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.

A procedural method for modeling the purkinje fibers of the heart.

The Purkinje fibers are located in the ventricular walls of the heart, just beneath the endocardium and conduct excitation from the right and left bundle branches to the ventricular myocardium. Recently, anatomists succeeded in photographing the Purkinje fibers of a sheep, which clearly showed the mesh structure of the Purkinje fibers. In this study, we present a technique for modeling the mesh structure of Purkinje fibers semiautomatically using an extended L-system. The L-system is a formal grammar that defines the growth of a fractal structure by generating rules (or rewriting rules) and an initial structure. It was originally formulated to describe the growth of plant cells, and has subsequently been applied for various purposes in computer graphics such as modeling plants, buildings, streets, and ornaments. For our purpose, we extended the growth process of the L-system as follows: 1) each growing branch keeps away from existing branches as much as possible to create a uniform distribution, and 2) when branches collide, we connect the colliding branches to construct a closed mesh structure. We designed a generating rule based on observations of the photograph of Purkinje fibers and manually specified three terminal positions on a three-dimensional (3D) heart model: those of the right bundle branch, the anterior fascicle, and the left posterior fascicle of the left branch. Then, we grew fibers starting from each of the three positions based on the specified generating rule. We achieved to generate 3D models of Purkinje fibers of which physical appearances closely resembled the real photograph. The generation takes a few seconds. Variations of the Purkinje fibers could be constructed easily by modifying the generating rules and parameters.

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.

His-Purkinje lineages and development.

The heartbeat is initiated and coordinated by a multi-component set of specialized muscle tissues collectively referred to as the pacemaking and conduction system. Over the last few years, impetus has gathered into unravelling the cellular and molecular processes that regulate differentiation and integration of this essential cardiac network. One focus of our collective work has been the developmental history of cells comprising His-Purkinje tissues of the conduction system. This interest in part arose from studies of the expression of connexins in periarterial Purkinje fibres of the chick heart. Using lineage-tracing strategies, including those based on replication-defective retroviruses and adenoviruses, it has been shown that conduction cells are derived from multipotent, cardiomyogenic progenitors in the tubular heart. Moreover, heterogeneity within myocardial clones has indicated that the elaboration of the conduction system in the chick embryo occurs by progressive, localized recruitment from within this pool of cardiomyogenic cells. Cell birth dating has revealed that inductive conscription of cells to central elements of the conduction system (e.g. the His bundle) precedes recruitment to the peripheral components of the network (i.e. subendocardial and periarterial Purkinje fibres). Birth dating studies in rodents suggest an analogous recruitment process is occurring in this species. In addition to summarizing earlier work, this chapter provides information on ongoing studies of cell-cell signalling and transcriptional mechanisms that may regulate the development of His-Purkinje tissues.

Differential developmental effects of acute hypoxia on the rabbit atrioventricular conduction axis.

The differential developmental effects of hypoxia on antegrade fast and slow and retrograde conduction through the atrioventricular junction are unknown. This study describes the effects of hypoxia on fast and slow antegrade atrioventricular node, infra-Hisian and retrograde conduction in immature and mature hearts during premature pacing protocols in excise, perfused adult and neonatal rabbits. The results are: (1) antegrade conduction delay through the atrioventricular node is the same developmentally, but delay through the His-Purkinje system is greater in adults; (2) hypoxia reduces the extra delay in the His-Purkinje system in adults; (3) fast atrioventricular node conduction is more sensitive to hypoxia in neonates than in adults, and slow atrioventricular node conduction is more sensitive to hypoxia in adults than in neonates, and (4) retrograde atrioventricular node conduction is more resistant to hypoxia in neonates than in adults.

[Tendon of Todaro in the human heart]. / Sciegno Todara w sercu ludzkim.

