Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked with potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite over a decade of investigation. Post-translational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel post-translational modifications and disease. We recently identified a novel pathway for post-translational regulation of the primary cardiac voltage-gated Na+ channel (Nav1.5) by CaMKII. However, a role for this pathway in cardiac disease has not been evaluated.
Methods and Results
We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Nav1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Nav1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Nav1.5 resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large animal model of acquired heart disease and in failing human myocardium.
We identify the mechanism for two human arrhythmia variants that affect Nav1.5 channel activity through direct effects on channel post-translational modification. We propose that the CaMKII phosphorylation motif in the Nav1.5 DI-DII cytoplasmic loop is a critical nodal point for pro-arrhythmic changes to Nav1.5 in congenital and acquired cardiac disease.
arrhythmia (mechanisms); calmodulin dependent protein kinase II; heart failure; ion channels; long-QT syndrome; myocardial infarction
Cardiac Na channel remodeling provides a critical substrate for generation of reentrant arrhythmias in border zones of the infarcted canine heart. Recent studies show that Nav1.5 assembly and function are linked to ankyrin-G, gap, and mechanical junction proteins. In this study our objective is to expound the status of the cardiac Na channel, its interacting protein ankyrinG and the mechanical and gap junction proteins at two different times post infarction when arrhythmias are known to occur; that is, 48 hr and 5 day post coronary occlusion. Previous studies have shown the origins of arrhythmic events come from the subendocardial Purkinje and epicardial border zone. Our Purkinje cell (Pcell) voltage clamp study shows that INa and its kinetic parameters do not differ between Pcells from the subendocardium of the 48hr infarcted heart (IZPCs) and control non-infarcted Pcells (NZPCs). Immunostaining studies revealed that disturbances of Nav1.5 protein location with ankyrin-G are modest in 48 hr IZPCs. Therefore, Na current remodeling does not contribute to the abnormal conduction in the subendocardial border zone 48 hr post myocardial infarction as previously defined. In addition, immunohistochemical data show that Cx40/Cx43 co-localize at the intercalated disc (IDs) of control NZPCs but separate in IZPCs. At the same time, Purkinje cell desmoplakin and desmoglein2 immunostaining become diffuse while plakophilin2 and plakoglobin increase in abundance at IDs. In the epicardial border zone 5 days post myocardial infarction, immunoblot and immunocytochemical analyses showed that ankyrin-G protein expression is increased and re-localized to submembrane cell regions at a time when Nav1.5 function is decreased. Thus, Nav1.5 and ankyrin-G remodeling occur later after myocardial infarction compared to that of gap and mechanical junctional proteins. Gap and mechanical junctional proteins remodel in IZPCs early, perhaps to help maintain Nav1.5 subcellular location position and preserve its function soon after myocardial infarction.
The transient outward potassium current (Ito) plays important roles in action potential (AP) morphology and dynamics; however, its role in the genesis of early afterdepolarizations (EADs) is not well understood. We aimed to study the effects and mechanisms of Ito on EAD genesis in cardiac cells using combined experimental and computational approaches.
Methods and results
We first carried out patch-clamp experiments in isolated rabbit ventricular myocytes exposed to H2O2 (0.2 or 1 mM), in which EADs were induced at a slow pacing rate. EADs were eliminated by either increasing the pacing rate or blocking Ito with 2 mM 4-aminopyridine. In addition to enhancing the L-type calcium current (ICa,L) and the late sodium current, H2O2 also increased the conductance, slowed inactivation, and accelerated recovery from the inactivation of Ito. Computer simulations showed that Ito promoted EADs under the condition of reduced repolarization reserve, consistent with the experimental observations. However, EADs were only promoted in the intermediate ranges of the Ito conductance and the inactivation time constant. The underlying mechanism is that Ito lowers the AP plateau voltage into the range at which the time-dependent potassium current (namely IKs) activation is further slowed and ICa,L is available for reactivation, leading to voltage oscillations to manifest EADs. Further experimental studies in cardiac cells of other species validated the theoretical predictions.
