Xiao-Yan Qi

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 (Ca2+) handling contributes to the CHF-related atrial arrhythmogenic substrate. Methods and Results—CHF was induced in dogs by ventricular tachypacing (240 bpm ×2 weeks). Cellular Ca2+-handling properties and expression/phosphorylation status of key Ca2+ handling and myofilament proteins were assessed in control and CHF atria. CHF decreased cell shortening but increased left atrial diastolic intracellular Ca2+ concentration ([Ca2+]i), [Ca2+]i transient amplitude, and sarcoplasmic reticulum (SR) Ca2+ load (caffeine-induced [Ca2+]i release). SR Ca2+ overload was associated with spontaneous Ca2+ transient events and triggered ectopic activity, which was suppressed by the inhibition of SR Ca2+ release (ryanodine) or Na+/Ca2+ exchange. Mechanisms underlying abnormal SR Ca2+ handling were then studied. CHF increased atrial action potential duration and action potential voltage clamp showed that CHF-like action potentials enhance Ca2+i loading. CHF increased calmodulin-dependent protein kinase II phosphorylation of phospholamban by 120%, potentially enhancing SR Ca2+ uptake by reducing phospholamban inhibition of SR Ca2+ ATPase, but it did not affect phosphorylation of SR Ca2+-release channels (RyR2). Total RyR2 and calsequestrin (main SR Ca2+-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 Ca2+ transients. Complex phosphorylation changes were explained by enhanced calmodulin-dependent protein kinase II&dgr; expression and function and type-1 protein-phosphatase activity but downregulated regulatory protein kinase A subunits. Conclusions—CHF causes profound changes in Ca2+-handling and -regulatory proteins that produce atrial fibrillation–promoting atrial cardiomyocyte Ca2+-handling abnormalities, arrhythmogenic triggered activity, and contractile dysfunction.

Kir3-Based Inward Rectifier Potassium Current Potential Role in Atrial Tachycardia Remodeling Effects on Atrial Repolarization and Arrhythmias

Background— We previously characterized a novel K+ current (IKH) with properties of constitutively active acetylcholine-related current in dog atrium. IKH is sensitive to tertiapin-Q (IC50 ≈10 nmol/L), a highly selective Kir3 current blocker. This study assessed the role of IKH in atrial tachycardia (AT)–remodeled canine left atrium (LA) with the use of tertiapin-Q as a probe. Methods and Results— Dogs were subjected to 7 to 13 days of AT (400 bpm). Coronary-perfused LA preparations were studied intact or subjected to cardiomyocyte isolation. IKH was recorded with patch-clamp methods. AT pacing increased time-dependent hyperpolarization-activated current (IKH) at −110 mV from −1.8±0.3 (control) to −3.4±0.5 pA/pF (AT) and the 100-nmol/L tertiapin-sensitive component from −1.5±0.4 (control) to −3.3±0.6 pA/pF (AT). Prolonged atrial tachyarrhythmias could be induced with single extrastimuli in AT-remodeled, but not control, preparations, reflecting the atrial fibrillation–promoting effects of AT remodeling. In AT-remodeled preparations, tachyarrhythmia duration averaged 11.0±5.2 seconds, with a cycle length of 108±6 ms. Tertiapin-Q decreased tachyarrhythmia duration (to 0.6±0.1 second; P<0.001) and increased tachyarrhythmia cycle length (to 175±10 ms; P<0.001). Atrial action potential duration (APD) was increased 65±6% by tertiapin in AT-remodeled hearts versus 19±2% (P<0.001) in control. In 2 AT-remodeled preparations, tachyarrhythmia lasted uninterrupted for >20 minutes; tertiapin-Q slowed and then terminated arrhythmia in both. Tertiapin had no effect on left ventricular cardiomyocyte currents or APD. Conclusions— AT remodeling increases IKH, and a highly selective Kir3 current antagonist, tertiapin-Q, increases APD and suppresses atrial tachyarrhythmias in AT-remodeled preparations without affecting ventricular electrophysiology. Constitutive acetylcholine-related K+ current contributes to AT-remodeling effects in dogs and is a potentially interesting antiarrhythmic target.