The material consists of 50 human hearts without either pathological changes or congenital malformations. Macroscopic and microscopic methods were applied. Histological specimens were stained alternatively with hematoxylin-eosin and according to van Giesson and Masson. In each heart the tendon of Todaro was dissected from its portion situated between the inferior vena cava and coronary sinus up to central part of the membranous septum (Fig. 1). The hearts were divided into 3 groups according to age: fetuses (20 hearts), infants (15) and adults (15) (Table I). In all hearts of fetuses and infants the tendon of Todaro was found to be a well-developed cylindrical structure covered with endocardium. In histologic specimens the tendon was s solid structure well-separated from other tissues (Fig. 3, Fig. 4). In hearts of adults (17-45 years old) the tendon of Todaro was less evident. However, in histological specimens it was present as a compact connective tissue band (Fig. 2). In hearts of subjects older than 50, the endocardium of this area was not elevated and not distinguishable even by palpation. The connective tissue band was formed by rather dissipated fibres, poorly separated from surrounding structures. As a result of our study one may conclude that the tendon of Todaro is present in each human heart. It is well-developed in hearts of fetuses and infants. Later it diminishes gradually becoming almost inconspicuous in old subjects.

Developmental differences in the electrophysiological response of isolated canine Purkinje fibers to adrenergic stimulation during simulated ischemia.

Developmental differences in adrenergic responsiveness may cause age-related changes in the cellular response to ischemia. Standard microelectrode techniques were used in isolated young and adult canine Purkinje fibers to determine the effect of simulated ischemia ([K+]o = 10 mM, pH 6.7, pO2 < 25 mm Hg) alone or with adrenergic stimulation on the rhythmic activity in spontaneously beating Purkinje fibers and on transmembrane potentials and delayed afterdepolarizations in paced Purkinje fibers (basic cycle length = 800-300 ms). The adrenergic agonists used were phenylephrine (5 x 10(-8) M) and isoproterenol (1 x 10(-6) M). For all automatic fibers studied, the control maximum diastolic potential in adults (-96 +/- 1 mV, n = 37) and in the young (-98 +/- 1 mV, n = 36) went to -62 +/- 1 mV during ischemia in both groups and returned to -96 +/- 2 mV with reperfusion. The incidence of rhythmic activity (expressed as percent) during ischemia alone was similar at both ages: adults, 22%; young, 25%. The incidence of ectopic activity with phenylephrine superfusion during ischemia for adults was 63%, an effect blocked by prazosin (1 x 10(-6) M) but not by propranolol (2 x 10(-7) M); the incidence for the young was 25%. Isoproterenol caused ectopic rhythms in 86% of young fibers and 17% of adult fibers (p < 0.05 young vs. adult). During reperfusion the return to control rhythm was slower in adults after ischemia alone or ischemia + alpha-adrenergic stimulation with phenylephrine. There were no age-related differences in the transmembrane potential response of paced fibers to ischemia or reperfusion, and there were no delayed afterdepolarizations with interruption of pacing at 800, 500, or 300 ms in either group. These data suggest that age-related differences in adrenergic responses alter the cellular response to an ischemic insult. To the extent that an ectopic beat may initiate an abnormal rhythm, these differences in sensitivity to adrenergic agonists may lead to developmental differences in arrhythmogenic potential during ischemia.

The developmental electrophysiologic effects of moricizine HCl on the canine cardiac Purkinje fiber.

Using standard microelectrode techniques, the developmental cellular electrophysiologic effects of moricizine HCl on adult and neonatal canine Purkinje fibers were studied. Steady state and rate-related changes in the transmembrane action potentials produced by moricizine HCl in both age groups were characterized and compared. Also, the rate of barium-induced abnormal automaticity before and after drug was also investigated in neonatal and adult Purkinje fibers. The major findings of this study are as follows. (1) The steady state and rate-related depressant effects of moricizine HCl on Vmax were similar in both age groups. (2) Moricizine HCl shortened APD90 in the adult fibers to a greater extent than in the neonate. (3) The concentration of moricizine HCl required to significantly reduce the rate of abnormal automaticity was less in the neonate than in the adult. The effect of moricizine HCl on APD90 of individual Purkinje fibers is influenced both by their control APD90 value as well as by maturational factors. It is less clear whether developmental differences in the effects of moricizine HCl on abnormal automaticity are solely a result of differences in control rates of abnormal automaticity between the two age groups.

Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development.

