Age-dependent changes in electrophysiology and calcium handling: implications for pediatric cardiac research.

Rodent models are frequently employed in cardiovascular research, yet our understanding of pediatric cardiac physiology has largely been deduced from more simplified two-dimensional cell studies. Previous studies have shown that postnatal development includes an alteration in the expression of genes and proteins involved in cell coupling, ion channels, and intracellular calcium handling. Accordingly, we hypothesized that postnatal cell maturation is likely to lead to dynamic alterations in whole heart electrophysiology and calcium handling. To test this hypothesis, we employed multiparametric imaging and electrophysiological techniques to quantify developmental changes from neonate to adult. In vivo electrocardiograms were collected to assess changes in heart rate, variability, and atrioventricular conduction (Sprague-Dawley rats). Intact, whole hearts were transferred to a Langendorff-perfusion system for multiparametric imaging (voltage, calcium). Optical mapping was performed in conjunction with an electrophysiology study to assess cardiac dynamics throughout development. Postnatal age was associated with an increase in the heart rate (181 ± 34 vs. 429 ± 13 beats/min), faster atrioventricular conduction (94 ± 13 vs. 46 ± 3 ms), shortened action potentials (APD80: 113 ± 18 vs. 60 ± 17 ms), and decreased ventricular refractoriness (VERP: 157 ± 45 vs. 57 ± 14 ms; neonatal vs. adults, means ± SD, P < 0.05). Calcium handling matured with development, resulting in shortened calcium transient durations (168 ± 18 vs. 117 ± 14 ms) and decreased propensity for calcium transient alternans (160 ± 18- vs. 99 ± 11-ms cycle length threshold; neonatal vs. adults, mean ± SD, P < 0.05). Results of this study can serve as a comprehensive baseline for future studies focused on pediatric disease modeling and/or preclinical testing.NEW & NOTEWORTHY This is the first study to assess cardiac electrophysiology and calcium handling throughout postnatal development, using both in vivo and whole heart models.

Embryology of the Cardiac Conduction System Relevant to Arrhythmias.

Embryogenesis of the heart involves the complex cellular differentiation of slow-conducting primary myocardium into the rapidly conducting chamber myocardium of the adult. However, small areas of relatively undifferentiated cells remain to form components of the adult cardiac conduction system (CCS) and nodal tissues. Further investigation has revealed additional areas of nodal-like tissues outside of the established CCS. The embryologic origins of these areas are similar to those of the adult CCS. Under pathologic conditions, these areas can give rise to important clinical arrhythmias. Here, we review the embryologic basis for these proarrhythmic structures within the heart.

Development of the cardiac conduction system in zebrafish.

The cardiac conduction system (CCS) propagates and coordinates the electrical excitation that originates from the pacemaker cells, throughout the heart, resulting in rhythmic heartbeat. Its defects result in life-threatening arrhythmias and sudden cardiac death. Understanding of the factors involved in the formation and function of the CCS remains incomplete. By transposon assisted transgenesis, we have developed enhancer trap (ET) lines of zebrafish that express fluorescent protein in the pacemaker cells at the sino-atrial node (SAN) and the atrio-ventricular region (AVR), termed CCS transgenics. This expression pattern begins at the stage when the heart undergoes looping morphogenesis at 36 h post fertilization (hpf) and is maintained into adulthood. Using the CCS transgenics, we investigated the effects of perturbation of cardiac function, as simulated by either the absence of endothelium or hemodynamic stimulation, on the cardiac conduction cells, which resulted in abnormal compaction of the SAN. To uncover the identity of the gene represented by the EGFP expression in the CCS transgenics, we mapped the transposon integration sites on the zebrafish genome to positions in close proximity to the gene encoding fibroblast growth homologous factor 2a (fhf2a). Fhf2a is represented by three transcripts, one of which is expressed in the developing heart. These transgenics are useful tools for studies of development of the CCS and cardiac disease.

Irx3 is required for postnatal maturation of the mouse ventricular conduction system.

