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Synapse-associated protein 97 (SAP97) is a scaffolding protein crucial for the functional expression of several cardiac ion channels and therefore proper cardiac excitability. Alterations in the functional expression of SAP97 can modify the ionic currents underlying the cardiac action potential and consequently confer susceptibility for arrhythmogenesis. In this study, we generated a murine model for inducible, cardiac-targeted Sap97 ablation to investigate arrhythmia susceptibility and the underlying molecular mechanisms. Furthermore, we sought to identify human SAP97 ( DLG1 ) variants that were associated with inherited arrhythmogenic disease. The murine model of cardiac-specific Sap97 ablation demonstrated several ECG abnormalities, pronounced action potential prolongation subject to high incidence of arrhythmogenic afterdepolarizations and notable alterations in the activity of the main cardiac ion channels. However, no DLG1 mutations were found in 40 unrelated cases of genetically elusive long QT syndrome (LQTS). Instead, we provide the first evidence implicating a gain of function in human DLG1 mutation resulting in an increase in Kv4.3 current ( I to ) as a novel, potentially pathogenic substrate for Brugada syndrome (BrS). In conclusion, DLG1 joins a growing list of genes encoding ion channel interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. Dysfunction in these critical components of cardiac excitability can potentially result in fatal cardiac disease. NEW & NOTEWORTHY The gene encoding SAP97 ( DLG1 ) joins a growing list of genes encoding ion channel-interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. In this study we provide the first data supporting DLG1- encoded SAP97's candidacy as a minor Brugada syndrome susceptibility gene.

Background: The mechanisms underlying spontaneous atrial fibrillation (AF) associated with atrial ischemia/infarction are incompletely elucidated. Here, we investigate the mechanisms underlying spontaneous AF in an ovine model of left atrial myocardial infarction (LAMI)., Methods and Results: LAMI was created by ligating the atrial branch of the left anterior descending coronary artery. ECG loop recorders were implanted to monitor AF episodes. In 7 sheep, dantrolene-a ryanodine receptor blocker-was administered in vivo during the 8-day observation period (LAMI-D, 2.5 mg/kg, IV, BID). LAMI animals experienced numerous spontaneous AF episodes during the 8-day monitoring period that were suppressed by dantrolene (LAMI, 26.1±5.1; sham, 4.3±1.1; LAMI-D, 2.8±0.8; mean±SEM episodes per sheep, P <0.01). Optical mapping showed spontaneous focal discharges (SFDs) originating from the ischemic/normal-zone border. SFDs were calcium driven, rate dependent, and enhanced by isoproterenol (0.03 µmol/L, from 210±87 to 3816±1450, SFDs per sheep) but suppressed by dantrolene (to 55.8±32.8, SFDs per sheep, mean±SEM). SFDs initiated AF-maintaining reentrant rotors anchored by marked conduction delays at the ischemic/normal-zone border. NOS1 (NO synthase-1) protein expression decreased in ischemic zone myocytes, whereas NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase and xanthine oxidase enzyme activities and reactive oxygen species (DCF [6-carboxy-2',7'-dichlorodihydrofluorescein diacetate]-fluorescence) increased. CaM (calmodulin) aberrantly increased [ 3 H]ryanodine binding to cardiac RyR2 (ryanodine receptors) in the ischemic zone. Dantrolene restored the physiological binding of CaM to RyR2., Conclusions: Atrial ischemia causes spontaneous AF episodes in sheep, caused by SFDs that initiate reentry. Nitroso-redox imbalance in the ischemic zone is associated with intense reactive oxygen species production and altered RyR2 responses to CaM. Dantrolene administration normalizes the CaM response, prevents LAMI-related SFDs, and AF initiation. These findings provide novel insights into the mechanisms underlying ischemia-related atrial arrhythmias., (© 2018 American Heart Association, Inc.)

Atrial fibrillation (AF) is by far the most common sustained tachyarrhythmia, affecting 1% to 2% of the general population. AF prevalence and the total annual cost for treatment are alarming, emphasizing the need for an urgent attention to the problem. Thus, having up-to-date information on AF risk factors and appreciating how they promote maintenance of AF maintenance are essential. This article presents a simplified examination of AF risk factors, including emerging genetic risks., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Epicardial adiposity and plasma levels of free fatty acids (FFAs) are elevated in atrial fibrillation, heart failure and obesity, with potentially detrimental effects on myocardial function. As major components of epicardial fat, FFAs may be abnormally regulated, with a potential to detrimentally modulate electro-mechanical function. The cellular mechanisms underlying such effects of FFAs are unknown., Objective: To determine the mechanisms underlying electrophysiological effects of palmitic (PA), stearic (SA) and oleic (OA) FFAs on sheep atrial myocytes., Methods: We used electrophysiological techniques, numerical simulations, biochemistry and optical imaging to examine the effects of acutely (≤ 15 min), short-term (4-6 hour) or 24-hour application of individual FFAs (10 μM) on isolated ovine left atrial myocytes (LAMs)., Results: Acute and short-term incubation in FFAs resulted in no differences in passive or active properties of isolated left atrial myocytes (LAMs). 24-hour application had differential effects depending on the FFA. PA did not affect cellular passive properties but shortened (p<0.05) action potential duration at 30% repolarization (APD30). APD50 and APD80 were unchanged. SA had no effect on resting membrane potential but reduced membrane capacitance by 15% (p<0.05), and abbreviated APD at all values measured (p≤0.001). OA did not significantly affect passive or active properties of LAMs. Measurement of the major voltage-gated ion channels in SA treated LAMs showed a ~60% reduction (p<0.01) of the L-type calcium current (ICa-L) and ~30% reduction (p<0.05) in the transient outward potassium current (ITO). A human atrial cell model recapitulated SA effects on APD. Optical imaging showed that SA incubated for 24 hours altered t-tubular structure in isolated cells (p<0.0001)., Conclusions: SA disrupts t-tubular architecture and remodels properties of membrane ionic currents in sheep atrial myocytes, with potential implications in arrhythmogenesis.

