Accepted Manuscript

Intrinsic regulation of sinoatrial node function and the zebrafish as a model of stretch
effects on pacemaking

Eilidh A. MacDonald, Matthew R. Stoyek, Robert A. Rose, T. Alexander Quinn

PII: S0079-6107(17)30041-X
DOI: 10.1016/j.pbiomolbio.2017.07.012
Reference: JPBM 1243

To appear in: Progress in Biophysics and Molecular Biology

Received Date: 15 February 2017

Revised Date: 17 July 2017
Accepted Date: 21 July 2017

Please cite this article as: MacDonald, E.A., Stoyek, M.R., Rose, R.A., Quinn, T.A., Intrinsic regulation
of sinoatrial node function and the zebrafish as a model of stretch effects on pacemaking, Progress in
Biophysics and Molecular Biology (2017), doi: 10.1016/j.pbiomolbio.2017.07.012.

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 ACCEPTED MANUSCRIPT
TITLE

Intrinsic Regulation of Sinoatrial Node Function and the Zebrafish as a Model of Stretch Effects on

Pacemaking

PAPER TYPE

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Original Paper

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AUTHORS

Eilidh A MacDonald1

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Matthew R Stoyek1

Robert A Rose2

T Alexander Quinn1,3
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AFFILIATIONS
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1
Department of Physiology and Biophysics, Dalhousie University, Halifax, Canada
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2
Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada
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School of Biomedical Engineering, Dalhousie University, Halifax, Canada
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CORRESPONDING AUTHOR

T Alexander Quinn, PhD

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Department of Physiology and Biophysics

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Dalhousie University

5850 College St, Lab 3F

Halifax, NS B3H 4R2

Phone: +1 902 494 4349

Fax: +1 902 494 1685

Email: [email protected]

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KEY WORDS

Autoregulation; Heart Rate; Intracardiac Nervous System; Mechano-Electric Coupling; Natriuretic

Peptides; Stretch-Activated Channels

ABBREVIATIONS

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action potential AP

atrial natriuretic peptide ANP

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β-adrenergic receptors β-AR

B-type natriuretic peptide BNP

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beating rate BR

C-type natriuretic peptide CNP

calcium
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cation non-selective stretch-activated channels SACNS

data acquisition DAQ

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diastolic depolarisation DD
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electrocardiogram ECG
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‘funny’ current If

heart rate variability HRV

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hyperpolarization-activated cyclic nucleotide-gated HCN

intracardiac nervous system ICNS

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long-lasting L-type
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long-lasting Ca2+ current ICa,L

natriuretic peptide NP

natriuretic peptide receptor NPR

outward potassium currents Iz

potassium K+

ring-deleted atrial natriuretic peptide analogue cANF

sarcoplasmic reticulum SR

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sinoatrial node SAN

sodium Na+

sodium-calcium exchanger current INCX

transient T-type

transient Ca2+ current ICa,T

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transmembrane potential Vm

type 1 angiotensin II receptors At1

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ABSTRACT

Excitation of the heart occurs in a specialised region known as the sinoatrial node (SAN). Tight

regulation of SAN function is essential for the maintenance of normal heart rhythm and the response

to (patho-)physiological changes. The SAN is regulated by extrinsic (central nervous system) and

intrinsic (neurons, peptides, mechanics) factors. The positive chronotropic response to stretch in

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particular is essential for beat-by-beat adaptation to changes in hemodynamic load. Yet, the

mechanism of this stretch response is unknown, due in part to the lack of an appropriate experimental

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model for targeted investigations. We have been investigating the zebrafish as a model for the study of

intrinsic regulation of SAN function. In this paper, we first briefly review current knowledge of the

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principal components of extrinsic and intrinsic SAN regulation, derived primarily from experiments in

mammals, followed by a description of the zebrafish as a novel experimental model for studies of

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intrinsic SAN regulation. This mini-review is followed by an original investigation of the response of
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the zebrafish isolated SAN to controlled stretch. Stretch causes an immediate and continuous increase

in beating rate in the zebrafish isolated SAN. This increase reaches a maximum part way through a
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period of sustained stretch, with the total change dependent on the magnitude and direction of stretch.
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This is comparable to what occurs in isolated SAN from most mammals (including human),
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suggesting that the zebrafish is a novel experimental model for the study of mechanisms involved in

the intrinsic regulation of SAN function by mechanical effects.