The ventricular conduction system (VCS) orchestrates the harmonious contraction in every heartbeat. Defects in the VCS are often associated with life-threatening arrhythmias and also promote adverse remodeling in heart disease. We have previously established that the Irx3 homeobox gene regulates rapid electrical propagation in the VCS by modulating the transcription of gap junction proteins Cx40 and Cx43. However, it is unknown whether other factors contribute to the conduction defects observed in Irx3 knockout (Irx3(-/-)) mice. In this study, we show that during the early postnatal period, Irx3(-/-) mice develop morphological defects in the VCS which are temporally dissociated from changes in gap junction expression. These morphological defects were accompanied with progressive changes in the cardiac electrocardiogram including right bundle branch block. Hypoplastic VCS was not associated with increased apoptosis of VCS cardiomyocytes but with a lack of recruitment and maturation of ventricular cardiomyocytes into the VCS. Computational analysis followed by functional verification revealed that Irx3 promotes VCS-enriched transcripts targeted by Nkx2.5 and/or Tbx5. Altogether, these results indicate that, in addition to ensuring the appropriate expression of gap junctional channels in the VCS, Irx3 is necessary for the postnatal maturation of the VCS, possibly via its interactions with Tbx5 and Nkx2.5.

Gene regulatory networks in cardiac conduction system development.

The cardiac conduction system is a specialized tract of myocardial cells responsible for maintaining normal cardiac rhythm. Given its critical role in coordinating cardiac performance, a detailed analysis of the molecular mechanisms underlying conduction system formation should inform our understanding of arrhythmia pathophysiology and affect the development of novel therapeutic strategies. Historically, the ability to distinguish cells of the conduction system from neighboring working myocytes presented a major technical challenge for performing comprehensive mechanistic studies. Early lineage tracing experiments suggested that conduction cells derive from cardiomyocyte precursors, and these claims have been substantiated by using more contemporary approaches. However, regional specialization of conduction cells adds an additional layer of complexity to this system, and it appears that different components of the conduction system utilize unique modes of developmental formation. The identification of numerous transcription factors and their downstream target genes involved in regional differentiation of the conduction system has provided insight into how lineage commitment is achieved. Furthermore, by adopting cutting-edge genetic techniques in combination with sophisticated phenotyping capabilities, investigators have made substantial progress in delineating the regulatory networks that orchestrate conduction system formation and their role in cardiac rhythm and physiology. This review describes the connectivity of these gene regulatory networks in cardiac conduction system development and discusses how they provide a foundation for understanding normal and pathological human cardiac rhythms.

Iroquois homeodomain transcription factors in heart development and function.

Numerous cardiac transcription factors play overlapping roles in both the specification and proliferation of the cardiac tissues and chambers during heart development. It has become increasingly apparent that cardiac transcription factors also play critical roles in the regulation of expression of many functional genes in the prenatal and postnatal hearts. Accordingly, mutations of cardiac transcription factors cannot only result in congenital heart defects but also alter heart function thereby predisposing to heart disease and cardiac arrhythmias. In this review, we summarize the roles of Iroquois homeobox (Irx) family of transcription factors in heart development and function. In all, 6 Irx genes are expressed with distinct and overlapping patterns in the mammalian heart. Studies in several animal models demonstrate that Irx genes are important for the establishment of ventricular chamber properties, the ventricular conduction system, as well as heterogeneity of the ventricular repolarization. The molecular mechanisms by which Irx proteins regulate gene expression and the clinical relevance of Irx functions in the heart are discussed.

The emerging genetic landscape underlying cardiac conduction system function.

Proper function of an organized Cardiac Conduction System (CCS) is vital to the survival of metazoans ranging from fly to man. The routine use of non-invasive electrocardiogram measures in the diagnosis and monitoring of cardiovascular health has established a trove of reliable CCS functional data in both normal and diseased cardiac states. Recent combination of echocardiogram (ECG) data with genome-wide association studies has identified genomic regions implicated in ECG variability which impact CCS function. In this study, we review the substantial recent progress in this area, highlighting the identification of novel loci, confirming the importance of previously implicated loci in CCS function, and exploring potential links between genes with important roles in developmental processes and variation in function of the CCS.

Developmental origin, growth, and three-dimensional architecture of the atrioventricular conduction axis of the mouse heart.

RATIONALE: The clinically important atrioventricular conduction axis is structurally complex and heterogeneous, and its molecular composition and developmental origin are uncertain. OBJECTIVE: To assess the molecular composition and 3D architecture of the atrioventricular conduction axis in the postnatal mouse heart and to define the developmental origin of its component parts. METHODS AND RESULTS: We generated an interactive 3D model of the atrioventricular junctions in the mouse heart using the patterns of expression of Tbx3, Hcn4, Cx40, Cx43, Cx45, and Nav1.5, which are important for conduction system function. We found extensive figure-of-eight rings of nodal and transitional cells around the mitral and tricuspid junctions and in the base of the atrial septum. The rings included the compact node and nodal extensions. We then used genetic lineage labeling tools (Tbx2(+/Cre), Mef2c-AHF-Cre, Tbx18(+/Cre)), along with morphometric analyses, to assess the developmental origin of the specific components of the axis. The majority of the atrial components, including the atrioventricular rings and compact node, are derived from the embryonic atrioventricular canal. The atrioventricular bundle, including the lower cells of the atrioventricular node, in contrast, is derived from the ventricular myocardium. No contributions to the conduction system myocardium were identified from the sinus venosus, the epicardium, or the dorsal mesenchymal protrusion. CONCLUSIONS: The atrioventricular conduction axis comprises multiple domains with distinctive molecular signatures. The atrial part proliferates from the embryonic atrioventricular canal, along with myocytes derived from the developing atrial septum. The atrioventricular bundle and lower nodal cells are derived from ventricular myocardium.

