Gregory E. Morley

Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43.

Abstract— Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1±0.13 versus 1.66±0.06;P <0.01). This novel genetic murine model of primary sudden cardiac death defines gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate.

Characterization of Conduction in the Ventricles of Normal and Heterozygous Cx43 Knockout Mice Using Optical Mapping

Conduction in Normal and C×43+/− Mice. Introduction: Gap junction channels are important determinants of conduction in the heart and may play a central role in the development of lethal cardiac arrhythmias. The recent development of a C×43‐deficient mouse has raised fundamental questions about the role of specific connexin isoforms in intercellular communication in the heart. Although a homozygous null mutation of the C×43 gene (C×43−/−) is lethal, the heterozygous (Cx43+/−) animals survive to adulthood. Reports on the cardiac electrophysiologic phenotype of the C×43+/− mice are contradictory. Thus, the effects of a null mutation of a single C×43 allele require reevaluation.

Reentry and fibrillation in the mouse heart. A challenge to the critical mass hypothesis.

The idea that fibrillation is only possible in hearts exceeding a critical size was introduced by W. Garrey >80 years ago and has since been generally accepted. In ventricular tissue, this critical size was originally estimated to be 400 mm(2). Recent estimates suggest that the critical size required for sustained reentry is approximately 100 to 200 mm(2), whereas 6 times this area is required for ventricular fibrillation. According to these estimates, fibrillation is not possible in the mouse heart, where the ventricular surface area is approximately 100 mm(2). To test whether sustained ventricular fibrillation could be induced in such an area, we used a high-speed video imaging system and a voltage-sensitive dye to quantify electrical activity on the epicardial surface of the Langendorff-perfused adult mouse heart. In 6 hearts, measurements during ventricular pacing at a basic cycle length (BCL) of 120 ms yielded maximum and minimum conduction velocities (CV(max) and CV(min)) of 0.63+/-0.04 and 0.38+/-0.02 mm/ms, respectively. At a BCL of 80 ms, CV(max) and CV(min) changed to 0.55+/-0.03 and 0. 34+/-0.02 mm/ms. Action potential durations (APDs), measured at 70% repolarization at those pacing frequencies were found to be 44.5+/-2. 9 and 40.4+/-2.6 ms, respectively. The wavelengths (CVxAPD) were calculated to be 28.6+/-3.4 mm in the CV(max) direction and 16.8+/-1. 5 mm in the CV(min) direction at BCL 120 ms. Wavelengths were significantly reduced (P<0.05) at BCL 80 ms (CV(max), 22.2+/-1.8 mm; CV(min), 13.7+/-0.9 mm). In 5 hearts, stationary vortex-like reentry organized by single rotors (4 of 5 hearts) or by pairs of rotors (1 of 5 hearts) was induced by burst pacing. In the ECG, the activity manifested as sustained monomorphic tachycardia. Detailed analysis showed that the local CVs were reduced in the vicinity of the rotor center, which allowed the reentry to take place within a smaller area than was calculated from wavelength measurements during pacing. In 4 of 7 hearts, burst pacing resulted in a polymorphic ECG pattern indistinguishable from ventricular fibrillation. These data challenge the critical mass hypothesis by demonstrating that ventricular tissue with an area as small as 100 mm(2) is capable of undergoing sustained fibrillatory activity.

High-Resolution Optical Mapping of the Right Bundle Branch in Connexin40 Knockout Mice Reveals Slow Conduction in the Specialized Conduction System

Connexin40 (Cx40) is a major gap junction protein that is expressed in the His-Purkinje system and thought to be a critical determinant of cell-to-cell communication and conduction of electrical impulses. Video maps of the ventricular epicardium and the proximal segment of the right bundle branch (RBB) were obtained using a high-speed CCD camera while simultaneously recording volume-conducted ECGs. In Cx40–/– mice, the PR interval was prolonged (47.4±1.4 in wild-type [WT] [n=6] and 57.5±2.8 in Cx40–/– [n=6];P <0.01). WT ventricular epicardial activation was characterized by focused breakthroughs that originated first on the right ventricle (RV) and then the left ventricle (LV). In Cx40–/– hearts, the RV breakthrough occurred after the LV breakthrough. Additionally, Cx40–/– mice showed RV breakthrough times that were significantly delayed with respect to QRS complex onset (3.7±0.7 ms in WT [n=6] and 6.5±0.7 ms in Cx40–/– [n=6];P <0.01), whereas LV breakthrough times did not change. Conduction velocity measurements from optical mapping of the RBB revealed slow conduction in Cx40–/– mice (74.5±3 cm/s in WT [n=7] and 43.7±6 cm/s in Cx40–/– [n=7];P <0.01). In addition, simultaneous ECG records demonstrated significant delays in Cx40–/– RBB activation time with respect to P time (P-RBB time; 41.6±1.9 ms in WT [n=7] and 55.1±1.3 ms in [n=7];P <0.01). These data represent the first direct demonstration of conduction defects in the specialized conduction system of Cx40–/– mice and provide new insight into the role of gap junctions in cardiac impulse propagation.

