Vertebrate hearts have evolved into multichambered structures requiring coordinated beating of their chambers to achieve antegrade blood flow throughout the organism. Unidirectional blood flow is achieved through two specialized structures that are unique to vertebrates: cardiac valves and the specialized cardiac conduction system (CCS). In the adult heart, the initial electrical impulses are generated in the slow pacemaker sino-atrial (SA) node and then propagated across the atrium. This electrical impulse is delayed at the atrioventricular (AV) boundary through specialized slow conducting AV node cardiomyocytes. After the delay at the AV node, electrical propagation travels rapidly through the fast conduction network comprised of the His-Purkinje system, which coordinates ventricular activation to occur from the apex to the base of the heart. This apex-to-base activation allows for efficient ejection of blood from the ventricles into the outflow tracts (OFTs) at the base of the heart [1
Despite extensive knowledge of the anatomy and physiology of the adult vertebrate CCS, the cellular and molecular events that govern the development of this specialized tissue remain unclear. Lineage tracing studies have revealed that the CCS is derived from cardiomyocyte progenitors [2
]. Myocardial factors that regulate the specification of the CCS include Nkx2.5 and Tbx5 [2
]. Loss of either transcriptional regulator leads to defects in the maturation and maintenance of the AV conduction system and subsequent AV heart block and bundle branch block.
Additional studies have revealed the requirement of the endocardium for cardiomyocyte specification to form the fast conduction network within the ventricle [5
]. Secreted factors from endocardial as well as other cardiac endothelial cells, such as Endothelin 1 and Neuregulin, are able to induce cardiac conduction markers in cultured embryonic cardiomyocytes and cultured hearts [7
]. Furthermore, hemodynamic changes regulate the secretion of Endothelin 1 from endocardial cells, thereby affecting the development of the fast conduction pathway [6
]. More recently, the role of the endocardium for the development of AV conduction delay has been investigated further using the zebrafish cloche
], which lacks endothelial cells among other defects [10
]. That study concluded that Neuregulin but not Endothelin 1 is required for the induction of AV conduction delay.
Optical mapping of cardiac excitation using voltage- and calcium-sensitive dyes has allowed the spatiotemporal analysis of electrical excitation wave dynamics, not only advancing our understanding of the electrical activity during cardiac arrhythmias but also allowing for further analysis of CCS development [11
]. However, the use of voltage- and calcium-sensitive dyes is associated with serious shortcomings, including a lack of cellular targeting, limited live animal experimentation, the need for physical loading of these indicators into cells, and cellular toxicity. To circumvent these problems, fluorescent calcium indicator proteins have begun to replace voltage- and calcium-sensitive dyes for physiologic in vivo analysis of tissue/organ electrical activity in different animal model systems including fly and mouse [12
]. Yet, optical mapping of mouse hearts is currently limited due to explantation for ex vivo analysis. Thus, we have taken advantage of the external fertilization and translucency of zebrafish embryos to create a cardiac-specific fluorescent calcium indicator transgenic line, Tg
, to perform in vivo optical mapping analyses throughout the stages of heart development.
Here we describe a multidisciplinary approach using the zebrafish toward understanding CCS development. Utilizing the Tg(cmlc2:gCaMP)s878 optical mapping system, we identified four distinct physiologic developmental stages of the CCS that correspond to cellular and anatomical changes of the developing zebrafish heart. (1) Initially, a linear conduction travels across the heart tube from the sinus venosus to the OFT (20–24 hours postfertilization (hpf)); (2) next, a significant AV conduction delay develops during cardiac chamber formation (36–48 hpf); (3) as the heart loops and develops ventricular trabeculations (72–96 hpf), an immature fast conduction network develops within the ventricle; (4) finally, this fast conduction network fully matures to an apex-to-base activation pattern when the ventricular apex has formed.
Furthermore, to identify regulators of CCS development, we performed a diploid ethylnitrosourea (ENU) mutagenesis screen and recovered several novel as well as known cardiovascular conduction/rhythm mutants, which we have analyzed using in vivo optical imaging techniques and classified according to the affected physiologic developmental stage of the CCS. Positional cloning of hobgoblin (hob), a novel mutant with AV heart block, reveals that tcf2, a homeobox transcription factor gene involved in mature onset diabetes of the young, also regulates conduction between the atrium and the ventricle. Thus, these detailed electrophysiologic and genetic analyses of wild-type and mutant hearts provide further insights into the development of the vertebrate CCS and will lead to a better understanding of the pathogenesis of cardiac arrhythmias.