeneity was confirmed by both bipolar electrode mapping in vivo and optical mapping. Optical mapping demonstrated stable border zone reentry in all 9 MI preparations but in none of 6 shams. Border zone tissue showed increased fibrous tissue content. CONCLUSIONS: Chronic atrial ischemia/infarction creates substrates for both spontaneous ectopy (Ca(2+)-release events, increased Na(+)-Ca(2+) exchange current) and sustained reentry (conduction abnormalities that anchor reentry). Thus, chronic atrial infarction in dogs promotes both AF triggers and the substrate for AF maintenance. These results provide novel insights into potential AF mechanisms in patients with coronary artery disease.

Role of constitutively active acetylcholine-mediated potassium current in atrial contractile dysfunction caused by atrial tachycardia remodelling.

AIMS: Atrial fibrillation (AF)-induced contractile dysfunction contributes importantly to thrombo-embolic stroke, the most serious AF complication. Atrial cardiomyocytes have a constitutively active acetylcholine-regulated K(+)-current (I(KAChc)) that is enhanced by atrial tachycardia (AT). I(KAChc) contributes to action potential duration (APD) shortening in AT-remodelled atrial cardiomyocytes; APD regulates contractility by controlling Ca(2+)-loading and systolic Ca(2+)-release. This study investigated the potential role of I(KAChc) in AF-related contractile dysfunction. METHODS AND RESULTS: Dogs were divided into two groups: (i) unpaced control (CTL); (ii) AT (400 bpm for at least 7 days). Tertiapin-Q (TQ), a selective I(KAChc) blocker, was used to define I(KAChc) contributions to contractility. Single-cell left atrial (LA) intracellular Ca(2+)-transients (CaTrs), cell-shortening (CS), and whole LA tissue tension-generation were measured. Atrial tachycardia increased I(KAChc). Whole LA contractility was decreased in AT (0.17 ± 0.05 g) compared with CTL (0.40 ± 0.09 g), with significant reversal (0.30 ± 0.06 g) after TQ administration. Ca(2+)-transient amplitude and CS in single-cell were decreased by AT compared with CTL (167 ± 14 vs. 88 ± 10 nM; 10.3 ± 1.3 vs. 1.7 ± 0.3 µm, respectively; P < 0.001). The AT-induced reductions in single-cell CaTr amplitude and CS were partly reversed by TQ administration (88 ± 10 vs. 112 ± 16 nM; P < 0.001; 1.7 ± 0.3 vs. 3.6 ± 0.7 µm; P < 0.01). We then measured CaTr and CS with carbachol and/or TQ to vary I(KACh) at various extracellular [Ca(2+)]. The CaTr-CS relationship was linear and AT results fell on the regression line, indicating that AT-remodelling effects on contractility are attributable to reduced CaTr. CONCLUSION: Up-regulated I(KAChc) contributes to AF-related contractile dysfunction and could be a novel target to prevent hypocontractility-related thrombo-embolic complications.

Multiple potential molecular contributors to atrial hypocontractility caused by atrial tachycardia remodeling in dogs.

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.

Mechanisms by which adenosine restores conduction in dormant canine pulmonary veins.

