Background-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 ␤-galactosidase in CCS-lacZ transgenic mice and the connexin40 gap junction protein. Key Words: cell adhesion molecules Ⅲ electrophysiology Ⅲ genetics Ⅲ Purkinje fiber P urkinje fibers (PFs) are the most distal component of the cardiac conduction system (CCS), first described by Purkinje in 1839 as gray, flat, gelatin-like ramifications, running under the endocardium. 1 Some 70 years later, Tawara 2 more fully characterized the Purkinje system, identifying the left (anterior, septal, and posterior) and right fascicular strands, which served to connect the distal PFs to the bundle branches proper. Tawara was also the first to correctly suggest the functional role of the Purkinje system in rapidly transmitting the electric wave of excitation to the ventricular muscle. Clinical Perspective on p 194PFs appear to play a prominent role in the genesis of ventricular arrhythmias, (reviewed in Reference 3). 3 PF and anterior or posterior left fascicular triggers have been implicated in the initiation of monomorphic ventricular tachycardia in post-myocardial infarction patients, as demonstrated by cure after focal ablation of Purkinje fiber or fascicular potentials. 4 -6 PF-based triggers have also been described in patients with ventricular tachycardia associated with dilated forms of cardiomyopathy, 7 as well as idiopathic ventricular fibrillation (VF), in which ablation of premature beats arising from the PF network resulted in significant reductions in the recurrence of VF. 8 PF-dependent triggering of arrhythmias has also been proposed in inherited syndromes including catecholaminergic polymorphic VT, 9 Brugada syndrome, and long-QT syndrome. 10 Despite growing evidence implicating PFs in ventricular arrhythmogenesis, our understanding of the cellular mechanisms underlying PF-dependent diseases is hampered by the lack of knowledge of the developmental biology of individual Purkinje cells (PCs), their patterning into a network of highly coupled cells, and their adaptive and maladaptive responses to pathological stimuli. To some extent, this gap in knowledge reflects the anatomic complexity of the PF network, which includes branching cells that couple not only with neighboring PCs but also with working myocytes at Purkinje- In recent years, a number of "...

Objective: 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 Ca 2+ dependent and is prevented by apamin, which blocks small conductance Ca 2+ -activated K + channels (SK Ca ). The present study investigates the ionic and molecular mechanisms whereby two apamin-sensitive SK Ca channels, SK2 and SK3, might contribute to the regional APD changes. Methods: 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. Results: 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. Conclusions: 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 Ca 2+ -activated K + currents in atrial remodeling and provides a possible basis for evolution of an arrhythmogenic substrate.

Background 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. Conclusions 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.

Background-Anisotropic reentrant excitation occurs in the remodeled substrate of the epicardial border zone (EBZ) of the 5-day infarcted canine heart. Reentry is stabilized because of the formation of functional lines of block. We hypothesized that regional differences of ionic currents in cells of the EBZ form these lines of block. Therefore, we first mapped reentrant circuits of sustained tachycardias, then dispersed cells (infarct zone cells, IZs) from the central common pathway of the circuit (IZc) as well as from the other side of the line of block (outer pathway, IZo) for study. Methods and Results-We mapped reentrant circuits in the EBZ of infarcted hearts during sustained ventricular tachycardias (Ͼ30 seconds, nϭ17 episodes, cycle lengthsϭ218Ϯ7.9 ms). I Na density was reduced in both IZc and IZo, and the kinetic properties of IZc I Na were markedly altered versus IZo. Structural remodeling of the sodium channel protein Na v 1.5 occurred in IZs, with cell surface localization differing from normal cells. Both IZc and IZo have similar but reduced I CaL , whereas IZc showed changes in Ca 2ϩ current kinetics with an acceleration of current decay. Computer simulations of the 2D EBZ showed that incorporating only differences between I Na in IZc and IZo prevented stability of the reentrant circuit. Incorporating only differences between I CaL in the IZc and IZo cells also prevented stability of the circuit. However, incorporating both I Na and I CaL current differences stabilized the simulated reentrant circuit, and lines of block formed between the 2 distinct regions. Conclusions-Despite differences in I Na and I CaL properties in cells of the center and outer pathways of a reentrant circuit, the resulting changes in effective refractory periods tend to stabilize reentry in this remodeled substrate.

Background: PP2A regulates cardiac excitability and physiology. Results: PP2A regulation in heart occurs through integrative transcriptional, translational, and post-translational control of three classes of subunits (17 genes) to control holoenzyme synthesis, localization, and maintenance; pathways are mechanistically altered in heart disease. Conclusion:Multiple mechanisms are present for acute and chronic regulation of specific PP2A populations. Significance: Results provide molecular insight into cardiac PP2A regulation.