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1. SINOATRIAL NODE FUNCTION

Excitation of the heart, which is responsible for its pumping action, begins within the organ itself -

originating from specialised tissue known as the sinoatrial node (SAN). It was more than 135 years

ago that Walter Gaskell first established the ‘myogenic’ origin of cardiac rhythm generation (Gaskell,

1882), and twenty-five years later that its anatomical location was identified (Keith and Flack, 1907).

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Yet, even more than a century on, mechanisms of spontaneous pacemaker activity are still debated

(DiFrancesco and Noble, 2012; Maltsev and Lakatta, 2012; Rosen et al., 2012).

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What is clear, derived primarily from mammalian studies, is that cardiac pacemaking involves a

robust, flexible system, integrating multiple contributors that allow rapid adaptation to changes in

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circulatory demand (Irisawa et al., 1993). Normal automaticity requires spontaneous diastolic

depolarisation (DD) of SAN cells. This involves a positive inward current (‘funny’ current, If), passing

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through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, acting in early diastole
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against reducing outward potassium (K+) currents (IK) (DiFrancesco, 2010). As diastole progresses,

depolarisation rate increases as trans-sarcolemmal calcium (Ca2+) flux through CaV3.1 transient (T-
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type) Ca2+ channels (ICa,T) and CaV1.3 long-lasting (L-type) Ca2+ channels (ICa,L) becomes dominant
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(Mesirca et al., 2015). Once transmembrane potential (Vm) crosses the activation threshold of CaV1.2
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ICa,L, an action potential (AP) is initiated (Mesirca et al., 2015). As this system supports rhythmic

spontaneous AP generation, it has been dubbed the ‘membrane-clock’ (Maltsev et al., 2006). SAN
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automaticity is further supported by spontaneous and CaV1.3 ICa,L-triggered (Torrente et al., 2016) Ca2+

release from the sarcoplasmic reticulum (SR), which results in an additional depolarising current,
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through the sodium (Na+)-Ca2+ exchanger (INCX, with one Ca2+ ion removed from the cell as three Na+
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ions enter) (Lakatta et al., 2008). As Ca2+ cycling remains rhythmic even after clamping of Vm, this

has been conceptualised as an independent pacemaker mechanism, and is referred to as the ‘Ca2+-

clock’ (Maltsev et al., 2006). While both of these ‘clocks’ have been shown to be independently

sufficient for SAN automaticity, they do not operate in isolation (the Ca2+-clock relies on trans-

sarcolemmal ion fluxes to alter Vm; the membrane-clock includes Ca2+ cycling), and thus they are now

considered as a coupled system (Vm/Ca2+-system), whose interactions contribute to robust pacemaking

even when individual mechanisms are impeded (Lakatta et al., 2010).

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2. SINOATRIAL NODE REGULATION

Studies in mammals have also shown that regulation of the membrane/Ca2+-system by factors

extrinsic (external or extracardiac) and intrinsic (internal or intracardiac) to the heart is essential for

the maintenance of normal SAN function and its response to physiological and pathophysiological

changes.

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2.1 Extrinsic Regulation

The primary extrinsic mediator of SAN activity is the central nervous system, acting through direct

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extracardiac sympathetic and parasympathetic innervation of intracardiac neural circuits and SAN

cells (Gordan et al., 2015). Terminals of sympathetic postganglionic neurons projecting from spinal

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cord nuclei to SAN cells release norepinephrine, which stimulates Gαs-coupled β-adrenergic receptors

(β-AR), causing an increase in intracellular cAMP levels inside the cell. As a result, the activity of

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cAMP-regulated effectors, such as HCN, delayed rectifier, and L- and (possibly) T-type Ca2+ channels
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are augmented, increasing the rate of DD and thus the frequency of SAN excitation and beating rate

(BR) (DiFrancesco, 2006; Irisawa et al., 1993). In contrast, terminals of parasympathetic neurons
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projecting from brainstem vagal motor nuclei to postganglionic parasympathetic neurons within the
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heart (which comprise the intracardiac nervous system, ICNS, involved in intrinsic regulation – see
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below) release acetylcholine. This release in turn stimulates intracardiac neuronal terminals to release

acetylcholine, activating Gαi/o-coupled cholinergic M2 muscarinic receptors on SAN cells, which

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reduces intracellular cAMP concentration, causing a decrease in depolarising currents and BR

(Fischmeister et al., 2006). At the same time, Gβγ subunits also contribute to a reduction in BR by
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directly activating G-protein-coupled inward rectifying potassium channels, resulting in membrane

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hyperpolarization (DiFrancesco, 1993).

