Faculty Bibliography

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose towards arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization, but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiologic effects of GJR. We paced wildtype (normal Cx43 abundance) and heterozygous Cx43 knockout mice (Cx43(+/-), 66% mean reduction in Cx43) for six hours at 10-15% above their average sinus rate. We investigated the electrophysiologic effects of pacing on the whole animal using programmed electrical stimulation, and in isolated ventricular myocytes with patch clamp studies. Cx43(+/-) myocytes had significantly shorter action potential durations (APD) and increased steady state and inward rectifier potassium currents (Iss and IK1, respectively) compared to wildtype littermate cells. In Cx43(+/-) hearts, pacing resulted in significant prolongation of ventricular effective refractory period and action potential duration, and significant diminution of Iss compared to unpaced Cx43(+/-) hearts. However, these changes were not seen in paced wildtype mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiologic changes which may contribute to the worsened prognosis often associated with pacing in the failing heart. Key words: Connexin43, ventricular myocytes, mouse, gap junction

The cardiac conduction system initiates and maintains electrical activity that must pass through every cell of the heart to trigger contraction. How are these ""specialized"" cells differentiated from working cardiomyocytes? The question is simple, the story complex, and the answers remain elusive. Discrepancies between morphological studies and functional data leave genetics and molecular biology to fill the gaps in our understanding. In this chapter, we travel the path from the early gross anatomy discoveries to high-resolution optical mapping studies, focusing on the latest work from both murine and avian systems. We speculate on the future direction of investigation into conduction system development and highlight the clinical significance of this work.

BACKGROUND: The atrioventricular (AV) node is essential for the sequential excitation and optimized contraction of the adult multichambered heart; however, relatively little is known about its formation from the embryonic AV canal. A recent study demonstrated that signaling by Alk3, the type 1a receptor for bone morphogenetic proteins, in the myocardium of the AV canal was required for the development of both the AV valves and annulus fibrosus. To test the hypothesis that bone morphogenetic protein signaling also plays a role in AV node formation, we investigated conduction system function and AV node morphology in adult mice with conditional deletion of Alk3 in the AV canal. METHODS AND RESULTS: High-resolution optical mapping with correlative histological analysis of 28 mutant hearts revealed 4 basic phenotypic classes based on electrical activation patterns and volume-conducted ECGs. The frequency of AV node conduction and morphological abnormalities increased from no detectable anomalies (class I) to severe defects (class IV), which included the presence of bypass tracts, abnormal ventricular activation patterns, fibrosis of the AV node, and twin AV nodes. CONCLUSIONS: The present findings demonstrate that bone morphogenetic protein signaling is required in the myocardium of the AV canal for proper AV junction development, including the AV node

Pacemaker cells in the heart generate periodic electrical signals that are conducted to the working myocardium via the specialized conduction system. Effective cell-to-cell communication is critical for rapid, uniform conduction of cardiac action potentials-- a prerequisite for effective, synchronized cardiac contraction. Local circuit currents form the basis of the depolarization wave front in the working myocardium. These currents flow from cell to cell via gap junction channels. In this chapter, we trace the path of the action potential from its generation in the sinus node to propagation through the working myocardium, with a detailed discussion of the role of gap junctions. First, we review the transmembrane ionic currents and the basic principles of conduction of the action potential to the working myocardium via the specialized tissues of the heart. Next, we consider the relative contribution of cell geometry, size, and gap junction conductance. These factors are examined in terms of their source-to-sink relationships. Lastly, we will discuss new insights into the importance of gap junctions in cardiac conduction in health and disease which have been gained from high resolution optical mapping in connexin-deficient mice