The understanding of ionic mechanisms underlying cardiac rhythm disturbances (arrhythmias) is an issue of significance in the medical science community. Several advances in molecular, cellular, and optical techniques in the past few decades have substantially increased our knowledge of ionic mechanisms that are thought to underlie arrhythmias. The application of these techniques in the study of ion channel biophysics and regulatory properties has provided a wealth of information, with some important therapeutic implications for dealing with the disease. In this review, we briefly consider the cellular and tissue manifestations of a number of cardiac rhythm disturbances, while focusing on our current understanding of the ionic current mechanisms that have been implicated in such rhythm disturbances., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Atrial fibrillation (AF) is by far the most common sustained tachyarrhythmia, affecting 1% to 2% of the general population. AF prevalence and the total annual cost for treatment are alarming, emphasizing the need for an urgent attention to the problem. Thus, having up-to-date information on AF risk factors and appreciating how they promote maintenance of AF maintenance are essential. This article presents a simplified examination of AF risk factors, including emerging genetic risks., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Persistent atrial fibrillation (PAF) results in electromechanical and structural remodeling by mechanisms that are poorly understood. Myofibroblast proliferation and fibrosis are major sources of structural remodeling in PAF. Myofibroblasts also interact with atrial myocytes via direct physical contact and release of signaling molecules, which may contribute to remodeling., Objective: To determine whether myofibroblasts contribute to atrial myocyte electromechanical remodeling via direct physical contact and platelet-derived growth factor (PDGF) signaling., Methods: Myofibroblasts and myocytes from adult sheep atria were co-cultured for 24 hours. Alternatively adult sheep atrial myocytes were exposed to 1 ng/mL recombitant PDGF AB peptide for 24 hours., Results: Myocytes making contact with myofibroblasts demonstrated significant reduction (P ≤ .05) in peak L-type calcium current density, shortening of action potential duration (APD), and reduction in calcium transients. These effects were blocked by pretreatment with a PDGF-AB neutralizing anti-body. Heterocellular contact also severely disturbed the localization of the L-type calcium channel. Myocytes exposed to recombinant PDGF-AB peptide for 24 hours demonstrated reduced APD50, APD80 and Peak L-type calcium current. Pretreatment with a PDGF-AB neutralizing antibody prevented these effects. Finally, while control atrial myocytes did not respond in a 1:1 manner to pacing frequencies of 3 Hz or higher, atrial myocytes from hearts that were tachypaced for 2 months and normal myocytes treated with PDGF-AB for 24 hours could be paced up to 10 Hz., Conclusions: In addition to leading to fibrosis, atrial myofibroblasts contribute to electromechanical remodeling of myocytes via direct physical contact and release of PDGF-AB, which may be a factor in PAF-induced remodeling., (Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Collecting electrophysiological and molecular data from the murine conduction system presents technical challenges. Thus, only little advantage has been taken of numerous genetically engineered murine models to study excitation through the cardiac conduction system of the mouse., Objective: To develop an approach for isolating murine cardiac Purkinje cells (PCs), to characterize major ionic currents and to use the data to simulate action potentials (APs) recorded from PCs., Methods: Light microscopy was used to isolate and identify PCs from apical and septal cells. Current and voltage clamp techniques were used to record APs and whole cell currents. We then simulated a PC AP on the basis of our experimental data., Results: APs recorded from PCs were significantly longer than those recorded from ventricular cells. The prominent plateau phase of the PC AP was very negative (≈-40 mV). Spontaneous activity was observed only in PCs. The inward rectifier current demonstrated no significant differences compared to ventricular myocytes (VMs). However, sodium current density was larger, and the voltage-gated potassium current density was significantly less in PCs compared with myocytes. T-type Ca(2+) currents (I(Ca,T)) were present in PCs but not VMs. Computer simulations suggest that I(Ca,T) and cytosolic calcium diffusion significantly modulate AP profile recorded in PCs, as compared to VMs., Conclusions: Our study provides the first comprehensive ionic profile of murine PCs. The data show unique features of PC ionic mechanisms that govern its excitation process. Experimental data and numerical modeling results suggest that a smaller voltage-gated potassium current and the presence of I(Ca,T) are important determinants of the longer and relatively negative plateau phase of the APs., (Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Rationale: Kv1.5 (KCNA5) is expressed in the heart, where it underlies the I(Kur) current that controls atrial repolarization, and in the pulmonary vasculature, where it regulates vessel contractility in response to changes in oxygen tension. Atrial fibrillation and hypoxic pulmonary hypertension are characterized by downregulation of Kv1.5 protein expression, as well as with oxidative stress. Formation of sulfenic acid on cysteine residues of proteins is an important, dynamic mechanism for protein regulation under oxidative stress. Kv1.5 is widely reported to be redox-sensitive, and the channel possesses 6 potentially redox-sensitive intracellular cysteines. We therefore hypothesized that sulfenic acid modification of the channel itself may regulate Kv1.5 in response to oxidative stress., Objective: To investigate how oxidative stress, via redox-sensitive modification of the channel with sulfenic acid, regulates trafficking and expression of Kv1.5., Methods and Results: Labeling studies with the sulfenic acid-specific probe DAz and horseradish peroxidase-streptavidin Western blotting demonstrated a global increase in sulfenic acid-modified proteins in human patients with atrial fibrillation, as well as sulfenic acid modification to Kv1.5 in the heart. Further studies showed that Kv1.5 is modified with sulfenic acid on a single COOH-terminal cysteine (C581), and the level of sulfenic acid increases in response to oxidant exposure. Using live-cell immunofluorescence and whole-cell voltage-clamping, we found that modification of this cysteine is necessary and sufficient to reduce channel surface expression, promote its internalization, and block channel recycling back to the cell surface. Moreover, Western blotting demonstrated that sulfenic acid modification is a trigger for channel degradation under prolonged oxidative stress., Conclusions: Sulfenic acid modification to proteins, which is elevated in diseased human heart, regulates Kv1.5 channel surface expression and stability under oxidative stress and diverts channel from a recycling pathway to degradation. This provides a molecular mechanism linking oxidative stress and downregulation of channel expression observed in cardiovascular diseases.