In cardiac cells, Ito, with a proper conductance and inactivation speed, potentiates EADs by setting the AP plateau into the voltage range where ICa,L reactivation is facilitated and IKs activation is slowed.
Transient outward current; Early afterdepolarization; Cardiac arrhythmias; Computer model; Dynamic mechanisms
Reentry accounts for most life-threatening arrhythmias, complicating myocardial infarction, and therapies that consistently prevent reentry from occurring are lacking. In this study, we compare antiarrhythmic effects of gene transfer of green fluorescent protein (GFP; sham), the skeletal muscle sodium channel (SkM1), the liver-specific connexin (Cx32), and SkM1/Cx32 in the subacute canine infarct.
Methods and results
Immediately after ligation of the left anterior descending artery, viral constructs were implanted in the epicardial border zone (EBZ). Five to 7 days later, efficient restoration of impulse propagation (narrow QRS and local electrogram duration) occurred in SkM1, Cx32, and SkM1/Cx32 groups (P< 0.05 vs. GFP). Programmed electrical stimulation from the EBZ induced sustained ventricular tachycardia (VT)/ventricular fibrillation (VF) in 15/22 GFP dogs vs. 2/12 SkM1, 6/14 Cx32, and 8/10 SkM1/Cx32 (P< 0.05 SkM1 vs. GFP). GFP, SkM1, and SkM1/Cx32 had predominantly polymorphic VT/VF, whereas in Cx32 dogs, monomorphic VT predominated (P< 0.05 for Cx32 vs. GFP). Tetrazolium red staining showed significantly larger infarcts in Cx32- vs. GFP-treated animals (P< 0.05).
Whereas SkM1 gene transfer reduces the incidence of inducible VT/VF, Cx32 therapy to improve gap junctional conductance results in larger infarct size, a different VT morphology, and no antiarrhythmic efficacy.
Myocardial infarction; Arrhythmias; Na+ channels; Connexins; Gene therapy
We have shown reduced density and altered kinetics in slowly activating K+ currents (IKs) in epicardial border zone (EBZ) cells (IZs) of the 5-day-infarcted canine heart (Jiang M, Cabo, C, Yao J-A, Boyden PA, and Tseng G-N. Cardiovasc Res 48: 34–43, 2000). β-Adrenergic stimulation with isoproterenol increases IKs in normal cells (NZs). In this study, we used a voltage-clamp protocol with an external solution to isolate IKs from contaminating currents to determine the effects of 1 µM isoproterenol on IKs in IZs and NZs. Under our recording conditions, 10 µM azimilide-sensitive currents were stimulated with isoproterenol to compare responsiveness of IKs to isoproterenol in the two cell groups. IKs tail density was reduced 67% in IZs (group I, n = 26) compared with NZs (n = 24, P < 0.05). Isoproterenol-stimulated azimilide-sensitive tail currents were increased 1.72 ± 0.2-fold in NZs and 2.2 ± 0.3-fold in IZs (P > 0.05). In 33% of IZs (group II, n = 13), native currents showed no tail currents; however, isoproterenol-stimulated azimilide-sensitive currents were voltage dependent, fast activating, and large in amplitude compared with group I IZs, similar to “lone” KCNQ1 currents. Using short clamp pulses, we also found an increase in sustained currents sensitive to tetraethylammonium chloride (TEA) and no change in C-9356-sensitive currents in IZs with little or no transient outward current. In some IZs where IKs is downregulated, the effect of isoproterenol on IKs was similar to that on IKs in NZs. In others, the existence of lone KCNQ1-type currents, which are sensitive to β-adrenergic stimulation, is consistent with our findings of an increased KCNQ1-to-KCNE1 mRNA ratio (Jiang et al.). Accompanying altered IKs in IZs are an enhanced TEA-sensitive current and a normal C-9356-sensitive current.
epicardial border zone; delayed rectifier; tetraethylammonium; C-9356; isoproterenol
The border zone of healing myocardial infarcts is an arrhythmogenic substrate partly due to structural and functional remodeling of the ventricular gap junction protein, Connexin43 (Cx43). Cx43 in arrhythmogenic substrates is a potential target for antiarrhythmic therapy.