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/dtmax (by 34±10%) only in PV. Increased dV/dtmax was not due to direct effects on INa, 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 (IKAdo) 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 IKAdo. PVs with dormant conduction show less radiofrequency-induced depolarization than nondormant veins, allowing adenosine-induced hyperpolarization to restore excitability by removing voltage-dependent INa inactivation and explaining the restoration of conduction in dormant PVs.

MicroRNA-26 governs profibrillatory inward-rectifier potassium current changes in atrial fibrillation

Atrial fibrillation (AF) is a highly prevalent arrhythmia with pronounced morbidity and mortality. Inward-rectifier K+ current (IK1) is believed to be an important regulator of reentrant-spiral dynamics and a major component of AF-related electrical remodeling. MicroRNA-26 (miR-26) is predicted to target the gene encoding KIR2.1, KCNJ2. We found that miR-26 was downregulated in atrial samples from AF animals and patients and this downregulation was accompanied by upregulation of IK1/KIR2.1 protein. miR-26 overexpression suppressed expression of KCNJ2/KIR2.1. In contrast, miR-26 knockdown, inhibition, or binding-site mutation enhanced KCNJ2/KIR2.1 expression, establishing KCNJ2 as a miR-26 target. Knockdown of endogenous miR-26 promoted AF in mice, whereas adenovirus-mediated expression of miR-26 reduced AF vulnerability. Kcnj2-specific miR-masks eliminated miR-26-mediated reductions in Kcnj2, abolishing miR-26s protective effects, while coinjection of a Kcnj2-specific miR-mimic prevented miR-26 knockdown-associated AF in mice. Nuclear factor of activated T cells (NFAT), a known actor in AF-associated remodeling, was found to negatively regulate miR-26 transcription. Our results demonstrate that miR-26 controls the expression of KCNJ2 and suggest that this downregulation may promote AF.

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 Ca2+ 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 Ca2+ 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 Ca2+ 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.

Potassium Channel Subunit Remodeling in Rabbits Exposed to Long-Term Bradycardia or Tachycardia Discrete Arrhythmogenic Consequences Related to Differential Delayed-Rectifier Changes

Background— Sustained heart rate abnormalities produce electrical remodeling and susceptibility to arrhythmia. Uncontrolled tachycardia produces heart failure and ventricular tachyarrhythmia susceptibility, whereas bradycardia promotes spontaneous torsade de pointes (TdP). This study compared arrhythmic phenotypes and molecular electrophysiological remodeling produced by tachycardia versus bradycardia in rabbits. Methods and Results— We evaluated mRNA and protein expression of subunits underlying rapid (IKr) and slow (IKs) delayed-rectifier and transient-outward K+ currents in ventricular tissues from sinus rhythm control rabbits and rabbits with AV block submitted to 3-week ventricular pacing either at 60 to 90 bpm (bradypaced) or at 350 to 370 bpm (tachypaced). QT intervals at matched ventricular pacing rates were longer in bradypaced than tachypaced rabbits (eg, by ≈50% at 60 bpm; P<0.01). KvLQT1 and minK mRNA and protein levels were downregulated in both bradypaced and tachypaced rabbits, whereas ERG was significantly downregulated in bradypaced rabbits only. Kv4.3 and Kv1.4 were downregulated by tachypacing only. Patch-clamp experiments showed that IKs was reduced in both but IKr was decreased in bradypaced rabbits only. Continuous monitoring revealed spontaneous TdP in 75% of bradypaced but only isolated ventricular ectopy in tachypaced rabbits. Administration of dofetilide (0.02 mg/kg) to mimic IKr downregulation produced ultimately lethal TdP in all tachypaced rabbits. Conclusions— Sustained tachycardia and bradycardia downregulate IKs subunits, but bradycardia also suppresses ERG/IKr, causing prominent repolarization delays and spontaneous TdP. Susceptibility of tachycardia/heart failure rabbits to malignant tachyarrhythmias is induced by exposure to IKr blockers. These results point to a crucial role for delayed-rectifier subunit remodeling in TdP susceptibility associated with rate-related cardiac remodeling.