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I(K1)), which is important for maintenance of the cell resting membrane potential, and the sodium current (I(Na)), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I(K1) modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I(K1)-I(Na) interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I(K1)-I(Na) interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na(V)1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na(v)1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na(v)1.5-Kir2.1 interactions. The reciprocal modulation between Na(v)1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.

Background: Sodium channel α-subunits in ventricular myocytes (VMs) segregate either to the intercalated disc or to lateral membranes, where they associate with region-specific molecules., Objective: To determine the functional properties of sodium channels as a function of their location in the cell., Methods: Local sodium currents were recorded from adult rodent VMs and Purkinje cells by using the cell-attached macropatch configuration. Electrodes were placed either in the cell midsection (M) or at the cell end (area originally occupied by the intercalated disc [ID]). Channels were identified as tetrodotoxin (TTX)-sensitive (TTX-S) or TTX-resistant (TTX-R) by application of 100 nM of TTX., Results: Average peak current amplitude was larger in ID than in M and largest at the site of contact between attached cells. TTX-S channels were found only in the M region of VMs and not in Purkinje myocytes. TTX-R channels were found in both M and ID regions, but their biophysical properties differed depending on recording location. Sodium current in rat VMs was upregulated by tumor necrosis factor-alpha. The magnitude of current increase was largest in the M region, but this difference was abolished by application of 100 nM of TTX., Conclusions: Our data suggest that (a) a large fraction of TTX-R (likely Na(v)1.5) channels in the M region of VMs are inactivated at normal resting potential, leaving most of the burden of excitation to TTX-R channels in the ID region; (b) cell-cell adhesion increases functional channel density at the ID; and (c) TTX-S (likely non-Na(v)1.5) channels make a minimal contribution to sodium current under control conditions, but they represent a functional reserve that can be upregulated by exogenous factors., (Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Synapse-associated protein-97 (SAP97) is a membrane-associated guanylate kinase scaffolding protein expressed in cardiomyocytes. SAP97 has been shown to associate and modulate voltage-gated potassium (Kv) channel function. In contrast to Kv channels, little information is available on interactions involving SAP97 and inward rectifier potassium (Kir2.x) channels that underlie the classical inward rectifier current, I(K1). To investigate the functional effects of silencing SAP97 on I(K1) in adult rat ventricular myocytes, SAP97 was silenced using an adenoviral short hairpin RNA vector. Western blot analysis showed that SAP97 was silenced by approximately 85% on day 3 post-infection. Immunostaining showed that Kir2.1 and Kir2.2 co-localize with SAP97. Co-immunoprecipitation (co-IP) results demonstrated that Kir2.x channels associate with SAP97. Voltage clamp experiments showed that silencing SAP97 reduced I(K1) whole cell density by approximately 55%. I(K1) density at -100 mV was -1.45 +/- 0.15 pA/picofarads (n = 6) in SAP97-silenced cells as compared with -3.03 +/- 0.37 pA/picofarads (n = 5) in control cells. Unitary conductance properties of I(K1) were unaffected by SAP97 silencing. The major mechanism for the reduction of I(K1) density appears to be a decrease in Kir2.x channel abundance. Furthermore, SAP97 silencing impaired I(K1) regulation by beta(1)-adrenergic receptor (beta1-AR) stimulation. In control, isoproterenol reduced I(K1) amplitude by approximately 75%, an effect that was blunted following SAP97 silencing. Our co-IP data show that beta1-AR associates with SAP97 and Kir2.1 and also that Kir2.1 co-IPs with protein kinase A and beta1-AR. SAP97 immunolocalizes with protein kinase A and beta1-AR in the cardiac myocytes. Our results suggest that in cardiac myocytes SAP97 regulates surface expression of channels underlying I(K1), as well as assembles a signaling complex involved in beta1-AR regulation of I(K1).

Cardiac I(K1) and I(KACh) are the major potassium currents displaying classical strong inward rectification, a unique property that is critical for their roles in cardiac excitability. In the last 15 years, research on I(K1) and I(KACh) has been propelled by the cloning of the underlying inwardly rectifying potassium (Kir) channels, the discovery of the molecular mechanism of strong rectification and the linking of a number of disorders of cardiac excitability to defects in genes encoding Kir channels. Disease-causing mutations in Kir genes have been shown experimentally to affect one or more of the following channel properties: structure, assembly, trafficking, and regulation, with the ultimate effect of a gain- or a loss-of-function of the channel. It is now established that I(K1) and I(KACh) channels are heterotetramers of Kir2 and Kir3 subunits, respectively. Each homomeric Kir channel has distinct biophysical and regulatory properties, and individual Kir subunits often display different patterns of regional, cellular, and membrane distribution. These differences are thought to underlie important variations in the physiological properties of I(K1) and I(KACh). It has become increasingly clear that the contribution of I(K1) and I(KACh) channels to cardiac electrical activity goes beyond their long recognized role in the stabilization of resting membrane potential and shaping the late phase of action potential repolarization in individual myocytes but extends to being critical elements determining the overall electrical stability of the heart., (Copyright 2009 Elsevier Inc. All rights reserved.)