BACKGROUND: Adenosine acutely reconnects pulmonary veins (PVs) after radiofrequency application, revealing "dormant conduction" and identifying PVs at risk of reconnection, but the underlying mechanisms are unknown. METHODS AND RESULTS: Canine PV and left-atrial (LA) action potentials were recorded with standard microelectrodes and ionic currents with whole-cell patch clamp before and after adenosine perfusion. PVs were isolated with radiofrequency current application in coronary-perfused LA-PV preparations. Adenosine abbreviated action potential duration similarly in PV and LA but significantly hyperpolarized resting potential (by 3.9+/-0.5%; P<0.05) and increased dV/dt(max) (by 34+/-10%) only in PV. Increased dV/dt(max) was not due to direct effects on I(Na), which was reduced similarly by adenosine in LA and PV but correlated with resting-potential hyperpolarization (r=0.80). Adenosine induced larger inward rectifier K(+)current (I(KAdo)) in PV (eg, -2.28+/-0.04 pA/pF; -100 mV) versus LA (-1.28+/-0.16 pA/pF). Radiofrequency ablation isolated PVs by depolarizing resting potential to voltages positive to -60 mV. Adenosine restored conduction in 5 dormant PVs, which had significantly more negative resting potentials (-57+/-6 mV) versus nondormant (-46+/-5 mV, n=6; P<0.001) before adenosine. Adenosine hyperpolarized both, but more negative resting-potential values after adenosine in dormant PVs (-66+/-6 mV versus -56+/-6 mV in nondormant; P<0.001) were sufficient to restore excitability. Adenosine effects on resting potential and conduction reversed on washout. Spontaneous recovery of conduction occurring in dormant PVs after 30 to 60 minutes was predicted by the adenosine response. CONCLUSIONS: Adenosine selectively hyperpolarizes canine PVs by increasing I(KAdo). PVs with dormant conduction show less radiofrequency-induced depolarization than nondormant veins, allowing adenosine-induced hyperpolarization to restore excitability by removing voltage-dependent I(Na) inactivation and explaining the restoration of conduction in dormant PVs.

The calcium/calmodulin/kinase system and arrhythmogenic afterdepolarizations in bradycardia-related acquired long-QT syndrome.

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.

Ion channel subunit expression changes in cardiac Purkinje fibers: a potential role in conduction abnormalities associated with congestive heart failure.

Purkinje fibers (PFs) play key roles in cardiac conduction and arrhythmogenesis. Congestive heart failure (CHF) causes well-characterized atrial and ventricular ion channel subunit expression changes, but effects on PF ion channel subunits are unknown. This study assessed changes in PF ion channel subunit expression (real-time PCR, immunoblot, immunohistochemistry), action potential properties, and conduction in dogs with ventricular tachypacing-induced CHF. CHF downregulated mRNA expression of subunits involved in action potential propagation (Nav1.5, by 56%; connexin [Cx]40, 66%; Cx43, 56%) and repolarization (Kv4.3, 43%, Kv3.4, 46%). No significant changes occurred in KChIP2, KvLQT1, ERG, or Kir3.1/3.4 mRNA. At the protein level, downregulation was seen for Nav1.5 (by 38%), Kv4.3 (42%), Kv3.4 (57%), Kir2.1 (26%), Cx40 (53%), and Cx43 (30%). Cx43 dephosphorylation was indicated by decreased larger molecular mass bands (pan-Cx43 antibody) and a 57% decrease in Ser368-phosphorylated Cx43 (phospho-specific antibody). Immunohistochemistry revealed reduced Cx40, Cx43, and phospho-Cx43 expression at intercalated disks. Action potential changes were consistent with observed decreases in ion channel subunits: CHF decreased phase 1 slope (by 56%), overshoot (by 32%), and phase 0 dV/dt(max) (by 35%). Impulse propagation was slowed in PF false tendons: conduction velocity decreased significantly from 2.2+/-0.1 m/s (control) to 1.5+/-0.1 m/s (CHF). His-Purkinje conduction also slowed in vivo, with HV interval increasing from 35.5+/-1.2 (control) to 49.3+/-3.4 ms (CHF). These results indicate important effects of CHF on PF ion channel subunit expression. Alterations in subunits governing conduction properties may be particularly important, because CHF-induced impairments in Purkinje tissue conduction, which this study is the first to describe, could contribute significantly to dyssynchronous ventricular activation, a major determinant of prognosis in CHF-patients.

Funny current downregulation and sinus node dysfunction associated with atrial tachyarrhythmia: a molecular basis for tachycardia-bradycardia syndrome.