It is worth noting that the function of the SAN is also affected (albeit to a lesser degree) by peptides

produced in endocrine glands (Gordan et al., 2015). Circulating epinephrine and norepinephrine (and

dopamine, when converted to epinephrine or norepinephrine) released by the adrenal glands will cause

an increase in BR through a similar mechanism to neurotransmitters released by sympathetic neurons.

2.2 Intrinsic Regulation

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Intrinsic SAN regulation is similarly governed by neuronal (ICNS) and peptidic (myogenic

peptides) mechanisms, as well as mechanical factors (tissue stretch).

2.2.1 Intracardiac Nervous System

The ICNS comprises a collection of efferent, interconnecting (local circuit), and afferent neurons

embedded within the myocardium, which has traditionally been viewed only as a relay station for

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extrinsic neural input. Work in the last two decades, however, has demonstrated that this view may be

overly simplistic, given the neurochemical complexity (Leger et al., 1999; Steele and Choate, 1994),

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distributed nature (Armour et al., 1997; Leger et al., 1999; Pauza et al., 2000; Yuan et al., 1994), and

capability for local information processing of neurons within the ICNS, as well as interactions not

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fully explained by this traditional understanding (Armour, 2008; Rajendran et al., 2016). It is now

known that the ICNS contains parasympathetic, sympathetic, and non-noradrenergic, non-cholinergic

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transmitters and modulators, so along with being a target of extrinsic neurons, it may also form
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intracardiac circuits important for the internal processing and integration of extrinsic inputs and for

intracardiac reflex control of cardiac function (Ardell and Armour, 2016).

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Functional and anatomical data have shown that intracardiac neurons receive inputs from extrinsic
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efferent parasympathetic preganglionic and sympathetic postganglionic neurons, local interneurons,

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and afferent neurons from other locations within the heart (Gagliardi et al., 1988; Smith, 1999). As a

result, even after acute (Gagliardi et al., 1988) and chronic (Smith et al., 2001) isolation of the heart
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from the central nervous system (decentralisation), the ICNS remains responsive to changes in the

cardiac environment. In addition, by simultaneous recordings of parasympathetic (vagal) and

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sympathetic (cardiopulmonary) nerves, it has been demonstrated that they can be co-activated
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(Koizumi et al., 1982; Kollai and Koizumi, 1979), and that adrenergic-cholinergic interactions

augment activity of the ICNS in a manner that does not occur in the presence of only one input (Smith,

1999). This may be less surprising when one considers that it appears as though less than 20% of

intracardiac neurons receive direct inputs from extrinsic nerves, and instead are acting as

interconnecting neurons (Armour, 2008).

While it is now appreciated that the ICNS plays an important role in the intracardiac integration of

autonomic inputs and possibly in local reflex regulation of cardiac function, details of the anatomical

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and functional distribution and connectivity of neurons within the ICNS, and an understanding of its

mechanisms of actions, are incomplete. In mammals, intracardiac neurons are widely distributed in

ganglia throughout both atria (Armour et al., 1997; Leger et al., 1999; Pauza et al., 2000; Steele and

Choate, 1994; Steele et al., 1996), yet the projection patterns of subpopulations of intracardiac neurons

to specific targets remain unknown [except to some degree in hearts of rat (Pardini et al., 1987) and

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guinea pig (Steele and Choate, 1994)]. This is complicated by the large number of neurons involved;

in the human heart the ICNS encompasses more than 106 neurons (Pauza et al., 2000), in the dog heart

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more than 105 neurons (Armour and Ardell, 2004), and even in the heart of smaller animals, such as

that of the guinea pig, ~2000 neurons (Leger et al., 1999). An additional difficulty stems from the fact

