Adrenergic deficiency leads to impaired electrical conduction and increased arrhythmic potential in the embryonic mouse heart

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Abstract

To determine if adrenergic hormones play a critical role in the functional development of the cardiac pacemaking and conduction system, we employed a mouse model where adrenergic hormone production was blocked due to targeted disruption of the dopamine β-hydroxylase (Dbh) gene. Immunofluorescent histochemical evaluation of the major gap junction protein, connexin 43, revealed that its expression was substantially decreased in adrenergic-deficient (Dbh−/−) relative to adrenergic-competent (Dbh+/+ and Dbh+/−) mouse hearts at embryonic day 10.5 (E10.5), whereas pacemaker and structural protein staining appeared similar. To evaluate cardiac electrical conduction in these hearts, we cultured them on microelectrode arrays (8 × 8, 200 μm apart). Our results show a significant slowing of atrioventricular conduction in adrenergic-deficient hearts compared to controls (31.4 ± 6.4 vs. 15.4 ± 1.7 ms, respectively, p < 0.05). To determine if the absence of adrenergic hormones affected heart rate and rhythm, mouse hearts from adrenergic-competent and deficient embryos were cultured ex vivo at E10.5, and heart rates were measured before and after challenge with the β-adrenergic receptor agonist, isoproterenol (0.5 μM). On average, all hearts showed increased heart rate responses following isoproterenol challenge, but a significant (p < 0.05) 225% increase in the arrhythmic index (AI) was observed only in adrenergic-deficient hearts. These results show that adrenergic hormones may influence heart development by stimulating connexin 43 expression, facilitating atrioventricular conduction, and helping to maintain cardiac rhythm during a critical phase of embryonic development.

Highlights

► Adrenergic deficiency leads to slowed atrioventricular conduction in embryonic mouse heart. ► Adrenergic deficiency leads to decreased expression of Cx43 in the embryonic heart. ► Adrenergic deficiency does not alter Hcn4 or sarcomeric α-actinin expression in heart development. ► Adrenergic deficiency leads to increased susceptibility to arrhythmia in the embryonic mouse heart.

Introduction

Mice that lack the ability to produce the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to targeted disruption of the dopamine β-hydroxylase (Dbh) gene die at mid-gestation from apparent heart failure [25]. Structural formation of the heart was not markedly perturbed in the adrenergic-deficient embryos, though subtle abnormalities such as dilated atria and disorganized ventricular myofibrils were observed in the deficient group. In addition, blood pooling in major organs and slower in vivo heart rates led to the conclusion that heart failure was the likely cause of death in adrenergic-deficient embryos. The mechanism of action appears to be primarily through β-adrenergic receptor activation because isoproterenol (β-agonist) but not L-phenylephrine (α-agonist) could rescue the adrenergic-deficient (Dbh−/−) mouse embryos when supplied via the maternal drinking water [26]. Despite a relatively wide body of data on adrenergic mechanisms in the adult heart, only rudimentary information exists regarding adrenergic actions in the embryonic heart. A major gap in our current knowledge is how embryonic activation of β-adrenergic signaling specifically affects cardiac function and embryonic survival at these critical formative stages of development.

Independent studies have shown that the heart itself is a source of adrenergic hormones during early development [4], [8], [13], [14]. “Intrinsic Cardiac Adrenergic” (ICA) cells appear in the heart at about the time that it first starts to beat [4], [13]. There is a transient clustering of ICA cells in regions of the heart progressively associated with development of the cardiac pacemaking and conduction system, including the pacemaker cells in the sinoatrial node, the atrioventricular node, bundle of His, and Purkinje fibers [8]. Some of these transient ICA cells appear to differentiate into cardiac myocytes, including the specialized myocytes that serve as pacemaker cells in the sinoatrial and atrioventricular nodes as well as extensive labeling of myocytes throughout the ventricular conduction system [5]. These observations have led us to hypothesize that NE and/or EPI play a critical role in the embryonic development of the cardiac pacemaking and conduction system [6].

In the present study, we utilized the Dbh knockout mouse model to test the hypothesis that NE and EPI play a critical role in the development of the cardiac pacemaking and conduction systems. Our initial experiments evaluated the in situ expression of a key pacemaker channel protein, the hyperpolarization-activated cyclic nucleotide-modulated channel isoform 4 (Hcn4) [24], and a major gap junction protein responsible for fast ventricular conduction, connexin 43 (Cx43) [28], in adrenergic-competent and deficient embryos. We then used microelectrode arrays (MEAs) to evaluate electrical conduction, and videomicroscopy to examine heart rate and rhythm.

Section snippets

Animals

The Dbh mouse strain and collection of embryos used in this study has been described previously [25]. All animal procedures were performed in accordance with NIH guidelines and were approved by the University of Central Florida Animal Care and Use Committee. Most of our analyses were performed using embryonic day 10.5 (E10.5) mouse embryos because E10.5 is the latest stage of development when adrenergic-deficient embryos are still largely asymptomatic [25].

Immunofluorescence histochemistry

Dual immunofluorescent histochemical

Results

Since ICA cells have previously been identified in regions of the developing heart associated with conduction and pacemaking function [5], [8], we employed immunofluorescent histochemical staining to evaluate a key pacemaking protein (Hcn4) and a major gap junction protein (Cx43) important for the generation and propagation, respectively, of electrical signaling in adrenergic-competent and deficient embryonic hearts. As shown in Fig. 1, our results indicate that Cx43 immunofluorescent staining

Discussion

Initially, we aimed to examine protein staining intensity and distribution patterns for Cx43 and Hcn4 to determine if these were altered in adrenergic-deficient embryonic hearts around the time of their demise. Cx43 staining was less intense relative to that for sarcomeric α-actinin and Hcn4 in adrenergic-deficient hearts compared with competent controls at E10.5. Our co-immunofluorescent histochemical staining data show that was not true one day earlier at E9.5, where Cx43 expression was not

Acknowledgments

This work was supported by NIH Grants to SNE (HL078716) and JH (5RO1EB005459), and a postdoctoral fellowship (DGT) from the American Heart Association (0825395E).

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    1

    These two authors contributed equally to this work.

    2

    Department of Biology, Seminole State College of Florida, 100 Weldon Blvd., Sanford, FL 32773.

    3

    Wayne State University, Department of Pharmacology, 540 E. Canfield, Detroit, MI 48201.

    4

    Institute of Biology, Faculty of Natural Sciences, University of West Hungary, Károlyi Gáspár tér 4, Szombathely, H-9700, Hungary.

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