Abstract-We have investigated the subcellular spontaneous Ca 2ϩ events in canine Purkinje cells using laser scanning confocal microscopy. Three types of Ca 2ϩ transient were found: (1) nonpropagating Ca 2ϩ transients that originate directly under the sarcolemma and lead to (2) small Ca 2ϩ wavelets in a region limited to Ϸ6-m depth under the sarcolemma causing (3) large Ca 2ϩ waves that travel throughout the cell (CWWs). Immunocytochemical studies revealed 3 layers of Ca 2ϩ channels: (1) channels associated with type 1 IP 3 receptors (IP 3 R 1 ) and type 3 ryanodine receptors (RyR 3 ) are prominent directly under the sarcolemma; (2) type 2 ryanodine receptors (RyR 2 s) are present throughout the cell but virtually absent in a layer between 2 and 4 m below the sarcolemma (Sub-SL); (3) type 3 ryanodine receptors (RyR 3 ) is the dominant Ca 2ϩ release channel in the Sub-SL. Simulations of both nonpropagating and propagating transients show that the generators of Ca 2ϩ wavelets differ from those of the CWWs with the threshold of the former being less than that of the latter. Thus, Purkinje cells contain a functional and structural Ca 2ϩ system responsible for the mechanism that translates Ca 2ϩ release occurring directly under the sarcolemma into rapid Ca 2ϩ release in the Sub-SL, which then initiates large-amplitude long lasting Ca 2ϩ releases underlying CWWs. I n cells devoid of t tubules such as atrial and Purkinje cells (Pcells), excitation-contraction coupling (ECC) involves Ca2ϩ release from stores located near the sarcolemma and subsequent Ca 2ϩ -induced Ca 2ϩ release (CICR) along a lattice of sarcoplasmic reticulum (SR) enveloping the sarcomeres, which then activate myofibrils throughout the cell. [1][2][3][4][5] Drugs which affect SR function, such as thapsigargin and ryanodine, inhibit Ca 2ϩ activation of Pcells. 2,6 Conversely, spontaneous nonpropagating Ca 2ϩ release and Ca 2ϩ waves cause sarcolemma depolarization in both pacemaker cells and Pcells, which can lead to nondriven electrical activity even at normal [Ca 2ϩ ] o . 2,7-9 Abnormal Ca 2ϩ release in the network of Pcells may also be involved in lethal arrhythmias after myocardial infarction. 7,10 Previous observations suggested that micro Ca 2ϩ transients, spanning only a few micrometers and traveling over short distances, initiate cell-wide Ca 2ϩ waves (CWWs) which in turn induce nondriven electrical activity in a Pcell aggregate. 7 Here, we determined the mechanistic relationships between the different subcellular spontaneous Ca 2ϩ events in canine Pcells using confocal microscopy. Materials and MethodsEighteen aggregates of 2 to 6 cells were enzymatically dispersed from the Purkinje network of canine left ventricle (nϭ9) 7 and placed in a chamber on the stage of an inverted laser scanning confocal microscope (LSCM). Fluorescence was measured only in rod-shaped Pcells with typical junctional ends, clear striations, and membranes free of blebs. 2 Measurement and Analysis of Ca 2؉ TransientsConfocal line-scans were first positioned parallel t...

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.

Human ether-à-go-go-related gene (HERG) encodes a K channel similar to the rapid delayed rectifier channel current (I(Kr)) in cardiac myocytes. Modulation of I(Kr) by extracellular acidosis under pathological conditions may impact on cardiac electrical activity. Therefore, we studied the effects of extracellular acidification on I(Kr) function and the underlying mechanism, using HERG expressed in Xenopus oocytes as a model. Acidification [extracellular pH (pH(o)) 8.5-6.5] accelerated HERG deactivation (at -80 mV, the time constant tau of the major component of deactivation was 253 +/- 17, 158 +/- 10, and 65 +/- 5 ms at pH(o) 8.5, 7.5, and 6.5, respectively; n = 7-10 each), with no effects on other gating kinetics except a modest acceleration of recovery from inactivation (at -80 mV, tau of recovery was 4.7 +/- 0.3, 3.8 +/- 0.3, and 1.3 +/- 0.2 ms at pH(o) 8. 5, 7.5, and 6.5, respectively; n = 4-7 each). The following were ruled out as the underlying mechanisms: 1) voltage shift in channel activation, 2) pore blockade by protons, 3) protonation of histidines on the extracellular domain of HERG, 4) acceleration of recovery from C-type inactivation, and 5) interaction between an external H(+) binding site and the cytoplasmic NH(2)-terminal domain (a key determinant of HERG deactivation rate). Extracellular application of diethylpyrocarbonate caused an irreversible acceleration of HERG deactivation and prevented further acceleration by external acidification. Our data suggest that side chains accessible to the extracellular solution mediated the effects of elevating extracellular H(+) concentration on channel deactivation.