Biphasic development of the mammalian ventricular conduction system.

RATIONALE: The ventricular conduction system controls the propagation of electric activity through the heart to coordinate cardiac contraction. This system is composed of specialized cardiomyocytes organized in defined structures including central components and a peripheral Purkinje fiber network. How the mammalian ventricular conduction system is established during development remains controversial. OBJECTIVE: To define the lineage relationship between cells of the murine ventricular conduction system and surrounding working myocytes. METHODS AND RESULTS: A retrospective clonal analysis using the alpha-cardiac actin(nlaacZ/+) mouse line was carried out in three week old hearts. Clusters of clonally related myocytes were screened for conductive cells using connexin40-driven enhanced green fluorescent protein expression. Two classes of clusters containing conductive cells were obtained. Mixed clusters, composed of conductive and working myocytes, reveal that both cell types develop from common progenitor cells, whereas smaller unmixed clusters, composed exclusively of conductive cells, show that proliferation continues after lineage restriction to the conduction system lineage. Differences in the working component of mixed clusters between the right and left ventricles reveal distinct progenitor cell histories in these cardiac compartments. These results are supported by genetic fate mapping using Cre recombinase revealing progressive restriction of connexin40-positive myocytes to a conductive fate. CONCLUSIONS: A biphasic mode of development, lineage restriction followed by limited outgrowth, underlies establishment of the mammalian ventricular conduction system.

Expression of muscle segment homeobox genes in the developing myocardium.

Msx1 and Msx2 are essential for the development of many organs. In the heart, they act redundantly in development of the cardiac cushions. Additionally, Msx2 is expressed in the developing conduction system. However, the exact expression of Msx1 has not been established. We show that Msx1 is expressed in the cardiac cushions, but not in the myocardium. In Msx2-null mice, Msx1 is not ectopically expressed in the myocardium. The absence of myocardial defects in the Msx2 knock-out can therefore not be attributed to a redundant action of Msx1 in the myocardium.

Perinatal loss of Nkx2-5 results in rapid conduction and contraction defects.

Homeobox transcription factor Nkx2-5, highly expressed in heart, is a critical factor during early embryonic cardiac development. In this study, using tamoxifen-inducible Nkx2-5 knockout mice, we demonstrate the role of Nkx2-5 in conduction and contraction in neonates within 4 days after perinatal tamoxifen injection. Conduction defect was accompanied by reduction in ventricular expression of the cardiac voltage-gated Na+ channel pore-forming alpha-subunit (Na(v)1.5-alpha), the largest ion channel in the heart responsive for rapid depolarization of the action potential, which leads to increased intracellular Ca2+ for contraction (conduction-contraction coupling). In addition, expression of ryanodine receptor 2, through which Ca2+ is released from sarcoplasmic reticulum, was substantially reduced in Nkx2-5 knockout mice. These results indicate that Nkx2-5 function is critical not only during cardiac development but also in perinatal hearts, by regulating expression of several important gene products involved in conduction and contraction.

Postnatal development has a marked effect on ventricular repolarization in mice.

To better understand the mechanisms that underlie cardiac repolarization abnormalities in the immature heart, this study characterized and compared K(+) currents in mouse ventricular myocytes from day 1, day 7, day 20, and adult CD1 mice to determine the effects of postnatal development on ventricular repolarization. Current- and patch-clamp techniques were used to examine action potentials and the K(+) currents underlying repolarization in isolated myocytes. RT-PCR was used to quantify mRNA expression for the K(+) channels of interest. This study found that action potential duration (APD) decreased as age increased, with the shortest APDs observed in adult myocytes. This study also showed that K(+) currents and the mRNA relative abundance for the various K(+) channels were significantly greater in adult myocytes compared with day 1 myocytes. Examination of the individual components of total K(+) current revealed that the inward rectifier K(+) current (I(K1)) developed by day 7, both the Ca(2+)-independent transient outward current (I(to)) and the steady-state outward K(+) current (I(ss)) developed by day 20, and the ultrarapid delayed rectifier K(+) current (I(Kur)) did not fully develop until the mouse reached maturity. Interestingly, the increase in I(Kur) was not associated with a decrease in APD. Comparison of atrial and ventricular K(+) currents showed that I(to) and I(Kur) density were significantly greater in day 7, day 20, and adult myocytes compared with age-matched atrial cells. Overall, it appears that, in mouse ventricle, developmental changes in APD are likely attributable to increases in I(to), I(ss), and I(K1), whereas the role of I(Kur) during postnatal development appears to be less critical to APD.