Remodelling of cardiac repolarization: how homeostatic responses can lead to arrhythmogenesis

Cardiac action potentials (APs) are driven by ionic currents flowing through specific channels and exchangers across cardiomyocyte membranes. Once initiated by rapid Na(+) entry during phase 0, the AP time course is determined by the balance between inward depolarizing currents, carried mainly by Na(+) and Ca(2+), and outward repolarizing currents carried mainly by K(+). K(+) currents play a major role in repolarization. The loss of a K(+) current can impair repolarization, but there is a redundancy of K(+) currents so that when one K(+) current is dysfunctional, other K(+) currents increase to compensate, a phenomenon called repolarization reserve. Repolarization reserve protects repolarization under conditions that increase inward current or reduce outward current, threatening the balance that governs AP duration. This protection comes at the expense of reduced repolarization reserve, potentially resulting in unexpectedly large AP prolongation and arrhythmogenesis, when an additional repolarization-suppressing intervention is superimposed. The critical role of appropriate repolarization is such that cardiac rhythm stability can be impaired with either abnormally slow or excessively rapid repolarization. In cardiac disease states such as heart failure and atrial fibrillation (AF), changes in ion channel properties appear as part of an adaptive response to maintain function in the face of disease-related stress on the cardiovascular system. However, if the stress is maintained the adaptive ion channel changes may themselves lead to dysfunction, in particular cardiac arrhythmias. The present article reviews ionic remodelling of cardiac repolarization, and focuses on how potentially adaptive repolarization changes with congestive heart failure and AF can have arrhythmogenic consequences.

Heat shock proteins as molecular targets for intervention in atrial fibrillation

Atrial fibrillation (AF) is the most common sustained clinical tachyarrhythmia. AF is a progressive condition as demonstrated by the finding that maintenance of normal rhythm and contractile function becomes more difficult the longer AF exists. AF causes cellular stress, which induces atrial remodelling, involving reduction in the expression of L-type Ca(2+) channels and structural changes (myolysis), finally resulting in contractile dysfunction. Heat shock proteins (HSPs) comprise a family of proteins involved in the protection against different forms of cellular stress. Their classical function is the prevention of toxic protein aggregation by binding to (partially) unfolded proteins. Recent investigations reveal that HSPs prevent atrial remodelling and attenuate the promotion of AF in both cellular and animal experimental models. Furthermore, studies in humans suggest a protective role for HSPs against progression from paroxysmal AF to chronic, persistent AF. Therefore, manipulation of the HSP system may offer novel therapeutic approaches for the prevention of atrial remodelling. Such approaches may contribute to the maintenance or restoration of tissue integrity and contractile function. Ultimately, this concept may offer an additional treatment strategy to delay progression towards chronic AF and/or improve the outcome of cardioversion.

