Abstract-Short QT syndrome (SQTS) leads to an abbreviated QTc interval and predisposes patients to life-threatening arrhythmias. To date, two forms of the disease have been identified: SQT1, caused by a gain of function substitution in the HERG (I Kr ) channel, and SQT2, caused by a gain of function substitution in the KvLQT1 (I Ks ) channel. Here we identify a new variant, "SQT3", which has a unique ECG phenotype characterized by asymmetrical T waves, and a defect in the gene coding for the inwardly rectifying Kir2.1 (I K1 ) channel. The affected members of a single family had a G514A substitution in the KCNJ2 gene that resulted in a change from aspartic acid to asparagine at position 172 (D172N). Whole-cell patch-clamp studies of the heterologously expressed human D172N channel demonstrated a larger outward I K1 than the wild-type (PϽ0.05) at potentials between Ϫ75 mV and Ϫ45 mV, with the peak current being shifted in the former with respect to the latter (WT, Ϫ75 mV; D172N, Ϫ65 mV). Coexpression of WT and mutant channels to mimic the heterozygous condition of the proband yielded an outward current that was intermediate between WT and D172N. In computer simulations using a human ventricular myocyte model the increased outward I K1 greatly accelerated the final phase of repolarization, and shortened the action potential duration. Hence, unlike the known mutations in the two other SQTS forms (N588K in HERG and V307L in KvLQT1), simulations using the D172N and WT/D172N mutations fully accounted for the ECG phenotype of tall and asymmetrically shaped T waves. Although we were unable to test for inducibility of arrhythmia susceptibility due to lack of patients' consent, our computer simulations predict a steeper steady-state restitution curve for the D172N and WT/D172N mutation, compared with WT or to HERG or KvLQT1 mutations, which may predispose SQT3 patients to a greater risk of reentrant arrhythmias. (Circ Res. 2005;96:800-807.)

Background Human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electrical impulse propagation velocity and immature action potential profiles. Methods and Results Here we have identified an optimal extracellular matrix (ECM) for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal ECM combination have impulse propagation velocities ~2X faster than previously reported (43.6±7.0 cm·s−1 n=9) and have mature cardiomyocyte action potential profiles including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s, N=5 monolayers). In addition the optimal ECM promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1 and Connexin43) and myofilament markers (cTroponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase (FAK) activation prevented structural maturation. Conclusions Maturation of human stem cell derived cardiomyocyte monolayers is achieved in a one week period by plating cardiomyocytes on PDMS coverslips rather than on conventional 2D cell culture formats such as glass coverslips or plastic dishes. Activation of integrin signaling and FAK are essential for significant maturation of human cardiac monolayers.

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.

Abstract-Catecholaminergic polymorphic ventricular tachycardia (VT) is a lethal familial disease characterized by bidirectional VT, polymorphic VT, and ventricular fibrillation. Catecholaminergic polymorphic VT is caused by enhanced Ca 2ϩ release through defective ryanodine receptor (RyR2) channels. We used epicardial and endocardial optical mapping, chemical subendocardial ablation with Lugol's solution, and patch clamping in a knockin (RyR2/RyR2 R4496C ) mouse model to investigate the arrhythmogenic mechanisms in catecholaminergic polymorphic VT. In isolated hearts, spontaneous ventricular arrhythmias occurred in 54% of 13 RyR2/RyR2 R4496C and in 9% of 11 wild-type (Pϭ0.03) littermates perfused with Ca 2ϩ and isoproterenol; 66% of 12 RyR2/RyR2 R4496C and 20% of 10 wild-type hearts perfused with caffeine and epinephrine showed arrhythmias (Pϭ0.04). Epicardial mapping showed that monomorphic VT, bidirectional VT, and polymorphic VT manifested as concentric epicardial breakthrough patterns, suggesting a focal origin in the His-Purkinje networks of either or both ventricles. Monomorphic VT was clearly unifocal, whereas bidirectional VT was bifocal. Polymorphic VT was initially multifocal but eventually became reentrant and degenerated into ventricular fibrillation. Endocardial mapping confirmed the Purkinje fiber origin of the focal arrhythmias. Chemical ablation of the right ventricular endocardial cavity with Lugol's solution induced complete right bundle branch block and converted the bidirectional VT into monomorphic VT in 4 anesthetized RyR2/RyR2 R4496C mice. Under current clamp, single Purkinje cells from RyR2/RyR2 R4496C mouse hearts generated delayed afterdepolarization-induced triggered activity at lower frequencies and level of adrenergic stimulation than wild-type. Overall, the data demonstrate that the His-Purkinje system is an important source of focal arrhythmias in catecholaminergic polymorphic VT. 604772) is an inherited disease leading to arrhythmias and sudden cardiac death. 1 The autosomal dominant form has been linked to ryanodine receptor gene (RyR2) mutations, leading to increased spontaneous Ca 2ϩ release from the sarcoplasmic reticulum. 2 Typical arrhythmias are bidirectional ventricular tachycardia (BVT) and polymorphic ventricular tachycardia (PVT) that can degenerate into ventricular fibrillation (VF) and thus sudden cardiac death. 3 BVT is infrequent, characterized by beat-to-beat 180°alternation of the QRS of the ECG and occurs in CPVT, as well as in digitalis toxicity; thus, it has been inferred that arrhythmogenesis in CPVT is mediated by delayed afterdepolarization (DAD)-induced triggered activity (TA).Mice heterozygous for the R4496C mutation (RyR2/ RyR2 R4496C ) recapitulate the human phenotype of CPVT by developing BVT, PVT, and/or VF under adrenergic stimulation. 4 Recently, Liu et al 5 have demonstrated DADs in RyR2/RyR2 R4496C mouse ventricular myocytes both in control and in the presence of isoproterenol. However, it remains to be demonstrated whether the arrhythmia origin...