Methods and Results
We characterized Cx43 remodeling in the epicardial border zone (EBZ) of healing canine infarcts, 5 days after coronary occlusion and examined whether the gap junction specific agent, Rotigaptide, could reverse it. Cx43 remodeling in the EBZ was characterized by a decrease in Cx43 protein, lateralization and increased Cx43 phosphorylation at serine (S) 368. Rotigaptide partially reversed the loss of Cx43 but did not affect the increase in S368 phosphorylation nor did it reverse Cx43 lateralization. Rotigaptide did not prevent conduction slowing in EBZ nor did it decrease the induction of sustained ventricular tachycardia (SMVT) by programmed stimulation, although it did decrease the EBZ effective refractory period (ERP).
We conclude that partial reversal of Cx43 remodeling in healing infarct border zone may not be sufficient to restore normal conduction or prevent arrhythmias.
myocardial infarction; arrhythmias; gap junctions; remodeling
Purkinje fibers play an essential role in transmitting electrical impulses through the heart, but they may also serve as triggers for arrhythmias linked to defective intracellular calcium (Ca2+) regulation. Although prior studies have extensively characterized spontaneous Ca2+ release in nondriven Purkinje cells, little attention has been paid to rate-dependent changes in Ca2+ transients. Therefore we explored the behaviors of Ca2+ transients at pacing rates ranging from 0.125 to 3 Hz in single canine Purkinje cells loaded with fluo3 and imaged with a confocal microscope. The experiments uncovered the following novel aspects of Ca2+ regulation in Purkinje cells: 1) the cells exhibit a negative Ca2+-frequency relationship (at 2.5 Hz, Ca2+ transient amplitude was 66 ± 6% smaller than that at 0.125 Hz); 2) sarcoplasmic reticulum (SR) Ca2+ release occurs as a propagating wave at very low rates but is localized near the cell membrane at higher rates; 3) SR Ca2+ load declines modestly (10 ± 5%) with an increase in pacing rate from 0.125 Hz to 2.5 Hz; 4) Ca2+ transients show considerable beat-to-beat variability, with greater variability occurring at higher pacing rates. Analysis of beat-to-beat variability suggests that it can be accounted for by stochastic triggering of local Ca2+ release events. Consistent with this hypothesis, an increase in triggering probability caused a decrease in the relative variability. These results offer new insight into how Ca2+ release is normally regulated in Purkinje cells and provide clues regarding how disruptions in this regulation may lead to deleterious consequences such as arrhythmias.
Ca2+ transients; pacing rate; conduction system; Ca2+ sparks; Ca2+ waves
Cardiac membrane excitability is tightly regulated by an integrated network of membrane-associated ion channels, transporters, receptors, and signaling molecules. Membrane protein dynamics in health and disease are maintained by a complex ensemble of intracellular targeting, scaffolding, recycling, and degradation pathways. Surprisingly, despite decades of research linking dysfunction in membrane protein trafficking with human cardiovascular disease, essentially nothing is known regarding the molecular identity or function of these intracellular targeting pathways in excitable cardiomyocytes.
We sought to discover novel pathways for membrane protein targeting in primary cardiomyocytes.
Methods and Results
We report the initial characterization of a large family of membrane trafficking proteins in human heart. We employed a tissue-wide screen for novel ankyrin-associated trafficking proteins and identified four members of a unique Eps15 homology (EH) domain-containing protein family (EHD1, EHD2, EHD3, EHD4) that serve critical roles in endosome-based membrane protein targeting in other cell types. We show that EHD1-4 directly associate with ankyrin, provide the first information on the expression and localization of these molecules in primary cardiomyocytes, and demonstrate that EHD1-4 are co-expressed with ankyrin-B in the myocyte perinuclear region. Notably, the expression of multiple EHD proteins is increased in animal models lacking ankyrin-B, and EHD3-deficient cardiomyocytes display aberrant ankyrin-B localization and selective loss of Na/Ca exchanger expression and function. Finally, we report significant modulation of EHD expression following myocardial infarction, suggesting that these proteins may play a key role in regulating membrane excitability in normal and diseased heart.