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 Ca2+ handling, studying underlying mechanisms and consequences. Methods and Results—We measured ventricular cardiomyocyte [Ca2+]i (Indo1-AM), L-type Ca2+-current (ICaL) and APs (whole-cell perforated-patch), and Ca2+-handling protein expression (immunoblot). CAVB increased AP duration, cell shortening, systolic [Ca2+]i transients, and caffeine-induced [Ca2+]i release, and CAVB cells showed spontaneous early afterdepolarizations (EADs). ICaL density was unaffected by CAVB, but inactivation was shifted to more positive voltages, increasing the activation-inactivation overlap zone for ICaL window current. Ca2+-calmodulin–dependent protein kinase-II (CaMKII) autophosphorylation was enhanced in CAVB, indicating CaMKII activation. CAVB also enhanced CaMKII-dependent phospholamban-phosphorylation and accelerated [Ca2+]i-transient decay, consistent with phosphorylation-induced reductions in phospholamban inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase as a contributor to enhanced SR Ca2+ loading. The CaMKII-inhibitor KN93 reversed CAVB-induced changes in caffeine-releasable [Ca2+]i and ICaL inactivation voltage and suppressed CAVB-induced EADs. Similarly, the calmodulin inhibitor W7 suppressed CAVB-induced ICaL inactivation voltage shifts and EADs, and a specific CaMKII inhibitory peptide prevented ICaL inactivation voltage shifts. The SR Ca2+-uptake inhibitor thapsigargin and the SR Ca2+ release inhibitor ryanodine also suppressed CAVB-induced EADs, consistent with an important role for SR Ca2+ loading and release in arrhythmogenesis. AP-duration changes reached a maximum after 1 week of bradypacing, but peak alterations in CaMKII and [Ca2+]i required 2 weeks, paralleling the EAD time course. Conclusions—CAVB-induced remodeling enhances [Ca2+]i load and activates the Ca2+-calmodulin-CaMKII system, producing [Ca2+]i-handling abnormalities that contribute importantly to CAVB-induced arrhythmogenic afterdepolarizations.

Fibroblast Inward-Rectifier Potassium Current Upregulation in Profibrillatory Atrial Remodeling

RATIONALEnFibroblasts are involved in cardiac arrhythmogenesis and contribute to the atrial fibrillation substrate in congestive heart failure (CHF) by generating tissue fibrosis. Fibroblasts display robust ion currents, but their functional importance is poorly understood.nnnOBJECTIVEnTo characterize atrial fibroblast inward-rectifier K(+) current (IK1) remodeling in CHF and its effects on fibroblast properties.nnnMETHODS AND RESULTSnFreshly isolated left atrial fibroblasts were obtained from controls and dogs with CHF (ventricular tachypacing). Patch clamp was used to record resting membrane potential (RMP) and IK1. RMP was significantly increased by CHF (from -43.2±0.8 mV, control, to -55.5±0.9 mV). CHF upregulated IK1 (eg, at -90 mV from -1.1±0.2 to -2.7±0.5 pA/pF) and increased the expression of KCNJ2 mRNA (by 52%) and protein (by 80%). Ba(2+) (300 μmol/L) decreased the RMP and suppressed the RMP difference between controls and dogs with CHF. Store-operated Ca(2+) entry (Fura-2-acetoxymethyl ester) and fibroblast proliferation (flow cytometry) were enhanced by CHF. Lentivirus-mediated overexpression of KCNJ2 enhanced IK1 and hyperpolarized fibroblasts. Functional KCNJ2 suppression by lentivirus-mediated expression of a dominant negative KCNJ2 construct suppressed IK1 and depolarized RMP. Overexpression of KCNJ2 increased Ca(2+) entry and fibroblast proliferation, whereas the dominant negative KCNJ2 construct had opposite effects. Fibroblast hyperpolarization to mimic CHF effects on RMP enhanced the Ca(2+) entry. MicroRNA-26a, which targets KCNJ2, was downregulated in CHF fibroblasts. Knockdown of endogenous microRNA-26 to mimic CHF effects unregulated IK1.nnnCONCLUSIONSnCHF upregulates fibroblast KCNJ2 expression and currents, thereby hyperpolarizing RMP, increasing Ca(2+) entry, and enhancing atrial fibroblast proliferation. These effects are likely mediated by microRNA-26a downregulation. Remodeling-induced fibroblast KCNJ2 expression changes may play a role in atrial fibrillation promoting fibroblast remodeling and structural/arrhythmic consequences.