BACKGROUND: Sinoatrial node (SAN) dysfunction is frequently associated with atrial tachyarrhythmias (ATs). Abnormalities in SAN pacemaker function after termination of ATs can cause syncope and require pacemaker implantation, but underlying mechanisms remain poorly understood. This study examined the hypothesis that ATs impair SAN function by altering ion channel expression. METHODS AND RESULTS: SAN tissues were obtained from 28 control dogs and 31 dogs with 7-day atrial tachypacing (400 bpm). Ionic currents were measured from single SAN cells with whole-cell patch-clamp techniques. Atrial tachypacing increased SAN recovery time in vivo by approximately 70% (P<0.01), a change which reflects impaired SAN function. In dogs that underwent atrial tachypacing, SAN mRNA expression (real-time reverse-transcription polymerase chain reaction) was reduced for hyperpolarization-activated cyclic nucleotide-gated subunits (HCN2 and HCN4) by >50% (P<0.01) and for the beta-subunit minK by approximately 42% (P<0.05). SAN transcript expression for the rapid delayed-rectifier (I(Kr)) alpha-subunit ERG, the slow delayed-rectifier (I(Ks)) alpha-subunit KvLQT1, the beta-subunit MiRP1, the L-type (I(CaL)) and T-type (I(CaT)) Ca2+-current subunits Cav1.2 and Cav3.1, and the gap-junction subunit connexin 43 (were unaffected by atrial tachypacing. Atrial tachypacing reduced densities of the HCN-related funny current (I(f)) and I(Ks) by approximately 48% (P<0.001) and approximately 34% (P<0.01), respectively, with no change in voltage dependence or kinetics. I(Kr), I(CaL), and I(CaT) were unaffected. SAN cells lacked Ba2+-sensitive inward-rectifier currents, irrespective of AT. SAN action potential simulations that incorporated AT-induced alterations in I(f) accounted for slowing of periodicity, with no additional contribution from changes in I(Ks). CONCLUSIONS: AT downregulates SAN HCN2/4 and minK subunit expression, along with the corresponding currents I(f) and I(Ks). Tachycardia-induced remodeling of SAN ion channel expression, particularly for the "pacemaker" subunit I(f), may contribute to the clinically significant association between SAN dysfunction and supraventricular tachyarrhythmias.

Calpain mediates cardiac troponin degradation and contractile dysfunction in atrial fibrillation.

The self-perpetuation of atrial fibrillation (AF) is associated with atrial remodeling, including the degradation of the myofibril structure (myolysis). Myolysis is related to AF-induced activation of cysteine proteases and underlies loss of contractile function. In this study, we investigated which proteases are involved in the degradation of myofibrillar proteins during AF and whether their inhibition leads to preservation of contractile function after AF. In tachypaced HL-1 cardiomyocytes and atrial tissue from AF and control patients, degradation of myofibrillar proteins troponin (cTn) T, I, C, human cTnT and actin was investigated by Western blotting, and contractile function was analyzed by cell-shortening measurements. The role of major proteases was determined by applying specific inhibitors. Tachypacing of HL-1 cardiomyocytes induced a gradual and significant degradation of cTns but not actin, and caused contractile dysfunction. Both were prevented by inhibition of calpain but not by inhibition of caspases or the proteasome. In patients with persistent AF, a significant degradation of cTnT, cTnI and cTnC was found compared to sinus rhythm or paroxysmal AF, which correlated significantly with both calpain activity and the amount of myolysis. Additionally, by utilizing tachypaced human cTnT-transfected HL-1 cardiomyocytes, we directly showed that the degradation of human cTnT was mediated by calpain and not by caspases or proteasome. Our results suggest that calpain inhibition may therefore represent a key target in combating AF-related structural and functional remodeling.

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.

Differential interactions of Na+ channel toxins with T-type Ca2+ channels.

