Induction and patterning of the cardiac conduction system

The cardiac conduction system (CCS) is the component of the heart that initiates and maintains a rhythmic heartbeat. As the embryonic heart forms, the CCS must continue to develop and mature in a coordinated manner to ensure that proper pace making potential and distribution of action potential is maintained at all stages. This requires not only the formation of distinct and disparate components of the CCS, but the integration of these components into a functioning whole as the heart matures. Though research in this area of development may have lagged behind other areas of heart development, in recent years there has been much progress in understanding the ontogeny of the CCS and the developmental cues that drive its formation. This is largely due to studies on the avian heart as well as the use of molecular biology approaches. This review gives a perspective on advances in understanding the development of the vertebrate CCS, and reports new data illuminating the mechanism of conduction cell determination and maintenance in the mammalian heart. As much of our knowledge about the development of the CCS has been derived from the chick embryo, one important area facing the field is the relationship and similarities between the structure and development of avian and mammalian conduction systems. Specifically, the morphology of the distal elements of the mammalian CCS and the manner in which its components are recruited from working cardiomyocytes are areas of research that will, hopefully, receive more attention in the near future. A more general and outstanding question is how the disparate components of all vertebrate conduction systems integrate into a functional entity during embryogenesis. There is mounting evidence linking the patterning and formation of the CCS to instructive cues derived from the cardiac vasculature and, more specifically, to hemodynamic-responsive factors produced by cardiac endothelia. This highlights the need for a greater understanding of the biophysical forces acting on, and created by, the cardiovascular system during embryonic development. A better understanding of these processes will be necessary if therapeutics are to be developed that allow the regeneration of damaged cardiac tissues or the construction of biologically engineered heart tissues.     

Development of the cardiac conduction system

The cardiac conduction system (CCS) is a specialized tissue network that initiates and maintains a rhythmic heartbeat. The CCS consists of several functional subcomponents responsible for producing a pacemaking impulse and distributing action potentials across the heart in a coordinated manner. The formation of the distinct subcomponents of the CCS occurs within a precise temporal and spatial framework; thereby assuring that as the system matures from a tubular to a complex chambered organ, a rhythmic heartbeat is always maintained. Therefore, a defect in differentiation of any CCS component would lead to severe rhythm disturbances. Recent molecular, cell biological and physiological approaches have provided fresh and unexpected perspectives of the relationships between cell fate, gene expression and differentiation of specialized function within the developing myocardium. In particular, biomechanical forces created by the heartbeat itself have important roles in the inductive patterning and functional integration of the developing conduction system. This new understanding of the cellular origin and molecular induction of CCS tissues during embryogenesis may provide the foundation for tissue engineering, replacement and repair of these essential cardiac tissues in the future.     

Inducible gene deletion in the entire cardiac conduction system using Hcn4‐CreERT2 BAC transgenic mice

Developmental defects and disruption of molecular pathways of the cardiac conduction system (CCS) can cause life‐threatening cardiac arrhythmias. Despite decades of effort, knowledge about the development and molecular control of the CCS remains primitive. Mouse genetics, complementary to other approaches such as human genetics, has become a key tool for exploring the developmental processes of various organs and associated diseases. Genetic analysis using mouse models will likely provide great insights about the development of the CCS, which can facilitate the development of novel therapeutic strategies to treat arrhythmias. To enable genetic studies of the CCS, CCS‐associated Cre mouse models are essential. However, existing mouse models with Cre activity reported in the CCS have various limitations such as Cre leak, haploinsufficiency, and inadequate specificity of the Cre activity. To circumvent those limitations, we successfully generated Hcn4‐CreERT2 bacterial artificial chromosome (BAC) transgenic mice using BAC recombineering in which Cre activity was specifically detected in the entire CCS after tamoxifen induction. Our Hcn4‐CreERT2 BAC transgenic line will be an invaluable genetic tool with which to dissect the developmental control of CCS and arrhythmias. genesis 52:134–140. © 2013 Wiley Periodicals, Inc.     