Our findings identify and characterize a new class of cardiac trafficking proteins, define the first group of proteins associated with the ankyrin-based targeting network, and identify potential new targets to modulate membrane excitability in disease. Notably, these data provide the first link between EHD proteins and a human disease model.
trafficking; ion channel; ankyrin; EHD proteins; cytoskeleton; arrhythmia
Arrhythmias are benign or lethal depending on their sustainability and frequency. To determine why lethal arrhythmias are prone to occur in diseased hearts, usually characterized by non-uniform muscle contraction, we investigated the effect of non-uniformity on sustainability and frequency of triggered arrhythmias.
Methods and Results
Force, membrane potential, and intracellular Ca2+ concentration ([Ca2+]i) were measured in 51 rat ventricular trabeculae. Non-uniform contraction was produced by exposing a restricted region of muscle to a jet of 20 mmol/L 2,3-butanedione monoxime (BDM) or 20 μmol/L blebbistatin. Sustained arrhythmias (>10 s) could be induced by stimulus trains for 7.5 s only with the BDM or blebbistatin jet (100 nmol/L isoproterenol, 1.0 mmol/L [Ca2+]o, 24°C). During sustained arrhythmias, Ca2+ surges preceded synchronous increases in [Ca2+]i, while the stoppage of the BDM jet made the Ca2+ surges unclear and arrested sustained arrhythmias (n = 6). With 200 nmol/L isoproterenol, 2.5 mmol/L [Ca2+]o, and the BDM jet, lengthening or shortening of the muscle during sustained arrhythmias accelerated or decelerated their cycle both in the absence (n = 10) and the presence of 100 μmol/L streptomycin (n = 10), a stretch-activated channel blocker, respectively. The maximum rate of force relaxation correlated inversely with the change in cycle lengths (n = 14, P<0.01). Sustained arrhythmias with the BDM jet were significantly accelerated by 30 μmol/L SCH00013, a Ca2+ sensitizer of myofilaments (n = 10).
These results suggest that non-uniformity of muscle contraction is an important determinant of the sustainability and frequency of triggered arrhythmias due to the surge of Ca2+ dissociated from myofilaments in cardiac muscle.
triggered activity; contraction; calcium; non-uniform myocardium
Purkinje cells (PCs) comprise the most distal component of the cardiac conduction system and their unique electrophysiological properties and the anatomic complexity of the Purkinje fiber network may account for the prominent role these cells play in the genesis of various arrhythmic syndromes.
Methods and Results
– Differential transcriptional profiling of murine Purkinje fibers and working ventricular myocytes was performed to identify novel genes expressed in PCs. The most highly enriched transcript in Purkinje fibers encoded Contactin-2 (Cntn2), a cell adhesion molecule critical for neuronal patterning and ion channel clustering. Endogenous expression of Cntn2 in the murine ventricle was restricted to a subendocardial network of myocytes that also express β-galactosidase in CCS-lacZ transgenic mice and the connexin40 gap junction protein. Both Cntn2-lacZ knockin mice and Cntn2-EGFP BAC transgenic reporter mice confirmed expression of Cntn2 in the Purkinje fiber network, as did immunohistochemical staining of single canine Purkinje fibers. Whole-cell patch-clamp recordings and measurements of Ca2+ transients in Cntn2-EGFP+ cells revealed electrophysiological properties indicative of PCs and distinctive from those of cardiac myocytes, including prolonged action potentials and frequent afterdepolarizations.