Two types of voltage-dependent Ca(2+) channels have been identified in heart: high (I(CaL)) and low (I(CaT)) voltage-activated Ca(2+) channels. In guinea pig ventricular myocytes, low voltage-activated inward current consists of I(CaT) and a tetrodotoxin (TTX)-sensitive I(Ca) component (I(Ca(TTX))). In this study, we reexamined the nature of low-threshold I(Ca) in dog atrium, as well as whether it is affected by Na(+) channel toxins. Ca(2+) currents were recorded using the whole-cell patch clamp technique. In the absence of external Na(+), a transient inward current activated near -50 mV, peaked at -30 mV, and reversed around +40 mV (HP = -90 mV). It was unaffected by 30 microM TTX or micromolar concentrations of external Na(+), but was inhibited by 50 microM Ni(2+) (by approximately 90%) or 5 microM mibefradil (by approximately 50%), consistent with the reported properties of I(CaT). Addition of 30 microM TTX in the presence of Ni(2+) increased the current approximately fourfold (41% of control), and shifted the dose-response curve of Ni(2+) block to the right (IC(50) from 7.6 to 30 microM). Saxitoxin (STX) at 1 microM abolished the current left in 50 microM Ni(2+). In the absence of Ni(2+), STX potently blocked I(CaT) (EC(50) = 185 nM) and modestly reduced I(CaL) (EC(50) = 1.6 microM). While TTX produced no direct effect on I(CaT) elicited by expression of hCa(V)3.1 and hCa(V)3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni(2+) (IC(50) increased to 550 microM Ni(2+) for Ca(V)3.1 and 15 microM Ni(2+) for Ca(V)3.2); in contrast, 30 microM TTX directly inhibited hCa(V)3.3-induced I(CaT) and the addition of 750 microM Ni(2+) to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni(2+) alone. 1 microM STX directly inhibited Ca(V)3.1-, Ca(V)3.2-, and Ca(V)3.3-mediated I(CaT) but did not enhance the ability of Ni(2+) to block these currents. These findings provide important new implications for our understanding of structure-function relationships of I(CaT) in heart, and further extend the hypothesis of a parallel evolution of Na(+) and Ca(2+) channels from an ancestor with common structural motifs.

Antiarrhythmic properties of a rapid delayed-rectifier current activator in rabbit models of acquired long QT syndrome.

AIMS: Impaired repolarization in cardiac myocytes can lead to long QT syndrome (LQTS), with delayed repolarization and increased susceptibility to Torsades de Pointes (TdP) arrhythmias. Current pharmacological treatment of LQTS is often inadequate. This study sought to evaluate the antiarrhythmic effect of a novel compound (NS1643) that activates the rapid delayed-rectifier K+ current, I(Kr), in two rabbit models of acquired LQTS. METHODS AND RESULTS: We used two clinically relevant in vivo rabbit models of TdP in which we infused NS1643 or vehicle: (i) three-week atrioventricular block with ventricular bradypacing; (ii) dofetilide-induced I(Kr) inhibition in methoxamine-sensitized rabbits. In addition, we studied effects on ionic currents in cardiomyocytes with I(Kr) suppressed by bradycardia remodelling or dofetilide exposure. Bradypaced rabbits developed QT interval prolongation, spontaneous ventricular ectopy, and TdP. Infusion of NS1643 completely suppressed arrhythmic activity and shortened the QT interval; vehicle had no effect. NS1643 also suppressed ventricular tachyarrhythmias caused by infusion of dofetilide to methoxamine-sensitized rabbits, and reversed dofetilide-induced QT prolongation. NS1643 increased I(Kr) in cardiomyocytes isolated from normal and bradycardia-remodelled rabbits by approximately 75% and 50%, respectively (P < 0.001 for each). Similarly, NS1643 restored I(Kr) suppressed by 5 nmol/L dofetilide (tail current 0.28 +/- 0.03 pA/pF pre-dofetilide, 0.20 +/- 0.01 pA/pF in the presence of dofetilide, 0.27 +/- 0.02 pA/pF after adding NS1643 to dofetilide-containing solution, P < 0.01). CONCLUSION: Pharmacological activation of I(Kr) reverses acquired LQTS and TdP caused by bradycardic remodelling and I(Kr)-blocking drugs. I(Kr)-activating drug therapy could be a potentially interesting treatment approach for LQTS.

Pulmonary vein region ablation in experimental vagal atrial fibrillation: role of pulmonary veins versus autonomic ganglia.