Cardiac KATP channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice which express dominant negative Kir6.x pore-forming subunits (Kir6.1-AAA or Kir6.2-AAA) in cardiac myocytes by driving their expression with the alpha-myosin heavy chain promoter. Weight gain and development after birth of these mice were similar to wild-type mice, but an increased mortality was noted after the age of 4-5 months. Transgenic mice lacked cardiac KATP channel activity as assessed with patch clamp techniques. Consistent with a decreased current density observed at positive voltages, the action potential duration was increased in these mice. Some myocytes developed early afterdepolarizations following isoproterenol treatment. Hemodynamic measurements revealed no significant effects on ventricular function (apart from a slightly elevated heart rate) whereas in-vivo electrophysiological recordings revealed a prolonged ventricular effective refractory period in transgenic mice. The transgenic mice tolerated stress less well as evident from treadmill stress tests. The pro-arrhythmogenic features and lack of adaptation to a stress response in transgenic mice suggests that these features are intrinsic to the myocardium and that KATP channels in the myocardium have an important role in protecting the heart from lethal arrhythmias and adaptation to stress situations

Genetic studies in the mouse have demonstrated that conditional cardiac-resticted loss of connexin43 (Cx43), the major ventricular gap junction protein, is highly arrhythmogenic. However, whether more focal gap junction remodeling, as is commonly seen in acquired cardiomyopathies, influences the propensity for arrhythmogenesis is not known. We examined electrophysiological properties and the frequency of spontaneous and inducible arrhythmias in genetically engineered chimeric mice derived from injection of Cx43-deficient embryonic stem cells into wildtype recipient blastocysts. Chimeric mice had numerous well-circumscribed microscopic Cx43-negative foci in their hearts, comprising ~ 15% of the total surface area as determined by immunohistochemical analysis. Systolic function in the chimeric mice was significantly depressed as measured echocardiographically (19.0% decline in fractional shortening compared with controls, p < 0.05) and by invasive hemodynamics (17.6% reduction in dP/dT, p < 0.01). Chimeras had significantly more spontaneous arrhythmic events than controls (p < 0.01), including frequent runs of non-sustained ventricular tachycardia in some of the chimeric mice. However, in contrast to mice with conditional cardiac-resticted loss of Cx43 in the heart, no sustained ventricular tachyarrhythmias were observed. We conclude that focal areas of uncoupling in the myocardium increase the likelihood of arrhythmic triggers, but more widespread uncoupling is required to support sustained arrhythmias

BACKGROUND: Intercellular coupling via connexin40 (Cx40) gap junction channels is an important determinant of impulse propagation in the atria. METHODS AND RESULTS: We studied the role of Cx40 in intra-atrial excitation and propagation in wild-type (Cx40(+/+)) and knockout (Cx40(-/-)) mice using high-resolution, dual-wavelength optical mapping. On ECG, the P wave was significantly prolonged in Cx40(-/-) mice (13.4+/-0.5 versus 11.4+/-0.3 ms in Cx40(+/+)). In Cx40(+/+) hearts, spontaneous right atrial (RA) activation showed a focal breakthrough at the junction of the right superior vena cava, sulcus terminalis, and RA free wall, corresponding to the location of the sinoatrial node. In contrast, Cx40(-/-) hearts displayed ectopic breakthrough sites at the base of the sulcus terminalis, RA free wall, and right superior vena cava. Progressive ablation of such sites in 4 Cx40(-/-) mice resulted in ectopic focus migration and cycle length prolongation. In all Cx40(-/-) hearts the focus ultimately shifted to the sinoatrial node at a very prolonged cycle length (initial ectopic cycle length, 182+/-20 ms; postablation sinus cycle length, 387+/-44 ms). In a second group of experiments, epicardial pacing at 10 Hz revealed slower conduction in the RA free wall of 5 Cx40(-/-) hearts than in 5 Cx40(+/+) hearts (0.61+/-0.07 versus 0.94+/-0.07 m/s; P<0.05). Dominant frequency analysis in Cx40(-/-) RA demonstrated significant reduction in the area of 1:1 conduction at 16 Hz (40+/-10% versus 69+/-5% in Cx40(+/+)) and 25 Hz (36+/-11% versus 65+/-9% in Cx40(+/+)). CONCLUSIONS: This is the first demonstration of intra-atrial block, ectopic rhythms, and altered atrial propagation in the RA of Cx40(-/-) mice