Dystrophin transcripts were shown to be alternatively spliced in a pattern characteristic of both tissue type and developmental stage. Multiple novel spliced forms of dystrophin mRNA were identified in murine brain tissue, skeletal and cardiac muscle, diaphragm, and human cardiac Purkinje fibers. The transcript diversity was greatest in adult, non-skeletal muscle tissues. Sequence analysis revealed that four tandem exons of the murine gene are differentially spliced in at least 11 separate patterns to generate distinct isoforms. Two of these forms were observed in all tissues examined, while several others were uniquely observed in cardiac muscle and brain. Cardiac Purkinje fibers express an isoform primarily observed in brain tissue. Several spliced transcripts were observed only in postnatal development. Differential utilization of a fifth exon results in two mRNA splice forms that encode separate embryonic and adult C-termini of dystrophin. Comparison of murine with human dystrophin mRNAs showed that similar isoform expression patterns exist across species. These observations suggest that functionally distinct isoforms of the dystrophin protein are expressed in separate tissues and at different stages of development. These isoforms may be of significance in understanding the various tissue-specific effects produced by dystrophin gene mutations in Duchenne and Becker muscular dystrophy patients.

The morphology of the developing canine conducting system: bundle branch and Purkinje cell architecture from birth to week 12 of life.

This is a qualitative and quantitative study of dog bundle branch and Purkinje cell development from day 0 to week 12 of life; we correlate the morphologic data with changes observed in the functional properties of developing dog Purkinje tissue. The bundle branch itself has a roughly cylindrical shape and is surrounded by a collagen sheath covered with endocardium. Within the bundle, Purkinje cells are packed closely together in fascicles distributed evenly around a central artery. Cross-sectional area doubles in the right bundle and increases 5-fold in the left bundle system between day 0 and week 12 of life. About one third of the bundle by volume is Purkinje tissue; the rest is extracellular space containing an increasing amount of collagen as the animal ages. Purkinje cell cross-sectional area is constant during the first week of life, but its length doubles and the cell changes from a relatively round to a more cylindrical shape. Between day 7 and week 12, cell diameter doubles; Purkinje cell surface area increases 5-fold and its volume almost 10-fold. As a consequence, the surface to volume ratio halves and approaches the value reported for adult dogs by week 12 of life. The percent of the intercalated disc occupied by nexal junctions virtually doubles by week 12, the same period over which Purkinje fiber conduction velocity increases. The disc itself becomes less dominant as the cell enlarges; the total percent of sarcolemma involved in its formation decreases by a fourth and has achieved the adult value by week 12 of life. As this happens, the percent of cell membrane facing on clefts increases almost 6-fold, so that the total percent of sarcolemma facing on small spaces (approximately 340A wide) is constant over the age period studied. The paucity of clefts in newborn tissue compared with the value reported for the adult dog may help explain the relative lack of responsiveness to extracellular potassium concentration of the resting membrane potential described for fetal Purkinje tissue. Within the Purkinje cell itself, the percent by volume occupied by mitochondria remains relatively constant over the age span studied, while sarcomeric mass increases 3-fold over the same period of time; these data are consonant with the relative resistance of this tissue to hypoxia.

Developmental electrophysiologic effects of propafenone and 5-hydroxypropafenone on the canine cardiac Purkinje fiber.

Standard microelectrode techniques were used to investigate the in vitro developmental electrophysiologic effects of propafenone and 5-hydroxypropafenone on the adult and neonatal canine Purkinje fiber. The tonic and frequency-dependent depressant effects of these compounds on Vmax and amplitude were similar in both age groups. However, the ability of these compounds to shorten repolarization parameters was more pronounced in the adult. The extent of reduction of abnormal automaticity produced by propafenone was greater in the neonate compared to the adult, and 5-hydroxypropafenone significantly reduced automaticity only in the neonate. The sensitivity of neonatal abnormal automaticity to the effects of these compounds may prove to be important if the use of these agents is to be expanded into the realm of therapy for pediatric automatic rhythms.

Developmental cellular electrophysiologic effects of d-sotalol on canine cardiac Purkinje fibers.