Visualization and functional characterization of the developing murine cardiac conduction system

The cardiac conduction system is a complex network of cells that together orchestrate the rhythmic and coordinated depolarization of the heart. The molecular mechanisms regulating the specification and patterning of cells that form this conductive network are largely unknown. Studies in avian models have suggested that components of the cardiac conduction system arise from progressive recruitment of cardiomyogenic progenitors, potentially influenced by inductive effects from the neighboring coronary vasculature. However, relatively little is known about the process of conduction system development in mammalian species, especially in the mouse, where even the histological identification of the conductive network remains problematic. We have identified a line of transgenic mice where lacZ reporter gene expression delineates the developing and mature murine cardiac conduction system, extending proximally from the sinoatrial node to the distal Purkinje fibers. Optical mapping of cardiac electrical activity using a voltage-sensitive dye confirms that cells identified by the lacZ reporter gene are indeed components of the specialized conduction system. Analysis of lacZ expression during sequential stages of cardiogenesis provides a detailed view of the maturation of the conductive network and demonstrates that patterning occurs surprisingly early in embryogenesis. Moreover, optical mapping studies of embryonic hearts demonstrate that a murine His-Purkinje system is functioning well before septation has completed. Thus, these studies describe a novel marker of the murine cardiac conduction system that identifies this specialized network of cells throughout cardiac development. Analysis of lacZ expression and optical mapping data highlight important differences between murine and avian conduction system development. Finally, this line of transgenic mice provides a novel tool for exploring the molecular circuitry controlling mammalian conduction system development and should be invaluable in studies of developmental mutants with potential structural or functional conduction system defects.     

Characterization of embryonic cardiac pacemaker and atrioventricular conduction physiology in Xenopus laevis using noninvasive imaging

Congenital heart defects often include altered conduction as well as morphological changes. Model organisms, like the frog Xenopus laevis, offer practical advantages for the study of congenital heart disease. X. laevis embryos are easily obtained free living, and the developing heart is readily visualized. Functional and morphological evidence for a conduction system is available for adult frog hearts, but information on the normal properties of embryonic heart contraction is lacking, especially in intact animals. With the use of fine glass microelectrodes, we were able to obtain cardiac recordings and make standard electrophysiological measurements in 1-wk-old embryos (stage 46). In addition, a system using digital analysis of video images was adapted for measurement of the standard cardiac intervals and compared with invasive measurements. Video images were obtained of the heart in live, pharmacologically paralyzed, stage 46 X. laevis embryos. Normal values for the timing of the cardiac cycle were established. Intervals determined by video analysis (n = 53), including the atrial and ventricular cycle lengths (473 +/- 10 ms and 464 +/- 19 ms, respectively) and the atrioventricular interval (169 +/- 5 ms) were not statistically different from those determined by intrathoracic cardiac recordings. We also present the data obtained from embryos treated with standard medications that affect the human conduction system. We conclude that the physiology of embryonic X. laevis cardiac conduction can be noninvasively studied by using digital video imaging. Additionally, we show the response of X. laevis embryonic hearts to chronotropic agents is similar but not identical to the response of the human heart.     

降钙素基因相关肽在豚鼠冠脉血流量和心脏传导系统作用的比较

本文目的在于深入研究降钙素基因相关肽（ＣＧＲＰ）对豚鼠冠状血流量以及心脏传导系统各部分的作用。采用Ｌａｎｇｅｎｄｏｒｆｆ法灌流心脏,同步记录心脏表面电图和希氏束电活动。观察应用ＣＧＲＰ前后的冠脉流量、自主心率、在相同心房周期下的房室结（ＡＨ）及希浦系传导时间（ＨＶ）、心脏出现３：２文氏传导及２：１房室传导阻滞所需的最长起搏周期（ＰＣＬ３：２,ＰＣＬ２：１）。ＣＧＲＰ（３－３０ｎｍｏｌ／Ｌ）可显著增     

MURC, a muscle-restricted coiled-coil protein that modulates the Rho/ROCK pathway, induces cardiac dysfunction and conduction disturbance

We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias.     

Specification of the mouse cardiac conduction system in the absence of Endothelin signaling

Coordinated contraction of the heart is essential for survival and is regulated by the cardiac conduction system. Contraction of ventricular myocytes is controlled by the terminal part of the conduction system known as the Purkinje fiber network. Lineage analyses in chickens and mice have established that the Purkinje fibers of the peripheral ventricular conduction system arise from working myocytes during cardiac development. It has been proposed, based primarily on gain-of-function studies, that Endothelin signaling is responsible for myocyte-to-Purkinje fiber transdifferentiation during avian heart development. However, the role of Endothelin signaling in mammalian conduction system development is less clear, and the development of the cardiac conduction system in mice lacking Endothelin signaling has not been previously addressed. Here, we assessed the specification of the cardiac conduction system in mouse embryos lacking all Endothelin signaling. We found that mouse embryos that were homozygous null for both ednra and ednrb, the genes encoding the two Endothelin receptors in mice, were born at predicted Mendelian frequency and had normal specification of the cardiac conduction system and apparently normal electrocardiograms with normal QRS intervals. In addition, we found that ednra expression within the heart was restricted to the myocardium while ednrb expression in the heart was restricted to the endocardium and coronary endothelium. By establishing that ednra and ednrb are expressed in distinct compartments within the developing mammalian heart and that Endothelin signaling is dispensable for specification and function of the cardiac conduction system, this work has important implications for our understanding of mammalian cardiac development.     