Cntn2 is a novel marker of the specialized cardiac conduction system. Endogenous expression of Cntn2 as well as Cntn2-dependent transcriptional reporters provides a new tool through which Purkinje cell biology and pathophysiology can now more readily be deciphered. Expression of a contactin family member within the CCS may provide a mechanistic basis for patterning of the conduction system network and the organization of ion channels within Purkinje cells.
cell adhesion molecules; electrophysiology; genetics; Purkinje fiber
stem cells; cell therapy; arrhythmias; heart disease
Ion channel reorganization is a critical step in the pro-arrhythmogenic remodelling process that occurs in heart disease. Ankyrin-B (AnkB) is required for targeting and stabilizing ion channels, exchangers, and pumps. Despite a wealth of knowledge implicating the importance of AnkB in human cardiovascular physiology, nothing is known regarding the role of AnkB in common forms of acquired human disease.
Methods and results
We present the first report of AnkB regulation following myocardial infarction (MI). AnkB protein levels were reduced in the infarct border zone 5 days following coronary artery occlusion in the canine. We also observed a dramatic increase in AnkB mRNA levels 5 days post-occlusion. Surprisingly, the expression of the upstream AnkB cytoskeletal component β2-spectrin was unchanged in post-infarct tissues. However, protein levels and/or membrane expression of downstream AnkB-associated ion channels and transporters Na+/K+ ATPase, Na+/Ca2+ exchanger, and IP3 receptor were altered 5 days post-occlusion. Interestingly, protein levels of the protein phosphatase 2A, an AnkB-associated signalling protein, were significantly affected 5 days post-occlusion. AnkB and PP2A protein levels recovered by 14 days post-occlusion, whereas Na+/K+ ATPase levels recovered by 2 months post-occlusion.
These findings reveal the first evidence of ankyrin remodelling following MI and suggest an unexpected divergence point for regulation between ankyrin and the underlying cytoskeletal network. These findings suggest a logical, but unexpected, molecular mechanism underlying ion channel and transporter remodelling following MI.
Arrhythmia (mechanisms); Infarction; Remodelling; Signal transduction; Cytoskeleton
Calmodulin kinase II (CaMKII) mediates critical signaling pathways responsible for divergent functions in the heart including calcium cycling, hypertrophy and apoptosis. Dysfunction in the CaMKII signaling pathway occurs in heart disease and is associated with increased susceptibility to life-threatening arrhythmia. Furthermore, CaMKII inhibition prevents cardiac arrhythmia and improves heart function following myocardial infarction. Recently, a novel mechanism for oxidative CaMKII activation was discovered in the heart. Here, we provide the first report of CaMKII oxidation state in a well-validated, large-animal model of heart disease. Specifically, we observe increased levels of oxidized CaMKII in the infarct border zone (BZ). These unexpected new data identify an alternative activation pathway for CaMKII in common cardiovascular disease. To study the role of oxidation-dependent CaMKII activation in creating a pro-arrhythmia substrate following myocardial infarction, we developed a new mathematical model of CaMKII activity including both oxidative and autophosphorylation activation pathways. Computer simulations using a multicellular mathematical model of the cardiac fiber demonstrate that enhanced CaMKII activity in the infarct BZ, due primarily to increased oxidation, is associated with reduced conduction velocity, increased effective refractory period, and increased susceptibility to formation of conduction block at the BZ margin, a prerequisite for reentry. Furthermore, our model predicts that CaMKII inhibition improves conduction and reduces refractoriness in the BZ, thereby reducing vulnerability to conduction block and reentry. These results identify a novel oxidation-dependent pathway for CaMKII activation in the infarct BZ that may be an effective therapeutic target for improving conduction and reducing heterogeneity in the infarcted heart.