Developmental changes in heart photosensitivity of the isopod crustacean Ligia exotica.

During juvenile development, the cardiac pacemaker of the isopod crustacean Ligia exotica is transferred from the myocardium to the cardiac ganglion of the neurogenic heart. In adult, light stimulus decreases the beat frequency of the heart. To elucidate developmental changes in the photosensitivity of the juvenile Ligia heart, we examined the effect of a light stimulus on the semi-isolated heart of juveniles at various developmental stages by the recording membrane potential of the myocardium. We also examined the effect of hyperpolarizing current injection into the myocardium, because this causes different effects on the beat frequency between myogenic and neurogenic hearts. In newly hatched juveniles, beat frequency decreased upon current injection but exhibited no response to white light. In contrast, 10 days after hatching, beat frequency did not change upon current injection, but decreased in response to white light. The heart photoresponse of juveniles was reversibly eliminated by application of tetrodotoxin, which changes the heartbeat from neurogenic to myogenic by suppressing cardiac ganglion activity. The proportion of juveniles exhibiting a heart photoresponse increased gradually up to 100% during the period between 3 and 10 days after hatching. The results suggest that the heart photoresponse of L. exotica appears in association with transfer of the cardiac pacemaker from the myocardium to the cardiac ganglion during juvenile development.

Nkx2.5 cell-autonomous gene function is required for the postnatal formation of the peripheral ventricular conduction system.

The ventricular conduction system is responsible for rapid propagation of electrical activity to coordinate ventricular contraction. To investigate the role of the transcription factor Nkx2.5 in the morphogenesis of the ventricular conduction system, we crossed Nkx2.5(+/-) mice with Cx40(eGFP/+) mice in which eGFP expression permits visualization of the His-Purkinje conduction system. Major anatomical and functional disturbances were detected in the His-Purkinje system of adult Nkx2.5(+/-)/Cx40(eGFP/+) mice, including hypoplasia of eGFP-positive Purkinje fibers and the disorganization of the Purkinje fiber network in the ventricular apex. Although the action potential properties of the individual eGFP-positive cells were normal, the deficiency of Purkinje fibers in Nkx2.5 haploinsufficient mice was associated with abnormalities of ventricular electrical activation, including slowed and decremented conduction along the left bundle branch. During embryonic development, eGFP expression in the ventricular trabeculae of Nkx2.5(+/-) hearts was qualitatively normal, with a measurable deficiency in eGFP-positive cells being observed only after birth. Chimeric analyses showed that maximal Nkx2.5 levels are required cell-autonomously. Reduced Nkx2.5 levels are associated with a delay in cell cycle withdrawal in surrounding GFP-negative myocytes. Our results suggest that the formation of the peripheral conduction system is time- and dose-dependent on the transcription factor Nkx2.5 that is cell-autonomously required for the postnatal differentiation of Purkinje fibers.

Reduced sinoatrial cAMP content plays a role in postnatal heart rate slowing in the rabbit.