We examined the impact of coexpressing the inwardly rectifying potassium channel, Kir2.3, with the scaffolding protein, synapse-associated protein (SAP) 97, and determined that coexpression of these proteins caused an approximately twofold increase in current density. A combination of techniques was used to determine if the SAP97-induced increase in Kir2.3 whole cell currents resulted from changes in the number of channels in the cell membrane, unitary channel conductance, or channel open probability. In the absence of SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicular compartment with relatively little Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3, leading to prominent colocalization of Kir2.3 and SAP97 and a modest increase in cell surface Kir2.3. The median Kir2.3 single channel conductance in the absence of SAP97 was approximately 13 pS, whereas coexpression of SAP97 led to a wide distribution of channel events with three distinct peaks centered at 16, 29, and 42 pS. These changes occurred without altering channel open probability, current rectification properties, or pH sensitivity. Thus association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance. However, changes in single channel conductance play the major role in determining whole cell currents in this model system. We further suggest that the SAP97 effect results from SAP97 binding to the Kir2.3 COOH-terminal domain and altering channel conformation.

The mechanisms controlling the rotation frequency of functional reentry in ventricular fibrillation (VF) are poorly understood. It has been previously shown that Ba2+ at concentrations up to 50 mumol/L slows the rotation frequency in the intact guinea pig (GP) heart, suggesting a role of the inward rectifier current (I(K1)) in the mechanism governing the VF response to Ba2+. Given that other biological (e.g., sinoatrial node) and artificial systems display phase-locking behavior, we hypothesized that the mechanism for controlling the rotation frequency of a rotor by I(K1) blockade is phase-driven, i.e., the phase shift between transmembrane current and voltage remains constant at varying levels of I(K1) blockade. We measured whole-cell admittance in isolated GP myocytes and in transfected human embryonic kidney (HEK) cells stably expressing Kir 2.1 and 2.3 channels. The admittance phase, i.e., the phase difference between current and voltage, was plotted versus the frequency in control conditions and at 10 or 50 micromol/L Ba2+ (in GP heart cells) or 1 mM Ba2+ (in HEK cells). The horizontal distance between plots was called the "frequency shift in a single cell" and analyzed. The frequency shift in a single cell was -14.14 +/- 5.71 Hz (n = 14) at 10 microM Ba2+ and -18.51 +/- 4.00 Hz (n = 10) at 50 microM Ba2+, p < 0.05. The values perfectly matched the Ba2+-induced reduction of VF frequency observed previously in GP heart. A similar relationship was found in the computer simulations. The phase of Ba2+-sensitive admittance in GP cells was -2.65 +/- 0.32 rad at 10 Hz and -2.79 +/- 0.26 rad at 30 Hz. In HEK cells, the phase of Ba2+-sensitive admittance was 3.09 +/- 0.03 rad at 10 Hz and 3.00 +/- 0.17 rad at 30 Hz. We have developed a biological single-cell model of rotation-frequency control. The results show that although rotation frequency changes as a result of I(K1) blockade, the phase difference between transmembrane current and transmembrane voltage remains constant, enabling us to quantitatively predict the change of VF frequency resulting from I(K1) blockade, based on single-cell measurement.

Background: Data on pH regulation of the cardiac potassium current I(K1) suggest species-dependent differences in the molecular composition of the underlying Kir2 channel proteins., Objective: The purpose of this study was to test the hypothesis that the presence of the Kir2.3 isoform in heterotetrameric channels modifies channel sensitivity to pH., Methods: Voltage clamp was performed on HEK293 cells stably expressing guinea pig Kir2.1 and/or Kir2.3 isoforms and on sheep cardiac ventricular myocytes at varying extracellular pH (pH(o)) and in the presence of CO(2) to determine the sensitivity of macroscopic currents to pH. Single-channel activity was recorded from the HEK293 stables to determine the mechanisms of the changes in whole cell current., Results: Biophysical characteristics of whole-cell and single-channel currents in Kir2.1/Kir2.3 double stables displayed properties attributable to isoform heteromerization. Whole-cell Kir2.1/Kir2.3 currents rectified in a manner reminiscent of Kir2.1 but were significantly inhibited by extracellular acidification in the physiologic range (pK(a) approximately 7.4). Whole-cell currents were more sensitive to a combined extracellular and intracellular acidification produced by CO(2). At pH(o) = 6.0, unitary conductances of heteromeric channels were reduced. Ovine cardiac ventricular cell I(K1) was pH(o) and CO(2) sensitive, consistent with the expression of Kir2.1 and Kir2.3 in this species., Conclusion: Kir2.1 and Kir2.3 isoforms form heteromeric channels in HEK293. The presence of Kir2.3 subunit(s) in heteromeric channels confers pH sensitivity to the channels. The single and double stable cells presented in this study are useful models for studying physiologic regulation of heteromeric Kir2 channels in mammalian cells.