Calmodulin kinase II (CaMKII) is a multifunctional serine/threonine kinase that regulates diverse functions in heart. Recently, a novel pathway for CaMKII activation was discovered where oxidation of the kinase at specific methionine residues produces persistent activity. This alternative oxidation-dependent pathway has important implications for heart disease where oxidative stress is increased (e.g., heart failure and following myocardial infarction). We hypothesized that myocardial infarction caused by occlusion of a coronary artery would increase levels of oxidized CaMKII. Moreover, we hypothesized that oxidative CaMKII activation represents an important mechanistic link between increased oxidative stress and life-threatening heart rhythm disturbances (arrhythmias) in heart disease. We report a dramatic increase in levels of oxidized CaMKII following myocardial infarction in the canine. Based on these experimental data, we developed a novel mathematical model of CaMKII activity to study the role of oxidation-dependent CaMKII activation in regulating cardiac cell excitability. Our findings identify a novel role for oxidation-dependent CaMKII activation following myocardial infarction and provide a mechanistic link between oxidative stress and lethal cardiac arrhythmias in heart disease.
Triggered Purkinje ectopy can lead to the initiation of serious ventricular arrhythmias in post myocardial infarction (MI) patients. In the canine model, Purkinje cells from the subendocardial border of the healing infarcted heart can initiate ventricular arrhythmias. Intracellular Ca2+ abnormalities underlie these arrhythmias yet the subcellular reasons for these abnormalities remain unknown.
Methods and Results
Using 2D confocal microscopy, we directly quantify and compare typical spontaneous Ca2+ events in specific subcellular regions of normal Purkinje cells (NZPCs) with those Purkinje cells from the subendocardium of the 48hr infarcted canine heart (IZPCs). The Ca2+ event rate was higher in subsarcolemmal region (SSL) of IZPCs when compared to NZPCs, IZPC amplitudes were higher yet the spatial extents of these events were similar. The amplitude of Caffeine releasable Ca2+ in either the SSL or Core regions of IZPCs did not differ from NZPCs suggesting that Ca2+ overload was not related to the frequency change. In permeabilized Purkinje cells from both groups, the event rate was related to free [Ca2+] in both SSL and Core but in IZPCs this event rate was significantly increased at each free Ca2+ suggesting an enhanced sensitivity to Ca2+ release. Furthermore, decays of wide long lasting Ca2+ release events in IZPC's Core were significantly accelerated compared to those in NZPCs. JTV519 (K201) suppressed IZPC cell wide Ca2+ waves as well as normalized the enhanced event rate and its response to free Ca2+.
Increased spontaneous Ca2+ release events in IZPCs are due to uniform regionally increased Ca2+ release channel sensitivity to Ca2+ without a change in SR content. In addition, Ca2+ reuptake in IZPCs is accelerated. These properties would lower the threshold of Ca2+ release channels, setting the stage for the highly frequent arrhythmogenic cell wide Ca2+ waves observed in IZPCs.
myocardial infarction; calcium; arrhythmias; Purkinje cells; Ca2+ waves
Ca2+/calmodulin-dependent protein kinase II is a multifunctional serine/threonine kinase with diverse cardiac roles including regulation of excitation contraction, transcription, and apoptosis. Dynamic regulation of CaMKII activity occurs in cardiac disease and is linked to specific disease phenotypes through its effects on ion channels, transporters, transcription and cell death pathways. Recent mathematical models of the cardiomyocyte have incorporated limited elements of CaMKII signaling to advance our understanding of how CaMKII regulates cardiac contractility and excitability. Given the importance of CaMKII in cardiac disease, it is imperative that computer models evolve to capture the dynamic range of CaMKII activity. In this study, using mathematical modeling combined with biochemical and imaging techniques, we test the hypothesis that CaMKII signaling in the canine infarct border zone (BZ) contributes to impaired calcium homeostasis and electrical remodeling. We report that the level of CaMKII autophosphorylation is significantly increased in the BZ region. Computer simulations using an updated mathematical model of CaMKII signaling reproduce abnormal Ca2+ transients and action potentials characteristic of the BZ. Our simulations show that CaMKII hyperactivity contributes to abnormal Ca2+ homeostasis and reduced action potential upstroke velocity due to effects on INa gating kinetics. In conclusion, we present a new mathematical tool for studying effects of CaMKII signaling on cardiac excitability and contractility over a dynamic range of kinase activities. Our experimental and theoretical findings establish abnormal CaMKII signaling as an important component of remodeling in the canine BZ.