Abstract-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 ϩ ] 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 Ϫ30 mV (IϷ0 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 13.1Ϯ0.1 pS in 15 cells, which corresponded with I K1 in sheep atria (9.9Ϯ0.1 pS in 32 cells Key Words: Kir2 Ⅲ extracellular potassium Ⅲ heteromerization Ⅲ rectification I n the heart, the inwardly rectifying potassium (Kir) current (I K1 ) stabilizes the resting membrane potential and plays a major role during the final phase of action potential (AP) repolarization. 1-3 The Kir2.x channels mediate cardiac I K1 . 3 Previous studies have demonstrated that I K1 properties are different in atrial and ventricular myocytes. 1,[3][4][5][6] First, I K1 current density is higher in the ventricles than in the atria. 6,7 Second, ventricular I K1 has been described as having a more prominent negative slope conductance at depolarized potentials than atrial I K1 (ie, atrial I K1 does not rectify completely). 1,4,5 Also, the outward component of the background potassium current (I B; consisting mainly of I K1 ) is significantly increased in high extracellular potassium ([K ϩ ] o ) in ventricular but not atrial myocytes. 1 The molecular mechanisms underlying these I K1 differences are unknown.The Kir2.x channel expression patterns may determine outward I K1 properties. 8,9 Outward currents through Kir channels may play an important role in the dynamics of atrial and ventricular fibrillation, as studied in the sheep 10 and guinea pig, 11 respectively. However, outward current profiles of the individual Kir2.x isoforms have not been comparatively studied. Moreover, the effect of high [K ϩ ] o on outward currents of Kir2.x isoforms has also not been compared. It is possible that the properties of Kir2.x isoforms, existing either as homomers or heteromers, determine regional I K1 differences in the heart. Although recent studies have shown that Kir2.x subunits heteromerize, 12- .3 were cloned using the polymerase chain reaction and transiently transfected into human embryonic kidney 293 (HEK293) cells using the Qiagen Effectene protocol. Guinea pig and sheep cardiac myocytes were enzymatically dissociated using the Langendorff-retrograde perfusion method as described previously. 11 In...

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.reentry | scaffolding proteins | conduction velocity | protein trafficking I n the heart, the inward rectifying potassium current (I K1 ) is the major current responsible for the maintenance of the resting membrane potential (RMP), whereas the sodium current (I Na ) provides the largest fraction of the inward depolarizing current that flows during an action potential (1). It is well-known that a relationship exists between these two ionic currents that is crucial for proper cardiac electrical function; disruption of this balance results in changes in sodium channel availability, cell excitability, action potential duration, and conduction velocity (2). Accordingly, I K1 -I Na interactions are important in stabilizing and controlling the frequency of the electrical rotors that are responsible for the most dangerous cardiac arrhythmias, including ventricular tachycardia and fibrillation (3).Post synaptic density, discs large, and zonula occludens-1 (PDZ) domain proteins link different and in many cases, multiple proteins to macromolecular complexes through interactions with their various domains. More than 70 PDZ d...

Abstract-Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a "ball-and-chain" mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257

The results strongly support the hypothesis that IK1 plays an important role in rotor stabilization and VF dynamics.