Construction of the action potential duration (APD) restitution portrait allows visualization of multiple aspects of the dynamics of periodically paced myocytes at various basic cycle lengths (BCLs). For the first time, we obtained the restitution portrait of isolated rabbit and guinea pig cardiac ventricular myocytes and analyzed the time constant, tau, of APD accommodation and the slopes of different types of restitution curves, Sdyn and S12, measured at varying BCLs. Our results indicate that both tau and the individual slopes are species and pacing dependent. In contrast, the mutual relationship between slopes Sdyn and S12 does not depend on pacing history, being a generic feature of the species. In addition, the maximum slope S12, measured in the restitution portrait at the lowest BCL, predicts the onset of alternans. Further, we investigated the role of the L-type calcium current, ICa-L, in the restitution portrait. We found that ICa-L dramatically affects APD accommodation, as well as the individual slopes Sdyn and S12 measured in the restitution portrait. However, peak calcium current plays a role only at small values of BCL. In conclusion, the results demonstrate that the restitution portrait is a powerful technique to investigate restitution properties of periodically paced cardiac myocytes and the onset of alternans, in particular. Moreover, the data also show that ICa-L plays a crucial role in multiple aspects of cardiac dynamics measured through the restitution portrait.

Recent studies suggest that atrial fibrillation (AF) is maintained by fibrillatory conduction emanating from a small number of high-frequency reentrant sources (rotors). Our goal was to study the ionic correlates of a rotor during simulated chronic AF conditions. We utilized a two-dimensional (2-D), homogeneous, isotropic sheet (5 x 5 cm(2)) of human atrial cells to create a chronic AF substrate, which was able to sustain a stable rotor (dominant frequency approximately 5.7 Hz, rosette-like tip meander approximately 2.6 cm). Doubling the magnitude of the inward rectifier K(+) current (I(K1)) increased rotor frequency ( approximately 8.4 Hz), and reduced tip meander (approximately 1.7 cm). This rotor stabilization was due to a shortening of the action potential duration and an enhanced cardiac excitability. The latter was caused by a hyperpolarization of the diastolic membrane potential, which increased the availability of the Na(+) current (I(Na)). The rotor was terminated by reducing the maximum conductance (by 90%) of the atrial-specific ultrarapid delayed rectifier K(+) current (I(Kur)), or the transient outward K(+) current (I(to)), but not the fast or slow delayed rectifier K(+) currents (I(Kr)/I(Ks)). Importantly, blockade of I(Kur)/I(to) prolonged the atrial action potential at the plateau, but not at the terminal phase of repolarization, which led to random tip meander and wavebreak, resulting in rotor termination. Altering the rectification profile of I(K1) also slowed down or abolished reentrant activity. In combination, these simulation results provide novel insights into the ionic bases of a sustained rotor in a 2-D chronic AF substrate.

The inwardly rectifying potassium (Kir) 2.x channels mediate the cardiac inward rectifier potassium current (I(K1)). In addition to differences in current density, atrial and ventricular I(K1) have differences in outward current profiles and in extracellular potassium ([K+]o) dependence. The whole-cell patch-clamp technique was used to study these properties in heterologously expressed Kir2.x channels and atrial and ventricular I(K1) in guinea pig and sheep hearts. Kir2.x channels showed distinct rectification profiles: Kir2.1 and Kir2.2 rectified completely at potentials more depolarized than -30 mV (I approximately 0 pA). In contrast, rectification was incomplete for Kir2.3 channels. In guinea pig atria, which expressed mainly Kir2.1, I(K1) rectified completely. In sheep atria, which predominantly expressed Kir2.3 channels, I(K1) did not rectify completely. Single-channel analysis of sheep Kir2.3 channels showed a mean unitary conductance of 13.1+/-0.1 pS in 15 cells, which corresponded with I(K1) in sheep atria (9.9+/-0.1 pS in 32 cells). Outward Kir2.1 currents were increased in 10 mmol/L [K+]o, whereas Kir2.3 currents did not increase. Correspondingly, guinea pig (but not sheep) atrial I(K1) showed an increase in outward currents in 10 mmol/L [K+]o. Although the ventricles of both species expressed Kir2.1 and Kir2.3, outward I(K1) currents rectified completely and increased in high [K+]o-displaying Kir2.1-like properties. Likewise, outward current properties of heterologously expressed Kir2.1-Kir2.3 complexes in normal and 10 mmol/L [K+]o were similar to Kir2.1 but not Kir2.3. Thus, unique properties of individual Kir2.x isoforms, as well as heteromeric Kir2.x complexes, determine regional and species differences of I(K1) in the heart.

A major goal of basic research in cardiac electrophysiology is to understand the mechanisms responsible for ventricular fibrillation (VF). Here we review recent experimental and numerical results, from the ion channel to the organ level, which might lead to a better understanding of the cellular and molecular mechanisms of VF. The discussion centers on data derived from a model of stable VF in the Langendorff-perfused guinea pig heart that demonstrate distinct patterns of organization in the left (LV) and right (RV) ventricles. Analysis of optical mapping data reveals that VF excitation frequencies are distributed throughout the ventricles in clearly demarcated domains. The highest frequency domains are usually found on the anterior wall of the LV, demonstrating that a high frequency reentrant source (a rotor) that remains stationary in the LV is the mechanism that sustains VF in this model. Computer simulations predict that the inward rectifying potassium current (IK1) is an essential determinant of rotor stability and rotation frequency, and patch-clamp results strongly suggest that the outward component of the background current (presumably IK1) of cells in the LV is significantly larger in the LV than in the RV. These data have opened a new and potentially exciting avenue of research on the possible role played by inward rectifier channels in the mechanism of VF and may lead us toward an understanding of its molecular basis and hopefully lead to new preventative approaches.