Calcium/calmodulin-dependent protein kinase II; myocardial infarction; calcium handling; mathematical modeling; arrhythmia
Four voltage-gated potassium currents: Ito,f (KV4.2), Ito,s (KV1.4), IK,slow (KV1.5+KV2.1), and ISS (TASK1) govern murine ventricular repolarization. Although the accessory subunit, KChIP2, influences Ito,f expression, in preliminary experiments we found that action potential duration (APD) is maintained in KChIP2 knockout mice.
We tested the role of KChIP2 in regulating APD and studied the underlying ionic currents.
We used microelectrode techniques, whole-cell patch clamp studies, and real-time PCR amplification to characterize ventricular repolarization and its determinants in WT and KChIP2-/- mice.
Despite comparable baseline action potentials, APD was more markedly prolonged by 4-aminopyridine (4-AP) in KChIP2-/- preparations. Peak K+ current densities were similar in WT and KChIP2-/- cells (mean±sem IP: 28.3±2 (n=27) vs. 29.2±2 pA/pF (n=24), respectively; P>0.05). Heteropodatoxin-2 (HpTx-2, 1 μM) had no effect on current amplitude in KChIP2-/- myocytes. The current fractions sensitive to 4-AP (50 μM and 1 mM) were larger in KChIP2-/- than WT (P<0.05). Real-time PCR demonstrated absence of KChIP2 and increased KV1.5 expression in KChIP2-/- ventricular myocardium.
KChIP2 deficiency eliminated HpTx-2-sensitive Ito,f, but had little impact on total APD, secondary to upregulation of 4-AP-sensitive IK,slow in association with increased KV1.5 expression. There is increased sensitivity to 4-AP-mediated APD prolongation in KChIP2-/-. Thus, KChIP2 appears important for murine repolarization in circumstances of reduced repolarization reserve.
Potassium currents; action potentials; electrophysiology; animal models; repolarization
to determine whether IP3Rs contribute to the generation of wide long lasting perinuclear Ca2+ release events in canine Purkinje cells.
Spontaneous Ca2+ release events (elevations of basal [Ca2+] equivalent to F/F0 3.4SD over F0) were imaged using Fluo-4AM and 2D confocal microscope. Only cells free of Ca2+ waves were analyzed. Subsarcolemmal region (SSL) was defined as 5µm from cell edges. Core was the remaining cell.
The majority of events (94%, 0.0035 ± 0.0007 events(ev)/µm2/sec, n=34 cells) were detected within a single frame (typical events, TE). However, a subpopulation (6.0%, 0.00022±0.00005 ev/µm2/sec, n=41 cells: wide long lasting events, WLE) lasted for several frames, showed a greater spatial extent (51.0±3.9 vs. TE 9.0±0.3 µm2, P<0.01) and higher amplitude (F/F0 1.38±0.02 vs. TE 1.20±0.003, P<0.01). WLE event rate was increased by phenylephrine (10µM, P<0.01), inhibited by 2APB and U73122 (P<0.05), and abolished by tetracaine (1mM) and ryanodine (100µM). While SSL WLEs were scattered randomly, Core WLEs (n=69 events) were predominantly distributed longitudinally 18.2±1.6 ìm from the center of nuclei. Immunocytochemistry showed that IP3R1s were located not only at SSL region but also near both ends of nucleus overlapping with RyRs.
In Purkinje cells, wide long lasting Ca2+ release events occur in SSL and in specific perinuclear regions. They are likely due to RyRs and IP3R1s evoked Ca2+ release and may play a role in Ca2+ dependent nuclear processes.