1. Decreasing heart rate during development is known to be the result of parasympathetic nervous system maturation that depresses the pacemaker current (If) by acetylcholine (ACh). However, a direct effect of ACh on If has been ruled out and the involvement of other secondary messengers, such as cAMP, was verified in previous studies. Therefore, we hypothesized that reduced basal cAMP production in sinoatrial (SA) nodal cells may contribute to the slowing of heart rate after birth. 2. The electrocardiogram and heart rate variability (HRV) were documented and measured in vivo and in vitro (in isolated perfused Langendorff preparations) for rabbits aged 2, 4, 6, 8 and 12 weeks. Sinoatrial node action potential (AP) recording and perforated patch-clamp analyses were used to investigate the spontaneous depolarization rate and pacemaker If currents. Concentrations of cAMP in SA nodal tissues were determined by radioimmunoassay. Relative expression of adenylate cyclases (ADCY1, 5) and phosphodiesterases (PDE1A, 4A and 8A) were quantified by real-time reverse transcription-polymerase chain reaction. 3. Significantly reduced heart rate, but unchanged HRV, was observed in perfused hearts in the older age groups, accompanied with a slowed phase 4 spontaneous depolarization rate (90.5 +/- 4.7 vs 49.6 +/- 2.6 mV/s for 2 week vs 4 week hearts, respectively; n = 5; P < 0.05), a negative shift of the If threshold potential (-45.5 +/- 3.0 vs -51.1 +/- 6.0 mV for 2 week vs 4 week hearts, respectively; n = 9; P < 0.05) and decreasing basal levels of SA nodal cAMP (0.31 +/- 0.05 vs 0.025 +/- 0.002 micromol/L for 2 week vs 4 week hearts, respectively; n = 6; P < 0.05). Gene expression levels of PDE1A, 4A and 8A were increased in the 12 week group compared with the 2 week group 1.5-, 2- and 1.8-fold, respectively (P < 0.05), with little change in ADCY1 and 5. 4. These data suggest that, in addition to autonomic innervation, slowing of heart rate during postnatal maturation can be attributed to a negative shift of the If activation caused by diminished baseline cAMP content in SA nodal cells.

Neural crest cells retain multipotential characteristics in the developing valves and label the cardiac conduction system.

Multipotent neural crest cells (NCCs) are a major extracardiac component of cardiovascular development. Although recognized as contributing cells to the arterial valves at early developmental stages, NCC persistence in the valves at later times or in the adult heart is controversial. We analyzed NCC persistence and contributions to both semilunar and atrioventricular (AV) valves in the mature heart. Two NCC-specific promoters driving Cre recombinase, Wnt1-Cre and P0-Cre, were mated with floxed reporter mice, R26R or CAG-CAT-EGFP, to map NCC fate. Hearts were analyzed before aorticopulmonary (AP) septation through adult stages. As previously demonstrated, strong NCC labeling was detected in ventral and dorsal outflow cushions before AP septation. In contrast to previous reports, we found that substantial numbers of labeled cells persisted in the semilunar valves in late fetal, neonatal, and adult hearts. Furthermore, NCCs were also found in the AV valves, almost exclusively in the septal leaflets. NCCs in the AV valves expressed melanocytic and neurogenic markers. However, cells labeled in the proximal cardiac conduction system exhibited neurogenic and gliagenic markers, whereas some NCCs expressed no differentiation specific markers. These results suggest that cardiac NCCs contribute to the mature valves and the cardiac conduction system and retain multipotent characteristics late in development.

Expression pattern and ontogenesis of thyroid hormone receptor isoforms in the mouse heart.

Nuclear thyroid hormone (T3) receptors (TR) play a critical role in mediating the effects of T3 on development, differentiation and normal physiology of many organs. The heart is a major target organ of T3, and recent studies in knockout mice demonstrated distinct effects of the different TR isoforms on cardiac function, but the specific actions of TR isoforms and their specific localization in the heart remain unclear. We therefore studied the expression of TRalpha1, TRalpha2 and TRbeta1 isoforms in the mouse heart at different stages of development, using monoclonal antibodies against TRalpha1, TRalpha2 and TRbeta1. In order to identify distinct components of the embryonic heart, in situ hybridization for cardiac-specific markers was used with the expression pattern of sarcoplasmic reticulum calcium-ATPase 2a as a marker of myocardial structures, while the pattern of expression of connexin40 was used to indicate the developing chamber myocardium and peripheral ventricular conduction system. Here we show that in the ventricles of the adult heart the TRbeta1 isoform is confined to the cells that form the peripheral ventricular conduction system. TRalpha1, on the other hand, is present in working myocardium as well as in the peripheral ventricular conduction system. In the atria and in the proximal conduction system (sinoatrial node, atrio-ventricular node), TRalpha1 and TRbeta1 isoforms are co-expressed. We also found the heterogeneous expression of the TRalpha1, TRalpha2 and TRbeta1 isoforms in the developing mouse heart, which, in the case of the TRbeta1 isoform, gradually revealed a dynamic expression pattern. It was present in all cardiomyocytes at the early stages of cardiogenesis, but from embryonic day 11.5 and into adulthood, TRbeta1 demonstrated a gradual confinement to the peripheral ventricular conduction system (PVCS), suggesting a specific role of this isoform in the formation of PVCS. Detailed knowledge of the distribution of TRalpha1 and TRbeta1 in the heart is of importance for understanding not only their mechanism of action in the heart but also the design and (clinical) use of TR isoform-specific agonists and antagonists.