Linda C. Baker

Enhanced Dispersion of Repolarization and Refractoriness in Transgenic Mouse Hearts Promotes Reentrant Ventricular Tachycardia

The heterogeneous distribution of ion channels in ventricular muscle gives rise to spatial variations in action potential (AP) duration (APD) and contributes to the repolarization sequence in healthy hearts. It has been proposed that enhanced dispersion of repolarization may underlie arrhythmias in diseases with markedly different causes. We engineered dominant negative transgenic mice that have prolonged QT intervals and arrhythmias due to the loss of a slowly inactivating K(+) current. Optical techniques are now applied to map APs and investigate the mechanisms underlying these arrhythmias. Hearts from transgenic and control mice were isolated, perfused, stained with di-4-ANEPPS, and paced at multiple sites to optically map APs, activation, and repolarization sequences at baseline and during arrhythmias. Transgenic hearts exhibited a 2-fold prolongation of APD, less shortening (8% versus 40%) of APDs with decreasing cycle length, altered restitution kinetics, and greater gradients of refractoriness from apex to base compared with control hearts. A premature impulse applied at the apex of transgenic hearts produced sustained reentrant ventricular tachycardia (n=14 of 15 hearts) that did not occur with stimulation at the base (n=8) or at any location in control hearts (n=12). In transgenic hearts, premature impulses initiated reentry by encountering functional lines of conduction block caused by enhanced dispersion of refractoriness. Reentrant VT had stable (>30 minutes) alternating long/short APDs associated with long/short cycle lengths and T wave alternans. Thus, optical mapping of genetically engineered mice may help elucidate some electrophysiological mechanisms that underlie arrhythmias and sudden death in human cardiac disorders.

Dispersion of repolarization and refractoriness are determinants of arrhythmia phenotype in transgenic mice with long QT.

Enhanced dispersion of repolarization (DR) and refractoriness may be a unifying mechanism central to arrhythmia genesis in the long QT (LQT) syndrome. The role of DR in promoting arrhythmias was investigated in several strains of molecularly engineered mice: (a) Kv4.2 dominant negative transgenic (Kv4.2DN) that lacks the fast component of the transient outward current, Ito,f, have action potential (AP) and QT prolongation, but no spontaneous arrhythmias, (b) Kv1.4 targeted mice (Kv1.4−/−) that lack the slow component of Ito (Ito,s), have no QT prolongation and no spontaneous arrhythmias, and (c) double transgenic (Kv4.2DN×Kv1.4−/−) mice that lack both Ito,f and Ito,s, have AP and QT prolongation, and spontaneous ventricular tachyarrhythmias. Hearts were perfused, stained with di‐4‐ANEPPS and optically mapped. Activation patterns and conduction velocities were similar between the strains but AP duration at 75% recovery (APD75) was longer in Kv4.2DN (28.0 ± 2.5 ms, P < 0.01, n= 6), Kv1.4−/− (28.4 ± 0.4 ms, P < 0.01, n= 5) and Kv4.2DN×Kv1.4−/− (34.3 ± 2.6 ms, P < 0.01, n= 6) mice than controls (20.3 ± 1.0 ms, n= 5). Dispersion of refractoriness between apex and base was markedly reduced in Kv4.2DN (0.3 ± 0.5 ms, n= 6, P < 0.05) but enhanced in Kv1.4−/− (14.2 ± 2.0 ms, n= 5, P < 0.05) and Kv4.2DN×Kv1.4−/− (15.0 ± 3 ms, n= 5, P < 0.5) mice compared with controls (10 ± 2 ms, n= 5). A premature pulse elicited ventricular tachycardia (VT) in Kv1.4−/− (n= 4/5) and Kv4.2DN×Kv1.4−/− hearts (n= 5/5) but not Kv4.2DN hearts (n= 0/6). Voltage‐clamp recordings showed that Ito,f was 30% greater in myocytes from the apex than base which may account for the absence of DR in Kv4.2DN mice. Thus, dispersion of repolarization (DR) appears to be an important determinant of arrhythmia vulnerability.