Objective: We tested the hypothesis that left atrial (LA) myocytes are more sensitive to acetylcholine (ACh) than right atrial (RA) myocytes, which results in a greater dose-dependent increase in LA than RA rotor frequency, increased LA-to-RA frequency gradient and increased incidence of wavelet formation during atrial fibrillation (AF)., Methods and Results: AF was induced in seven Langendorff-perfused sheep hearts in the presence of ACh (0.1-4.0 microM) and studied using optical mapping and bipolar recordings. Dominant frequencies (DFs) were determined in optical and electrical signals and phase movies were used to identify rotors and quantify their dynamics. DFs in both atria increased monotonically with ACh concentration until saturation, but the LA frequency predominated at all concentrations. Rotors were also seen more often in the LA, and although their life span decreased, their frequency and number of rotations increased. Patch-clamp studies demonstrated that ACh-activated potassium current (I(K,ACh)) density was greater in LA than RA sheep myocytes. Additionally, ribonuclease protection assay demonstrated that Kir3.4 and Kir3.1 mRNAs were more abundant in LA than in RA., Conclusions: A greater abundance of Kir3.x channels and higher I(K,ACh) density in LA than RA myocytes result in greater ACh-induced speeding-up of rotors in the LA than in the RA, which explains the ACh dose-dependent changes in overall AF frequency and wavelet formation.

Introduction: Stable high-frequency rotors sustain ventricular fibrillation (VF) in the guinea pig heart. We surmised that rotor stabilization in the left ventricle (LV) and fibrillatory conduction toward the right ventricle (RV) result from chamber-specific differences in functional expression of inward rectifier (Kir2.x) channels and unequal IK1 rectification in LV and RV myocytes. Accordingly, selective blockade of IK1 during VF should terminate VF., Methods and Results: Relative mRNA levels of Kir2.x channels were measured in LV and RV. In addition, LV (n = 21) and RV (n = 20) myocytes were superfused with BaCl2 (5-50 micromol/L) to study the effects on IK1. Potentiometric dye-fluorescence movies of VF were obtained in the presence of Ba2+ (0-50 micromol/L) in 23 Langendorff-perfused hearts. Dominant frequencies (DFs) were determined by spectral analysis, and singularity points were counted in phase maps to assess VF organization. mRNA levels for Kir2.1 and Kir2.3 were significantly larger in LV than RV. Concurrently, outward IK1 was significantly larger in LV than RV myocytes. Ba2+ decreased IK1 in a dose-dependent manner (LV change > RV change). In baseline control VF, the fastest DF domain (28-40 Hz) was located on the anterior LV wall and a sharp LV-to-RV frequency gradient of 21.2 +/- 4.3 Hz was present. Ba2+ significantly decreased both LV frequency and gradient, and it terminated VF in a dose-dependent manner. At 50 micromol/L, Ba2+ decreased the average number of wavebreaks (1.7 +/- 0.9 to 0.8 +/- 0.6 SP/sec x pixel, P < 0.05) and then terminated VF., Conclusion: The results strongly support the hypothesis that IK1 plays an important role in rotor stabilization and VF dynamics.

Studies have characterized conduction velocity in the right and left bundle branches (RBB, LBB) of normal and genetically engineered mice. However, no information is available on the action potential characteristics of the specialized conduction system (SCS). We have used microelectrode techniques to characterize action potential properties of the murine SCS, as well as epicardial and endocardial muscle preparations for comparison. In the RBB, action potential duration at 50%, 70%, and 90% repolarization (APD(50,70,90)) was 6+/-0.7, 35+/-6, and 90+/-7 ms, respectively. Maximum upstroke velocity (dV/dt(max)) was 153+/-14 V/s, and conduction velocity averaged 0.85+/-0.2 m/s. APD(90) was longer in the Purkinje network of fibers (web) than in the RBB (P<0.01). Web APD(50) was longer in the left than in the right ventricle (P<0.05). Yet, web APD(90) was longer in the right than in the left ventricle (P<0.001). APD(50,70) was significantly longer in the endocardial than in the epicardial (P<0.001; P<0.003). APD(90) in the epicardial and endocardial was shorter than in the RBB ( approximately 36 ms versus approximately 100 ms). Spontaneous electrical oscillations in phase 2 of the SCS occasionally resulted in early afterdepolarizations. These results demonstrate that APDs in the murine SCS are significantly ( approximately 2-fold) longer than in the myocardium and implicate the role of the murine SCS in arrhythmias. The differences should have important implications in the use of the mouse heart to study excitation, propagation, and arrhythmias.

Chemical regulation of connexin (Cx) 40 and Cx43 follows a ball-and-chain model, in which the carboxyl terminal (CT) domain acts as a gating particle that binds to a receptor affiliated with the pore. Moreover, Cx40 channels can be closed by a heterodomain interaction with the CT domain of Cx43 and vice versa. Here, we report similar interactions in the establishment of the unitary conductance and voltage-dependent profile of Cx40 in N2A cells. Two mean unitary conductance values ("lower conductance" and "main") were detected in wild-type Cx40. Truncation of the CT domain at amino acid 248 (Cx40tr248) caused the disappearance of the lower-conductance state. Coexpression of Cx40tr248 with the CT fragment of either Cx40 (homodomain interactions) or Cx43 (heterodomain interactions) rescued the unitary conductance profile of Cx40. In the N2A cells, the time course of macroscopic junctional current relaxation was best described by a biexponential function in the wild-type Cx40 channels, but it was reduced to a single-exponential function after truncation. However, macroscopic junctional currents recorded in the oocyte expression system were not significantly different between the wild-type and mutant channels. Concatenation of the CT domain of Cx43 to amino acids 1 to 248 of Cx40 yielded a chimeric channel with unitary conductance and voltage-gating profile indistinguishable from that of wild-type Cx40. We conclude that residence of Cx40 channels in the lower-conductance state involves a ball-and-chain type of interaction between the CT domain and the pore-forming region. This interaction can be either homologous (Cx40 truncation with Cx40CT) or heterologous (with the Cx43CT).

