Faculty Bibliography

Ventricular tachycardia is a common heart rhythm disorder and a frequent cause of sudden cardiac death. Aberrant cell-cell coupling through gap junction channels, a process termed gap junction remodeling, is observed in many of the major forms of human heart disease and is associated with increased arrhythmic risk in both humans and in animal models. Genetically engineered mice with cardiac-restricted knockout of Connexin43, the major cardiac gap junctional protein, uniformly develop sudden cardiac death, although a detailed electrophysiological understanding of their profound arrhythmic propensity is unclear. Using voltage-sensitive dyes and high resolution optical mapping techniques, we found that uncoupling of the ventricular myocardium results in ectopic sites of ventricular activation. Our data indicate that this behavior reflects alterations in source-sink relationships and paradoxical conduction across normally quiescent Purkinje-ventricular muscle junctions. The aberrant activation profiles are associated with wavefront collisions, which in the setting of slow conduction may account for the highly arrhythmogenic behavior of Connexin43-deficient hearts. Thus, the extent of gap junction remodeling in diseased myocardium is a critical determinant of cardiac excitation patterns and arrhythmia susceptibility

Endocardial cushions are precursors of mature atrioventricular (AV) valves. Their formation is induced by signaling molecules originating from the AV myocardium, including bone morphogenetic proteins (BMPs). Here, we hypothesized that BMP signaling plays an important role in the AV myocardium during the maturation of AV valves from the cushions. To test our hypothesis, we used a unique Cre/lox system to target the deletion of a floxed Alk3 allele, the type IA receptor for BMPs, to cardiac myocytes of the AV canal (AVC). Lineage analysis indicated that cardiac myocytes of the AVC contributed to the tricuspid mural and posterior leaflets, the mitral septal leaflet, and the atrial border of the annulus fibrosus. When Alk3 was deleted in these cells, defects were seen in the same leaflets, ie, the tricuspid mural leaflet and mitral septal leaflet were longer, the tricuspid posterior leaflet was displaced and adherent to the ventricular wall, and the annulus fibrosus was disrupted resulting in ventricular preexcitation. The defects seen in mice with AVC-targeted deletion of Alk3 provide strong support for a role of Alk3 in human congenital heart diseases, such as Ebstein's anomaly. In conclusion, our mouse model demonstrated critical roles for Alk3 signaling in the AV myocardium during the development of AV valves and the annulus fibrosus

Sarcomere protein gene mutations cause hypertrophic cardiomyopathy (HCM), a disease with distinctive histopathology and increased susceptibility to cardiac arrhythmias and risk for sudden death. Myocyte disarray (disorganized cell-cell contact) and cardiac fibrosis, the prototypic but protean features of HCM histopathology, are presumed triggers for ventricular arrhythmias that precipitate sudden death events. To assess relationships between arrhythmias and HCM pathology without confounding human variables, such as genetic heterogeneity of disease-causing mutations, background genotypes, and lifestyles, we studied cardiac electrophysiology, hypertrophy, and histopathology in mice engineered to carry an HCM mutation. Both genetically outbred and inbred HCM mice had variable susceptibility to arrhythmias, differences in ventricular hypertrophy, and variable amounts and distribution of histopathology. Among inbred HCM mice, neither the extent nor location of myocyte disarray or cardiac fibrosis correlated with ex vivo signal conduction properties or in vivo electrophysiologically stimulated arrhythmias. In contrast, the amount of ventricular hypertrophy was significantly associated with increased arrhythmia susceptibility. These data demonstrate that distinct somatic events contribute to variable HCM pathology and that cardiac hypertrophy, more than fibrosis or disarray, correlates with arrhythmic risk. We suggest that a shared pathway triggered by sarcomere gene mutations links cardiac hypertrophy and arrhythmias in HCM

The remodeling of ventricular gap junctions, as defined by changes in size, distribution, or function, is a prominent feature of diseased myocardium. However, the regulation of assembly and maintenance of gap junctions remains poorly understood. To investigate N-cadherin function in the adult myocardium, we used a floxed N-cadherin gene in conjunction with a cardiac-specific tamoxifen-inducible Cre transgene. The mutant animals appeared active and healthy until their sudden death approximately 2 months after deleting N-cadherin from the heart. Electrophysiologic analysis revealed abnormal conduction in the ventricles of mutant animals, including diminished QRS complex amplitude consistent with loss of electrical coupling in the myocardium. A significant decrease in the gap junction proteins, connexin-43 and connexin-40, was observed in N-cadherin-depleted myocytes. Perturbation of connexin function resulted in decreased ventricular conduction velocity, as determined by optical mapping. Our data suggest that perturbation of the N-cadherin/catenin complex in heart disease may be an underlying cause, leading to the establishment of the arrythmogenic substrate by destabilizing gap junctions at the cell surface