Altered Right Atrial Excitation and Propagation in Connexin40 Knockout Mice

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.

Connexins and impulse propagation in the mouse heart

Connexins and Propagation. Gap junction channels are essential for normal cardiac impulse propagation. Three gap Junction proteins, known as connexins, are expressed in the heart: Cx40, Cx43, and Cx45. Each of these proteins forms channels with unique biophysical and electrophysiologic properties, as well as spatial distribution of expression throughout the heart. However, the specific functional role of the individual connexins in normal and abnormal propagation is unknown. The availability of genetically engineered mouse models, together with new developments in optical mapping technology, makes it possible to integrate knowledge about molecular mechanisms of intercellular communication and its regulation with our growing understanding of the microscopic and global dynamics of electrical impulse propagation during normal and abnormal cardiac rhythms. This article reviews knowledge on the mechanisms of cardiac impulse propagation, with particular focus on the role of cardiac connexins in electrical communication between cells. It summarizes results of recent studies on the electrophysiologic consequences of defects in the functional expression of specific gap junction channels in mice lacking either the Cx43 or Cx40 gene. It also reviews data obtained in a transgenic mouse model in which cell loss and remodeling of gap junction distribution leads to increased susceptibility to arrhythmias and sudden cardiac death. Overall, the results demonstrate that these are potentially powerful strategies for studying fundamental mechanisms of cardiac electrical activity and for testing the hypothesis that certain cardiac arrhythmias involve gap junction or other membrane channel dysfunction. These new approaches, which permit one to manipulate electrical wave propagation at the molecular level, should provide new insight into the detailed mechanisms of initiation, maintenance, and termination of cardiac arrhythmias, and may lead to more effective means to treat arrhythmias and prevent sudden cardiac death.

Conditional Gene Targeting of Connexin43: Exploring the Consequences of Gap Junction Remodeling in the Heart

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.

Effects of 2,4-dinitrophenol or low [ATP]i on cell excitability and action potential propagation in guinea pig ventricular myocytes.

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.

Understanding conduction of electrical impulses in the mouse heart using high-resolution video imaging technology.

The conduction of electrical impulses in the heart depends on the ability to efficiently transfer excitatory current between individual myocytes. Several recent studies have focused on the use of optical mapping techniques to determine the electrophysiological consequences and the proarrhythmic effects of reducing intercellular coupling in newly developed connexin knockout mice. This work has begun to unravel important questions regarding the role of connexins in intercellular coupling and propagation of electrical impulses in the heart. The purpose of this review is to discuss the techniques and unique issues involved in imaging electrical wave propagation in the heart. In addition, we will review recent experimental studies that address the role of intercellular communication in the development of cardiac arrhythmias. Microsc. Res. Tech. 52:241–250, 2001.

Cardiac gap junction remodeling by stretch: Is it a good thing?

Cardiac cells contract and are also normally exposed to the mechanical events in their surroundings. It is now well established that both cardiac gene expression and protein synthesis are subject to regulation by mechanical forces, including stretch. For example, mechanical stretch is known to be one of the most important stimuli leading to cardiac hypertrophy,1 2 3 4 5 and recent studies indicate that cardiac myocyte hypertrophy is stimulated in vitro by specific directions and degrees of stretch.6 Similarly, certain stretch-sensitive sarcolemmal ion channels and exchangers have been found in cardiac myocytes7 and have been implicated in the mechanism of stretch-induced arrhythmias.8 However, whereas signal transduction induced by mechanical stretch involves activation of a wide variety of second messenger systems,9 it remains to be determined which molecules are directly affected by stretch and which are the processes whereby mechanical stimuli trigger intracellular signaling pathways to activate protein kinase cascades and produce changes in function. The process of filling and ejecting blood subjects the cells of the heart to repetitive pulsatile stress. Our understanding of the basic electrophysiology underlying the cardiac action potential and its propagation across cells is largely on the basis of patch clamp data and isolated tissue experiments in the absence of mechanical stress. On the other hand, whole-heart electrophysiological mapping studies are often carried out in in situ functioning hearts. In either case, the role of mechanical stress in impulse initiation and propagation has not been adequately addressed. In addition, although stretch is thought to play an important role in cardiac remodeling that is associated with heart failure, very little is known about its role in normal electrical function. The study by Zhuang et al10 in this issue of Circulation Research , which is the result of a successful collaboration between …