Effects of metabolic inhibition on conduction, Ca transients, and arrhythmia vulnerability in embryonic mouse hearts

Developing myocardium is more dependent on glycolysis than adult myocardium, yet the effects of selectively inhibiting glycolysis versus oxidative phosphorylation on embryonic heart function have not been well characterized. Accordingly, we investigated how selective metabolic inhibition affects membrane voltage and intracellular Ca (Ca(i)) transients in embryonic mouse hearts, including their susceptibility to arrhythmias. A total of 136 isolated embryonic mouse hearts were exposed to either 1) 2-deoxyglucose (2DG; 10 mM) or iodoacetate (IAA; 0.1 mM) with 10 mM pyruvate in place of glucose to selectively inhibit glycolysis or 2) the mitochondrial uncoupler protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 500 nM) with 10 mM glucose present to selectively inhibit oxidative phosphorylation. Using confocal imaging, we found that mitochondrial membrane potential monitored with tetramethylrhodamine methyl ester (200 nM) remained stable with 2DG or IAA but depolarized within 5 min after exposure to FCCP. IAA and FCCP decreased heart rate, inhibited Ca(i) transient amplitude, shortened action potential duration at 80% repolarization (APD(80)), and prolonged atrioventricular conduction time to similar extents. Although 2DG decreased heart rate and Ca(i) transient amplitude, it did not significantly affect APD(80) and AV conduction time. In addition, spontaneous arrhythmias occurred in 77 of 136 embryonic hearts (57%) after exposure to IAA (28/53) or FCCP (49/83). There were no significant differences in the types or incidence of arrhythmias induced by IAA and FCCP. These data support the idea that both glycolysis and oxidative phosphorylation play critical metabolic roles in regulating cardiac function in the embryonic mouse heart.     

Teratogenic effects of bis-diamine on the developing cardiac conduction system

BACKGROUND: Congenital heart defects, including conotruncal anomalies, are often associated with arrhythmias. Bis-diamine induces conotruncal anomalies in embryos when administered to pregnant female rats. To investigate the mechanism of arrhythmia in conotruncal anomalies, we histologically examined the development of the cardiac conduction system in this animal model. METHODS: A single dose of 200 mg of bis-diamine was administered to pregnant Wistar rats on ED 10.5 of pregnancy. The embryos were removed on each day from ED 11.5 to 15.5. Immunoexpression of HNK-1, connexin40, and connexin43 were examined in serial sections. The distribution pattern of TUNEL-positive cells around the conduction system was also examined. RESULTS: HNK-1 immunoreactivity was evident in interventricular septum, in both the control and the bis-diamine-treated embryos from ED 12.5. Although a chain of connexin40-immunoreactive cells from interventricular septum to trabeculae, corresponding to the His bundle and its branches, was demonstrated at ED 13.5 in the control embryos, this chain was first detected at ED 14.5 in the bis-diamine-treated embryos. Immunoexpression of connexin43 in the working myocardium was also less in the bis-diamine-treated embryos than in the control at ED 13.5. The number of TUNEL-positive cells in the interventricular septum was highest at ED 12.5 in the control and at ED 13.5 in the bis-diamine-treated embryos. Furthermore, these TUNEL-positive cells were HNK-1 negative, vimentin-positive, and alpha smooth muscle actin-positive. CONCLUSIONS: Bis-diamine disturbed the normal development of gap junctions and apoptosis of myofibroblasts around the HNK-1-positive conduction tissue through overall poor myocardial proliferation and growth.     

Electrophysiology and anatomy of embryonic rabbit hearts before and after septation

Mechanisms of cardiac pacemaking and conduction system (CPCS) development are difficult to study, in part because of the absence of models that are physiologically similar to humans in which we can label the entire CPCS. Investigations of the adult rabbit heart have provided insight into normal and abnormal cardiac conduction. The adult and the embryonic rabbit have an endogenous marker of the entire cardiac conduction system, neurofilament 160 (NF-160). Previous work suggested that ventricular septation correlates with critical phases in avian CPCS development, in contrast to the mouse CPCS. Combining high-resolution optical mapping with immunohistochemical analysis of the embryonic rabbit heart, we investigated the significance of ventricular septation in patterning the rabbit embryonic conduction system. We hypothesized that 1) completion of ventricular septation does not correlate with changes in the ventricular activation sequence in rabbit embryos and 2) CPCS anatomy determines the activation sequence of the embryonic heart. We found that preseptated (days 11-13, n = 13) and postseptated (day 15, n = 5) hearts had similar "apex-to-base" ventricular excitation. PR intervals were not significantly different in either group. CPCS anatomy revealed continuity of the NF-160-positive tract connecting the presumptive sinoatrial node, atrioventricular (AV) junction, and ventricular conduction system. The presence of collagen in the AV junction coincided with the appearance of an AV interval. We conclude that the apex-to-base ventricular activation sequence in the rabbit embryo is present before completion of ventricular septation. CPCS anatomy reflects global cardiac activation as demonstrated by high-resolution optical mapping.     

