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Dissecting Molecular Pathways for Cardiac Conduction System Development and Disease
Summary: Christine Seidman's laboratory discovers gene mutations that cause human disease, with a focus on cardiovascular conditions such as cardiomyopathy (hypertrophic and dilated), heart failure, and congenital heart malformations. Her laboratory also produces model organisms that carry human mutations, and uses these models to determine how responses to gene mutations perturb or influence myocardial structure and specialized heart functions.
The heart contains myocytes with specialized electrophysiologic functions, in addition to the more abundant population of myocytes that function in contraction and relaxation. These specialized electrophysiologic myocytes are organized into the cardiac conduction system, a complex three-dimensional structure located deep within the heart that both initiates and propagates electrical impulses, so as to coordinate cardiac contraction. The conduction system is subdivided into atrial components, which are highly conserved throughout vertebrate evolution, and ventricular components that have evolved more recently to support the mammalian four-chamber heart. Atrial components of the conduction system initiate electrical impulses and transmit them to the atrioventricular junction. The ventricular components then rapidly transmit impulses from the atrioventricular junction to the lowermost segment of the ventricles, so that cardiac contraction initiates at the apex of the heart. Ventricular components of the conduction system include the atrioventricular bundle and the left and right bundle branches.
Functional deterioration of either the atrial or ventricular components of the conduction system that occurs with aging or from myocardial disease is often recognized in patients by abnormal patterns on electrocardiograms. Although conduction system disease is sometimes silent, it can produce life-threatening arrhythmias. Therapies for conduction system disease remain limited to implantation of devices, such as pacemakers or internal defibrillators, as well as prophylactic medications to reduce arrhythmias.
To gain insights about the molecular properties that distinguish the specialized electrophysiologic myocytes, we performed transcriptional analyses of the mouse ventricular conduction system. Because the more prevalent nonconduction myocytes are intimately associated with conduction system myocytes, we used the expression of minK-LacZ to guide microdissection of these specialized tissues. (The minK gene encodes the voltage-gated cardiac potassium channel and is selectively expressed in conduction myocytes.) We isolated left bundle branches from neonatal mice, constructed a cDNA library, and analyzed this using SAGE (serial analysis of gene expression). More than 60,000 SAGE tags were sequenced, annotated, and compared to a SAGE library derived from the mouse left ventricle.
To validate that the left bundle branch library is enriched in conduction system myocytes, we first confirmed the presence of tags that correspond to previously defined molecular markers of the conduction system, including minK. Among differences of RNAs expressed in the conduction system and left ventricular libraries, we found significantly fewer tags corresponding to 13 genes encoding components of the myocyte sarcomere complex. This confirmed previous morphologic studies that demonstrated a poorly developed sarcomere apparatus in the conduction system compared to ventricular myocytes. In contrast, the conduction system library was enriched for tags corresponding to RNAs that were previously identified in neural tissues.
To focus studies on molecules that might be critical to differentiate conduction system myocytes from contractile myocytes, we examined transcription factors that are differentially expressed in these libraries. Expression of Id2, a member of the Id (inhibitor of DNA) family of transcriptional repressors, is increased in the conduction system library. Because Id2 has been demonstrated to block myogenic activity of basic HLH (helix-loop-helix) proteins, and when expressed ectopically can promote differentiation of a neural crest lineage, we hypothesized that Id2 might be central to specifying myocytes in the conduction system lineage. To test this model, we studied four Id-family members during heart development and found that only Id2 expression is localized to the tip of the interventricular septum, precisely where the ventricular conduction system emerges. We confirmed this localized expression by producing transgenic Id2-LacZ embryonic mice, experiments that evidenced the Id2 promoter is sufficient to act as a conduction system enhancer in vivo. Because Id2-null mice had been previously produced, but cardiac phenotypes had not been studied, we examined conduction system anatomy and cardiac electrophysiology in these mutant mice. We identified both abnormal structure of the ventricular conduction system and functional deficits. The electrocardiograms of Id2-null mice showed left bundle branch block, a pattern typical of human ventricular conduction system disease.
We next considered the mechanisms by which Id2 expression is targeted to myocytes destined to adopt a conduction system fate. Analyses of the Id2 promoter revealed multiple binding site motifs for Tbx5 and Nkx2-5, two transcription factors with critical roles in cardiac morphogenesis. Throughout development, cardiac expression of Tbx5 and Nkx2-5 partially overlaps, particularly in the domains of the presumptive conduction system. Hypothesizing that this overlap accounts for conduction system expression of Id2, we interrogated putative Tbx5- and Nkx2-5-binding sites identified in the Id2 promoter. Biochemical studies and reporter assays in cells and in vivo established cooperative transcriptional activation by Tbx5 and Nkx2-5 of Id2. Furthermore, mutation of one Tbx5-binding site abolished Id2 expression in the ventricular conduction system of the developing heart.
These findings have informed our previous studies of the genetic basis for human heart disease. Patients with TBX5 or NKX2-5 mutations have congenital heart malformations and conduction system disease. Conduction system deficits occur even in mutation carriers without structural heart malformations, implying that these transcription factors have independent roles in heart morphogenesis and cardiac electrophysiology. To explore the mechanisms by which human mutations affect the conduction system, we characterized the cardiac electrophysiologic system in mice with haploinsufficiency of Tbx5, Nkx2-5, or both. Haploinsufficiency of Tbx5 or Nkx2-5 significantly prolongs the time for propagation of electrophysiology impulses throughout the ventricular conduction system, deficits that increase in compound mutant mice. Abnormalities of the ventricular conduction system structure that were observed in Id2-null mice are remarkably similar to those found in mice with Tbx5 haploinsufficiency. And in embryonic mice with compound Tbx5 and Nkx2-5 haploinsufficiency, Id2 expression is abolished in the interventricular septum domain where the presumptive conduction system develops. Myocytes residing in this domain also fail to exit the cell cycle, a previously recognized marker that myocytes have adopted a conduction system fate.
These studies provide a framework for a transcriptional network that promotes development of the cardiac ventricular conduction system. Overlapping expression of Tbx5 and Nkx2-5 appears sufficient to promote localized Id2 expression in myocardial regions where the ventricular conduction system develops. Id2 expression may then affect subsequent myocyte differentiation—inhibiting cardiac muscle gene expression (including genes encoding sarcomere proteins) and promoting neural gene expression. These properties ultimately distinguish conduction system cells from the nonconduction myocytes in the heart. Our future studies to understand this transcriptional program are aimed at delineating genes regulated by Id2. Insights into the transcriptional program of specialized myocytes of the conduction system should lead to better approaches to preserve cardiac electrophysiological function.
Last updated: July 10, 2007
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