The proton and Zn2+ effects on the human ether-a-go-go related gene (HERG) channels were studied after expression in Xenopus oocytes and stable transfection in the mammalian L929 cell line. Experiments were carried out using the two-electrode voltage clamp at room temperature (oocytes) or the whole-cell patch clamp technique at 35 degrees C (L929 cells). In oocytes, during moderate extracellular acidification (pHo = 6.4), current activation was not shifted on the voltage axis, the time course of current activation was unchanged, but tail current deactivation was dramatically accelerated. At pHo < 6.4, in addition to accelerating deactivation, the time course of activation was slower and the midpoint voltage of current activation was shifted to more positive values. Protons and Zn2+ accelerated the kinetics of deactivation with apparent Kd values about one order of magnitude lower than for tail current inhibition. For protons, the Kd values for the effect on tail current amplitude versus kinetics were, respectively, 1.8 microM (pKa = 5.8) and 0.1 microM (pKa = 7.0). In the presence of Zn2+, the corresponding Kd values were, respectively, 1.2 mM and 169 microM. In L929 cells, acidification to pHo = 6.4 did not shift the midpoint voltage of current activation and had no effect on the time course of current activation. Furthermore, the onset and recovery of inactivation were not affected. However, the acidification significantly accelerated tail current deactivation. We conclude that protons and Zn2+ directly interact with HERG channels and that the interaction results, preferentially, in the regulation of channel deactivation mechanism.

Introduction: A cardiac culture cell line (AT-1) recently has been generated from transgenic mice. Initial studies have yielded opposing results as to the nature of the major repolarizing current(s) in these cells. The present study describes the ion selectivity, voltage dependence, and E4031 sensitivity of the major time-dependent outward current present in AT-1 cells. In addition, we have determined whether an outward current with the characteristics we observed could be capable of modulating action potential duration in a frequency-dependent manner (for stimulation cycle lengths between 250 and 1000 msec)., Methods and Results: Action potentials and membrane currents were recorded from nonconfluent cultures of quiescent AT-1 cells using the "perforated patch" technique. AT-1 cells showed a round appearance 1 or 2 days after plating. An E4031-insensitive transient outward current seemed to be absent in these cells. The main time-dependent outward current was a rapidly activating and rectifying potassium current with properties similar to those of IKr. Most of the potassium current was sensitive to the benzenesulfonamide E4031 (5 microM). The same concentration of E4031 led to a 38% increase in action potential duration. Action potential parameters were independent of the stimulation cycle length within the range of 250 to 1000 msec, thus suggesting that the membrane currents involved in the action potential of AT-1 cells are completely reset within a diastolic interval of approximately 150 msec., Conclusion: AT-1 cells present a unique electrophysiologic phenotype, which is clearly different from that reported for freshly dissociated adult atrial or ventricular myocytes from other species. AT-1 cells may be a good model to study IKr, since there seems to be minimal contamination by other outward conductances (such as IKs). In addition, the feasibility of culturing AT-1 cells provides us with a system where electrophysiologic experiments on IKr currents could be combined with biochemical or molecular biological studies requiring significant periods of incubation in a cell culture system.

Inhibition of aerobic metabolism leads to a major disruption of cardiac cell homeostasis. The purpose of the present study was twofold: 1) We determined the relative importance of junctional and nonjunctional membrane resistance (Rj and Rm, respectively) in the development of propagation failure during inhibition of aerobic metabolism in guinea pig ventricular cell pairs. 2) We used the patch-action potential clamp technique in single ventricular myocytes to study some of the properties of the membrane channels that are responsible for shortening of action potential duration and eventual failure of cell excitation after metabolic blockade. In most experiments, whole-cell patch pipettes were filled with a solution containing 1 mM EGTA, 5 mM HEPES, and 5 mM ATP. Our results in cell pairs showed that pharmacological inhibition of aerobic metabolism with the mitochondrial uncoupler 2,4-dinitrophenol (DNP) led to a drop in Rm followed by an increase in Rj. The increase in Rj was not sufficient to cause a measurable delay in cell-to-cell propagation, whereas the drop in Rm consistently led to failure of cell excitation. Similar results were obtained in additional experiments in which the EGTA concentration in the pipette was reduced to 50 microM. Similar results were also obtained by loading the recording patch pipettes with a solution containing only 0.1 mM ATP. Our patch-action potential clamp experiments, on the other hand, revealed that DNP induced the opening of time- and voltage-independent membrane channels, with a unitary conductance of 23 pS. The channels allowed for the passage of outward current in the voltage range of the action potential, and the increase in membrane patch conductance correlated with the observed shortening of action potential duration during DNP superfusion. Our experiments provide the first simultaneous recordings of action potentials and DNP-induced channel currents in guinea pig ventricular myocytes. Overall, the data provide new evidence for the understanding of the cellular and subcellular mechanisms involved in the development of slow conduction velocity and propagation block after metabolic blockade.