The prevalence of atrial conduction defects and sinus node dysfunction increases with age. These age-related changes may play a critical role in establishing the substrate for the development of atrial fibrillation (AF), the most common sustained arrhythmia. Despite the association between atrial arrhythmias and age, little is known of the mechanisms that underlie changes in atrial electrophysiological function. Ongoing studies in our laboratory are focused on determining the mechanisms of atrial conduction defects associated with aging and disease. The purpose of this report is to present some initial studies of the murine sinus node and the approach we have taken to quantify conduction at the site of impulse initiation

While cardiac function in the mature heart is dependent on a properly functioning His-Purkinje system, the early embryonic tubular heart efficiently pumps blood without a distinct specialized conduction system. Although His-Purkinje system precursors have been identified using immunohistological techniques in the looped heart, little is known whether these precursors function electrically. To address this question, we used high-resolution optical mapping and fluorescent dyes with two CCD cameras to describe the motion-corrected activation patterns of 76 embryonic chick hearts from tubular stages (stage 10) to mature septated hearts (stage 35). Ventricular activation in the tubular looped heart (stages 10-17) using both calcium-sensitive fluo-4 and voltage-sensitive di-4-ANEPPS shows sequentially uniform propagation. In late looped hearts (stages 18-22), domains of the dorsal and lateral ventricle are preferentially activated before spreading to the remaining myocardium and show alternating regions of fast and slow propagation. During stages 22-26, action potentials arise from the dorsal ventricle. By stages 27-29, action potential breakthrough is also observed at the right ventricle apex. By stage 31, activation of the heart proceeds from foci at the apex and dorsal surface of the heart. The breakthrough foci correspond to regions where putative conduction system precursors have been identified immunohistologically. To date, our study represents the most detailed electrophysiological characterization of the embryonic heart between the looped and preseptated stages and suggests that ventricular activation undergoes a gradual transformation from sequential to a mature pattern with right and left epicardial breakthroughs. Our investigation suggests that cardiac conduction system precursors may be electrophysiologically distinct and mature gradually throughout cardiac morphogenesis in the chick

In many experimental circumstances, heart dynamics are, to a good approximation, periodic. For this reason, it makes sense to use high-resolution methods in the frequency domain to visualize the spectrum of imaging data of the heart and to estimate the deterministic signal content and extract the periodic signal from background noise in experimental data. In this paper, we describe the first application of a new method that we call cardiac rhythm analysis which uses a combination of principal component analysis and multitaper harmonic analysis to extract periodic, deterministic signals from high-resolution imaging data of cardiac electrical activity, We show that this method significantly increases the signal-to-noise ratio of our recordings, allowing for better visualization of signal dynamics and more accurate quantification of the properties of electrical conduction. We visualize the spectra of three cardiac data sets of mouse hearts exhibiting sinus rhythm, paced rhythm and monomorphic tachycardia. Then, for pedagogical purposes, we investigate the tachycardia more closely, demonstrating the presence of two distinct periodicities in the re-entrant tachycardia. Analysis of the tachycardia shows that cardiac rhythm analysis not only allows for better visualization of electrical activity, but also provides new opportunities to study multiple periodicities in signal dynamics

The cardiac conduction system is a network of cells responsible for the rhythmic and coordinated excitation of the heart. Components of the murine conduction system, including the peripheral Purkinje fibers, are morphologically indistinguishable from surrounding cardiomyocytes, and a paucity of molecular markers exists to identify these cells. The murine conduction system develops in close association with the endocardium. Using the recently identified CCS-lacZ line of reporter mice, in which lacZ expression delineates the embryonic and fully mature conduction system, we tested the ability of several endocardial-derived paracrine factors to convert contractile cardiomyocytes into conduction-system cells as measured by ectopic reporter gene expression in the heart. In this report we show that neuregulin-1, a growth and differentiation factor essential for ventricular trabeculation, is sufficient to induce ectopic expression of the lacZ conduction marker. This inductive effect of neuregulin-1 was restricted to a window of sensitivity between 8.5 and 10.5 days postcoitum. Using the whole mouse embryo culture system, neuregulin-1 was shown to regulate lacZ expression within the embryonic heart, whereas its expression in other tissues remained unaffected. We describe the electrical activation pattern of the 9.5-days postcoitum embryonic mouse heart and show that treatment with neuregulin-1 results in electrophysiological changes in the activation pattern consistent with a recruitment of cells to the conduction system. This study supports the hypothesis that endocardial-derived neuregulins may be the major endogenous ligands responsible for inducing murine embryonic cardiomyocytes to differentiate into cells of the conduction system

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