Purkinje cells; nucleus; Ca2+ transients; Phenylephrine
Atrial fibrillation is often initiated by bursts of ectopic activity arising in the pulmonary veins. We have previously shown that a 3-h intermittent burst pacing protocol (BPP), mimicking ectopic pulmonary vein foci, shortens action potential duration (APD) locally at the pulmonary vein-atrial interface (PV) while having no effect elsewhere in rabbit atrium. This shortening is Ca2+ dependent and is prevented by apamin, which blocks small conductance Ca2+-activated K+ channels (SKCa). The present study investigates the ionic and molecular mechanisms whereby two apamin-sensitive SKCa channels, SK2 and SK3, might contribute to the regional APD changes.
Microelectrode and patch clamp techniques were used to record APDs and apamin-sensitive currents in isolated rabbit left atria and cells dispersed from PV and Bachmann’s bundle (BB) regions. SK2 and SK3 mRNA and protein levels were quantified, and immunofluorescence was used to observe channel protein distribution.
There was a direct relationship between APD shortening and apamin-sensitive current in burst-paced but not sham-paced PV. Moreover, apamin-sensitive current density increased in PV but not BB after BPP. SK2 mRNA, protein, and current were increased in PV after BPP, while SK2 immunostaining shifted from a perinuclear pattern in sham atria to predominance at sites near or at the PV membrane.
BPP-induced acceleration of repolarization in PV results from SK2 channel trafficking to the membrane, leading to increased apamin–sensitive outward current. This is the first indication of involvement of Ca2+-activated K+ currents in atrial remodeling and provides a possible basis for evolution of an arrhythmogenic substrate.
Voltage-gated Nav channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Nav1.5 is the predominant Nav channel, and Nav1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Nav1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Nav channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Nav1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Nav1.5 expression, abnormal Nav1.5 membrane targeting, and reduced Na+ channel current density. We define the structural requirements on ankyrin-G for Nav1.5 interactions and demonstrate that loss of Nav1.5 targeting is caused by the loss of direct Nav1.5–ankyrin-G interaction. These data are the first report of a cellular pathway required for Nav channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Nav channel organization in multiple excitable tissues.
Pulmonary veins (PV) and coronary sinus (CS) play pivotal roles in triggering some episodes of atrial fibrillation. In isolated rabbit right or left atrial preparations, a 3-hour intermittent burst pacing (IBP) protocol shortens action potential duration (APD) in CS and PV, but not sinus node (SN) and left Bachmann’s bundle (BB) regions1.
To use patch clamp techniques to study the rapidly inactivating (Ito) and sustained (Isus) K+ currents as well as Ca2+ currents (ICa) in cells dispersed from IBP and Sham PV, BB, CS and SN regions to determine whether changes in these currents contributed to the APD shortening. Methods: Real-time PCR was performed for transient outward K+ and Ca2+ channel subunit mRNAs to determine if IBP affected expression levels.
Ito densities were unaffected by IBP in PV and BB cells, and mRNA levels of Kv4.3, Kv4.2, Kv1.4 and KChIP2 subunits of Ito in both regions were stable. In CS cells, Ito densities in IBP>Sham (P<.05) but there were no parallel mRNA changes. ICa density of PV cells was reduced from 14.27±2.08 pA/pF(at -5mV) in Sham to 7.52±1.65 pA/pF in IBP PV cells (P<0.05) due to a significant shift in voltage dependence of activation. These results were seen in the absence of mRNA changes in α1C and α1D Ca2+ channel subunits. In contrast, IBP had no effect on Ca2+ current densities and kinetics of CS cells, but decreased α1C and α1D mRNA levels.
In conclusion, there is region-specific remodeling of Ito and ICa by IBP protocols in rabbit atrium. Increased Ito in CS cells could account for the APD shortening observed with IBP, while an IBP-induced shift in voltage dependence of activation may contribute to APD shortening in PV cells.
Atrial fibrillation; remodeling; ionic currents; pacing
thoracic veins; afterdepolarizations; action potentials; ablation; arrhythmias