The subcellular mechanism of cell-to-cell communication in the natural pacemaker region of the mammalian heart was studied using electrophysiological and immunofluorescence techniques in isolated pairs of rabbit sinus nodal cells. By measuring whole-cell currents using a double patch-clamp approach, it was demonstrated that communication in the sinus node is mediated through gap junctional channels similar to those in other types of adult cardiac cell pairs. Macroscopic sinus nodal junctional resistance had a mean value of 387.9 +/- 97.1 M omega (mean +/- SEM, n = 10) and was greatly increased by superfusion with alkanols. Single-channel junctional conductance could be resolved in three cell pairs. Given their high membrane resistance (1.16 +/- 0.32 G omega, n = 12), the electrical coupling provided by as few as three gap junctional channels between nodal cells will allow for pacemaker synchronization. Further evidence for the presence of the channels was obtained from immunofluorescent double-labeling of desmin and the gap junction protein (connexin43) in sinus nodal tissue as well as in cultured sinus nodal cells. Using antisera against residues 243-257 of the connexin43 protein, a specific staining at the site of cell-to-cell apposition was demonstrated. These data provide direct evidence in favor of electronic coupling as the means for achieving pacemaker synchronization in the rabbit sinus node.

We have studied delayed rectifier K+ currents (IK) in cells isolated from the sinoatrial node (SAN) region of the guinea pig. Using whole cell patch-clamp procedures, we measured the voltage dependence of IK activation and IK kinetics and the IK equilibrium potential in 4.8 mM extracellular K concentration solutions. Experiments were designed to contrast properties of guinea pig SAN IK with those of IK recorded from SAN cells of the rabbit. We find that guinea pig SAN IK differs from IK recorded from single rabbit SAN cells in its activation threshold, and in the absence of inactivation of whole cell currents recorded over a wide voltage range. These results, along with the relative insensitivity of guinea pig SAN IK to E-4031 and lanthanum, suggest that under our experimental conditions, a strongly rectifying IK component (IK,r) is not the major component of delayed rectification in the guinea pig SAN, as it appears to be in SAN cells of the rabbit.

The phase-resetting and entrainment properties of single pacemaker cells were studied using computer simulations in a model of the rabbit sinus nodal cell, as well as using the whole-cell patch-clamp (current-clamp) technique in isolated rabbit sinus nodal cells. Spontaneous electrical activity in the cell model was reconstructed using Hodgkin-Huxley-type equations describing time- and voltage-dependent membrane currents. In both simulations and experiments, single subthreshold current pulses (depolarizing or hyperpolarizing) were used to scan the spontaneous cycle of the cells. Such pulses perturbed the subsequent discharge, producing temporary phasic changes in pacemaker period, and enabled the construction of phase response curves. On the basis of these results, we studied entrainment characteristics of the cells. For example, application of repetitive pulses allowed for phasic changes in the spontaneous cycle and resulted in stable 1:1 entrainment at a range of basic cycle length around the spontaneous cycle, or a 2:1 pattern at basic cycle length values about half the spontaneous cycle length. Between the two entrainment zones, complex Wenckebach-like patterns (e.g., 5:4, 4:3, and 3:2) were observed. The experimental data from the isolated cell were further analyzed from a theoretical perspective, and the results showed that the topological characteristics of the phase-resetting behavior accounts for the experimentally observed patterns during repetitive stimulation of the cell. This first demonstration of phase resetting in single cells provides the basis for phenomena such as mutual entrainment between electrically coupled pacemaker cells, apparent intranodal conduction, and reflex vagal control of heart rate.

1. The electrophysiology of single myocytes isolated from the rabbit tricuspid valve was studied using the patch-clamp method (whole-cell configuration). Cell dispersion was achieved by collagenase treatment, using the Langendorff retrograde perfusion procedure. 2. After isolation, and while incubating in the recovery (Kraftbrühe) solution, cells had clear striations and were mostly spindle-shaped, or rod-like (less than 10%), with length varying from 35 microns to over 150 microns, and diameter from 3 to 10 microns. 3. Upon exposure to Tyrode solution, the calcium-tolerant cells were mostly rounded with smooth surfaces and well-defined borders. The mean diameter of these cells was 15 +/- 5 microns (S.D., n = 9). A smaller percentage (about 30%) retained the original elongated shape. 4. Patch pipette recordings showed the presence of spontaneous activity in about 30% of round cells, and less frequently in elongated cells. Maximum diastolic potentials (MDPs) in the round cells averaged -82 +/- 6 mV, with a take-off potential of -56 +/- 3 mV (n = 9), and an average maximum upstroke velocity (Vmax) value of 6.3 +/- 0.6 V/s (n = 4). In quiescent cells, the mean resting potential was 69 +/- 12 mV (n = 43). 5. Voltage clamp ramps revealed a steady-state I-V relation with a negative slope region. The mean input resistance value was 25 +/- 9 M omega (n = 16) for the elongated, and 883 +/- 481 M omega (n = 8) for the round cells. 6. Hyperpolarizing 5 s pulses (holding potential = -50 mV) occasionally revealed a slow, time-dependent inward current whose peak increased progressively as a function of clamp potential. The slowly activating current was sensitive to caesium 2 mM), indicating its similarity to the so-called 'pacemaker current' (iF). In alternate voltage- and current-clamp experiments, blocking of iF did not stop pacemaker activity, but there was up to a fourfold increase in pacemaker cycle length. 7. In some cells, 5 s hyperpolarizing steps from a holding potential of -40 or -50 mV produced large, inwardly directed and voltage-dependent current surges that decayed rapidly with time, similar to the inactivation described for the inward rectifier current, iK1. The current was very prominent at voltages more negative than -100 mV, and its decay process was best fitted by two time constants, one fast and one slow. For example, at -150 mV the time constants were 61 and 634 ms. The inward current was blocked by barium (1 mM).(ABSTRACT TRUNCATED AT 400 WORDS)