Transcription Enhancer Factor-1-Related Factor-Transgenic Mice Develop Cardiac Conduction Defects Associated With Altered Connexin Phosphorylation

Background—Conduction system defects and slowed ventricular conduction are common in patients with systolic dysfunction and contribute to arrhythmias and sudden death. In animal models of heart failure, cardiac &agr;1-adrenergic signaling is constitutively activated. Here, we report the effects of constitutive activation of &agr;1-adrenergic signaling on connexin phosphorylation and cardiac conduction. Methods and Results—Transgenic mice were generated with cardiac-specific overexpression of the transcription factor RTEF-1 (transcription enhancer factor-1–related factor), which mediates &agr;1-adrenergic signaling in cardiac myocytes. Surface and intracardiac ECGs revealed prolongation of the PR, QRS, and AH intervals and the appearance of progressive atrial arrhythmias in RTEF-1 mice. Optical mapping using voltage-sensitive dye revealed slower conduction velocities across the atrial and ventricular myocardium. Intercellular dye transfer between RTEF-1 transgenic cardiac myocytes confirmed impaired conduction at the cellular level. Conduction defects were correlated with dephosphorylation of connexin40 and connexin43 and upregulation of protein phosphatase 1&bgr; (PP1&bgr;). Overexpression of PP1&bgr; in HeLa cells dephosphorylated cardiac connexin. Confocal microscopy revealed increased levels of dephosphorylated connexin43 at the cardiac gap junctions in RTEF-1 mice, suggesting that defective conduction is a result of impaired gap-junction conductance rather than assembly. Conclusion—Constitutive activation of &agr;1-adrenergic signaling through the RTEF-1 transcription factor results in chronic elevation of PP1&bgr; expression and connexin dephosphorylation. This mechanism may underlie some defects in cardiac conduction.

Flow cytometric assays to detect platelet activation and aggregation in device‐implanted calves

Cardiovascular device development often relies upon large-animal models to assess blood biocompatibility prior to initiating clinical trials. Unfortunately, the amount of information gleaned from such trials is limited by simple assays that do not take full advantage of immunotechnological advances that increasingly are applied in clinical studies. Thus we have developed and tested new flow cytometric techniques for measuring circulating activated bovine platelets and platelet microaggregates. Monoclonal antibodies (MAbs) raised against both activated and quiescent bovine platelets were incubated with control and PMA-or ADP-stimulated whole blood. Selected MAbs detected activated bovine platelets and platelet microaggregates in vitro with flow cytometry. Five calves implanted with one of two designs of nonpulsatile ventricular-assist devices (VADs) were followed with these assays prior to and during VAD implantation. Circulating activated bovine platelets and microaggregates increased after implantation in all animals and, alternatively, remained elevated or returned toward preimplant levels. Platelet activation percentages as detected temporally by three MAbs were correlated with one another, and platelet activation was correlated with microaggregate formation. In summary, these new methods for the sensitive measurement of circulating activated bovine platelets and microaggregates may provide valuable information for the development and assessment of future cardiovascular device designs.

Simultaneous Atrial and Ventricular Anti‐Tachycardia Pacing as a Novel Method of Rhythm Discrimination

Background: Inappropriate shocks remain a problem in patients with defibrillators (ICD).

Arrhythmia phenotype in mouse models of human long QT.

Enhanced dispersion of repolarization (DR) was proposed as a unifying mechanism, central to arrhythmia genesis in the long QT (LQT) syndrome. In mammalian hearts, K+ channels are heterogeneously expressed across the ventricles resulting in ‘intrinsic’ DR that may worsen in long QT. DR was shown to be central to the arrhythmia phenotype of transgenic mice with LQT caused by loss of function of the dominant mouse K+ currents. Here, we investigated the arrhythmia phenotype of mice with targeted deletions of KCNE1 and KCNH2 genes which encode for minK/IsK and Merg1 (mouse homolog of human ERG) proteins resulting in loss of function of IKs and IKr, respectively. Both currents are important human K+ currents associated with LQT5 and LQT2. Loss of minK, a protein subunit that interacts with KvLQT1, results in a marked reduction of IKs giving rise to the Jervell and Lange–Nielsen syndrome and the reduced KCNH2 gene reduces MERG and IKr.Hearts were perfused, stained with di-4-ANEPPS and optically mapped to compare action potential durations (APDs) and arrhythmia phenotype in homozygous minK (minK−/−) and heterozygous Merg1 (Merg+/−) deletions and littermate control mice. MinK−/− mice has similar APDs and no arrhythmias (n = 4). Merg+/− mice had prolonged APDs (from 20 ± 6 to 32 ± 9 ms at the base, p < 0.01; from 18 ± 5 to 25 ± 9 ms at the apex, p < 0.01; n = 8), longer refractory periods (RP) (36 ± 14 to 63 ± 27 at the base, p < 0.01 and 34 ± 5 to 53 ± 21 ms at the apex, p < 0.03; n = 8), higher DR 10.4 ± 4.1 vs. 14 ± 2.3 ms, p < 0.02) and similar conduction velocities (n = 8). Programmed stimulation exposed a higher propensity to VT in Merg+/− mice (60% vs. 10%). A comparison of mouse models of LQT based on K+ channel mutations important to human and mouse repolarization emphasizes DR as a major determinant of arrhythmia vulnerability.