BACKGROUND: Pulmonary vein (PV) -encircling radiofrequency ablation frequently is effective in vagal atrial fibrillation (AF), and there is evidence that PVs may be particularly prone to cholinergically induced arrhythmia mechanisms. However, PV ablation procedures also can affect intracardiac autonomic ganglia. The present study examined the relative role of PVs versus peri-PV autonomic ganglia in an experimental vagal AF model. METHODS AND RESULTS: Cholinergic AF was studied under carbachol infusion in coronary perfused canine left atrial PV preparations in vitro and with cervical vagal stimulation in vivo. Carbachol caused dose-dependent AF promotion in vitro, which was not affected by excision of all PVs. Sustained AF could be induced easily in all dogs during vagal nerve stimulation in vivo both before and after isolation of all PVs with encircling lesions created by a bipolar radiofrequency ablation clamp device. PV elimination had no effect on atrial effective refractory period or its responses to cholinergic stimulation. Autonomic ganglia were identified by bradycardic and/or tachycardic responses to high-frequency subthreshold local stimulation. Ablation of the autonomic ganglia overlying all PV ostia suppressed the effective refractory period-abbreviating and AF-promoting effects of cervical vagal stimulation, whereas ablation of only left- or right-sided PV ostial ganglia failed to suppress AF. Dominant-frequency analysis suggested that the success of ablation in suppressing vagal AF depended on the elimination of high-frequency driver regions. CONCLUSIONS: Intact PVs are not needed for maintenance of experimental cholinergic AF. Ablation of the autonomic ganglia at the base of the PVs suppresses vagal responses and may contribute to the effectiveness of PV-directed ablation procedures in vagal AF.

Calcium-handling abnormalities underlying atrial arrhythmogenesis and contractile dysfunction in dogs with congestive heart failure.

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.

Adrenergic control of a constitutively active acetylcholine-regulated potassium current in canine atrial cardiomyocytes.

OBJECTIVES: Canine atrial cardiomyocytes display a constitutively active, acetylcholine-regulated, time-dependent K+ current (IKH) that contributes to atrial repolarization and atrial tachycardia-induced atrial-fibrillation promotion. Adrenergic stimulation favors atrial arrhythmogenesis but its effects on IKH are poorly understood. METHODS AND RESULTS: Adrenergic modulation of IKH was studied in isolated canine atrial cardiomyocytes with whole-cell patch-clamping, and action-potential consequences were assessed in multicellular preparations with fine-tipped microelectrodes. Isoproterenol increased IKH in a concentration-dependent manner (maximum 103+/-22% increase), an effect mimicked by forskolin and 8-bromo-cyclic AMP. Isoproterenol effects were prevented by propranolol and the selective beta1-adrenoceptor blocker CGP-20712A, but not the beta2-blocker ICI-118551. Isoproterenol enhancement was prevented by pipette-administered protein kinase A (PKA) inhibitor peptide or by superfusion of H89 (PKA blocker). Phenylephrine decreased IKH in a reversible, concentration-dependent way. This effect was blocked by the alpha-antagonist prazosin and the selective alpha1A-blocker niguldipine, but not the alpha1B-blocker chloroethylclonidine or the alpha1D inhibitor BMY-7378. Phenylephrine effects were prevented by the phospholipase C (PLC) inhibitor U73122 and the protein kinase C (PKC) inhibitor bisindolylmaleimide. The PKC-activating phorbol ester PDD (but not its inactive analogue alpha-PDD) mimicked phenylephrine effects. Action potential recordings in the presence and absence of the selective IKH blocker tertiapin indicated a functional role of alpha- and beta-adrenergic actions on IKH. Adrenergic regulation of cholinergic agonist-induced K+ current paralleled that of IKH. CONCLUSIONS: IKH is under dual regulation by the adrenergic system: beta1-adrenergic stimulation enhances IKH via cAMP-dependent PKA pathways, whereas alpha1A-adrenergic stimulation inhibits IKH via PLC-mediated PKC activation. Modulation of constitutive acetylcholine-regulated K+ current is a novel potential mechanism for adrenergic control of atrial repolarization.

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.

Comparison of Ca2+-handling properties of canine pulmonary vein and left atrial cardiomyocytes.

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.