Notch1b and neuregulin are required for specification of central cardiac conduction tissue

Normal heart function is critically dependent on the timing and coordination provided by a complex network of specialized cells: the cardiac conduction system. We have employed functional assays in zebrafish to explore early steps in the patterning of the conduction system that previously have been inaccessible. We demonstrate that a ring of atrioventricular conduction tissue develops at 40 hours post-fertilization in the zebrafish heart. Analysis of the mutant cloche reveals a requirement for endocardial signals in the formation of this tissue. The differentiation of these specialized cells, unlike that of adjacent endocardial cushions and valves, is not dependent on blood flow or cardiac contraction. Finally, both neuregulin and notch1b are necessary for the development of atrioventricular conduction tissue. These results are the first demonstration of the endocardial signals required for patterning central ;slow' conduction tissue, and they reveal the operation of distinct local endocardial-myocardial interactions within the developing heart tube.     

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.     

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.     

Tbx20 regulation of cardiac cell proliferation and lineage specialization during embryonic and fetal development in vivo

TBX20 gain-of-function mutations in humans are associated with congenital heart malformations and myocardial defects. However the effects of increased Tbx20 function during cardiac chamber development and maturation have not been reported previously. CAG-CAT-Tbx20 transgenic mice were generated for Cre-dependent induction of Tbx20 in myocardial lineages in the developing heart. βMHCCre-mediated overexpression of Tbx20 in fetal ventricular cardiomyocytes results in increased thickness of compact myocardium, induction of cardiomyocyte proliferation, and increased expression of Bmp10 and pSmad1/5/8 at embryonic day (E) 14.5. βMHCCre-mediated Tbx20 overexpression also leads to increased expression of cardiac conduction system (CCS) genes Tbx5, Cx40, and Cx43 throughout the ventricular myocardium. In contrast, Nkx2.5Cre mediated overexpression of Tbx20 in the embryonic heart results in reduced cardiomyocyte proliferation, increased expression of a cell cycle inhibitor, p21(CIP1), and decreased expression of Tbx2, Tbx5, and N-myc1 at E9.5, concomitant with decreased phospho-ERK1/2 expression. Together, these analyses demonstrate that Tbx20 differentially regulates cell proliferation and cardiac lineage specification in embryonic versus fetal cardiomyocytes. Induction of pSmad1/5/8 at E14.5 and inhibition of dpERK expression at E9.5 are consistent with selective Tbx20 regulation of these pathways in association with stage-specific effects on cardiomyocyte proliferation. Together, these in vivo data support distinct functions for Tbx20 in regulation of cardiomyocyte lineage maturation and cell proliferation at embryonic and fetal stages of heart development.     

Focal modification of electrical conduction in the heart by viral gene transfer

Modern treatment of cardiac arrhythmias is limited to pharmacotherapy, radiofrequency ablation, or implantable devices. Antiarrhythmic medications suppress arrhythmias, but their systemic effects are often poorly tolerated and their proarrhythmic tendencies increase mortality. Radiofrequency ablation can cure only a limited number of arrhythmias. Implantable devices can be curative for bradyarrhythmias and lifesaving for tachyarrhythmias, but require a lifetime commitment to repeated procedures, are a significant expense, and may lead to severe complications. One possibility is the use of gene therapy as an antiarrhythmic strategy. As an initial attempt to explore this option, we focused on genetic modification of the atrioventricular node. First, we developed an intracoronary perfusion model for gene delivery, building on our previous work in isolated cardiac myocytes and hearts perfused ex vivo. Using this method, we infected porcine hearts with Adbetagal (recombinant adenovirus expressing Escherichia coli beta-galactosidase) or with AdGi (adenovirus encoding the Galphai2 subunit). We hypothesized that excess Galphai2 would mimic the effects of beta-adreneric antagonists, in effect creating a localized beta-blockade. Galphai2 overexpression suppressed baseline atrioventricular conduction and slowed the heart rate during atrial fibrillation without producing complete heart block. In contrast, expression of the reporter gene beta-galactosidase had no electrophysiological effects. Our results demonstrate the feasibility of using myocardial gene transfer strategies to treat common arrhythmias.