d-Sotalol may be a clinically useful class III antiarrhythmic agent for controlling ventricular arrhythmias in children. Because age-related differences in repolarization currents may contribute to developmental differences in response to antiarrhythmic agents that primarily affect repolarization, the electrophysiologic effects of d-sotalol were compared in Purkinje fibers from neonatal and adult dogs. Significant age-related changes characterized the antiarrhythmic profile of d-sotalol. d-Sotalol (10(-4) M) significantly prolonged the action potential duration of adult Purkinje fibers (310 +/- 8 to 380 +/- 7 ms, p less than 0.01) and neonatal fibers (247 +/- 5 to 342 +/- 9 ms, p less than 0.01). However, the lengthening of action potential duration was significantly greater in the immature age group. d-Sotalol had no significant effect on maximum diastolic potential, action potential amplitude, or phase zero upstroke velocity in normally polarized fibers. In contrast, different electrophysiologic effects were observed in K(+)-depolarized Purkinje fibers. Superfusion of adult K(+)-depolarized fibers with d-sotalol suppressed excitability in five (38%) of 13 fibers and significantly decreased action potential amplitude (88 +/- 2 to 83 +/- 1 mV, p less than 0.05) and phase zero upstroke velocity (180 +/- 14 to 105 +/- 3 V/s, p less than 0.01) in the other eight fibers. The membrane depressant effects observed in the younger age group were significantly less [no suppression of excitability and a smaller decrease in phase zero upstroke velocity (121 +/- 22 to 101 +/- 23 V/s, p less than 0.05). The magnitude of action potential duration prolongation by d-sotalol in K(+)-depolarized fibers was less than in normally polarized fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in alpha adrenergic effects on canine Purkinje fiber automaticity.

Canine cardiac Purkinje fiber automaticity is modified by adrenergic agonists. Alpha adrenergic agonists slow and beta agonists increase the rate of automatic discharge. These effects vary with maturation and development. We used standard microelectrode techniques to study the effects of the alpha agonist, phenylephrine, on automaticity in isolated neonatal (less than 10 days) and adult canine cardiac Purkinje fibers. Fibers were superfused with 5 X 10(-8) M phenylephrine dissolved in Tyrode's solution. 75% (24/32) of adult and 52% (12/23) of neonatal fibers showed a decrease in rate. The magnitude of the decrease was similar at both ages and the effect was blocked during superfusion with 1 X 10(-9) M phentolamine. The remaining adult and neonatal fibers demonstrated a significant increase in rate in response to 5 X 10(-8) M phenylephrine. This effect was blocked by 5 X 10(-7) M propranolol in the adult group but not in the neonates. However, the increase in rate of fibers from 0- to 2-day neonates was blocked by phentolamine. As an indicator of adrenergic innervation of neonatal hearts, we assayed the myocardial norepinephrine concentration. There was a 6- to 8-fold increase in concentration during the first 10 days of neonatal life, suggesting that innervation was rapidly increasing during this time. In summary, neonatal Purkinje fibers can show an alpha adrenergic-induced acceleration of automatic rate. This alpha adrenergic acceleration is not seen in adults. The change in response from neonate to adult may be effected by growth and development of autonomic nerves and associated changes in receptor function.

Developmental changes in the cardiac effects of amrinone in the dog.

Amrinone, a bipyridine compound, has been shown to exert a positive inotropic effect on the heart, without producing cardiac arrhythmias. Because of preliminary observations suggesting that the actions of amrinone might change significantly with growth and development, we studied its effects on the contractility and electrophysiology of isolated cardiac muscle of 0- to 96-day-old beagles and Purkinje fibers of 5-year-old beagles. Amrinone's effects on ventricular muscle contractility were age-related. A significant decrease in contractility of right ventricular trabeculae and papillary muscles (not associated with changes in action potential characteristics) was observed in the 0- to 3-day newborn, whereas, by day 4-10, amrinone increased contractility. The magnitude of the increase became greater through day 96 of life. The negative inotropic effect of amrinone is unassociated with changes in the action potential plateau, suggesting that the slow inward current is not involved in this mechanism.

Developmental changes in impulse conduction in the canine heart.

We studied the age-related changes that occur in conduction in Purkinje fiber bundles (PF) from adult and 8-wk-old dogs. As PF are stretched, conduction velocity increases to a maximum when bundle length is approximately 1.5 times control. Conduction velocity in adult PF was significantly faster than that in 8-wk PF at all bundle lengths, the maximum being 2.32 +/- 0.45 m/s (mean +/- SD) in adult and 1.56 +/- 0.59 m/s in 8-wk-old dogs. At greater than 1.5 times control length, conduction velocity decreased, as did resting potential, action potential amplitude, and upstroke velocity. At 1.5 times control length, the ratio of PF to collagen in the bundles increased, and structural changes consistent with both unbuckling and unfolding of the cell membranes were seen. At greater degrees of stretch, cleft size diminished, and intracellular edema was noted. In sum, structural changes in cardiac PF can explain the changes in conduction velocity that occur with stretch. At all degrees of stretch, however, velocity is faster in PF from adult than from young dogs.