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that in mammals the ICNS is deeply embedded in, and distributed throughout, the myocardium, and

thus largely inaccessible for integrative studies. One solution to this problem is the use of reduced

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experimental preparations representing a subset of the ICNS, such as isolated tissue, to study the
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properties of control circuits in vitro (Ardell et al., 2014; Smith, 1999). This approach, however,

eliminates intracardiac neuronal connections, so allows only limited conclusions to be drawn

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regarding the role of localized circuitry in controlling overall cardiac function. Studies using
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alternative (non-mammalian) experimental models, such as the zebrafish, may provide a solution to
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this problem, as described below.

2.2.2 Myogenic Peptides

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Release of peptides contained in cardiac myocytes and endothelial cells within or in close

proximity to the SAN can act in a paracrine (or autocrine) fashion to modulate SAN function. The
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primary factors released by cells in the atria are natriuretic peptides (NPs), a family of hormones with
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a number of potent effects in the cardiovascular system (Kuhn, 2004; Potter et al., 2006). Several

members of the NP family of have been identified, including atrial NP (ANP), B-type NP (BNP) and

C-type NP (CNP). Each of these peptides is expressed (albeit to different extents) in the heart and are

present in both cardiomyocytes and cardiac fibroblasts (Moghtadaei et al., 2016).

Three NP receptors (NPR), called NPR-A, NPR-B and NPR-C, are responsible for mediating the

effects of NPs upon receptor binding and activation (Potter et al., 2006). NPR-A (which binds ANP

and BNP) and NPR-B (which is selectively activated by CNP) are guanylyl cyclase linked receptors

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that enhance cGMP signalling (Lucas et al., 2000). NPR-C (which binds all NPs with similar affinity)

is coupled to inhibitory G-proteins that inhibit adenylyl cyclase (and thus cAMP) signalling (Anand-

Srivastava, 2005; Rose and Giles, 2008).

NPs are best known for their capacity to regulate blood pressure and cardiovascular homeostasis as

a result of their ability to induce natriuresis, diuresis, and vasodilation and to modulate endothelial

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permeability (Kuhn, 2004; Potter et al., 2006). Yet, NPs have recently emerged as potent regulators of

cardiac electrophysiology and ion channel function (Perrin and Gollob, 2012), and have been

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implicated as potential contributors to arrhythmogenesis (Egom et al., 2015; Moghtadaei et al., 2016),

including inherited (due to mutation of the ANP gene) atrial fibrillation in humans (Abraham et al.,

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2010; Hodgson-Zingman et al., 2008; Hua et al., 2015).

In the SAN, NPs can potently regulate BR through direct effects on SAN cells (Azer et al., 2012;

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Rose et al., 2007; Springer et al., 2012). In isolated SAN myocytes, BNP and CNP each increase BR
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in association with increases in the rate of DD, but without differences in maximum diastolic Vm (the

effects on BR also occur in isolated hearts). Voltage clamp studies have demonstrated that the changes
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in BR with BNP or CNP administration result from increases in If and total ICa,L, along with shifts in
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the voltage dependence of channel activation. In NPR-C knockout mice, the stimulatory effects of
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BNP and CNP on BR, If, and ICa,L are indistinguishable from wildtype mice in basal conditions. In

contrast, the effects of BNP and CNP were reduced by the PDE3 inhibition, and the effects of BNP
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were antagonized by NPR-A blockade, illustrating that BNP and CNP increase SAN BR by activating

the guanylyl cyclase-linked NPR-A and NPR-B receptors and inhibiting PDE3 activity (Springer et
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al., 2012). Consistent with these findings, ANP has also been shown to elicit a cGMP-dependent
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increase in If in human atrial myocytes (Lonardo et al., 2004).

Although NPR-C does not affect SAN function in basal conditions, it does have effects in the

presence of β-AR activation (Azer et al., 2012; Springer et al., 2012). Specifically, in the presence of

isoproterentol, the ring-deleted ANP analogue cANF (which only activates NPR-C) dose-dependently

decreases BR by decreasing the rate of DD, effects that are absent in NPR-C knockout mice (Azer et

al., 2012). In contrast, BNP and CNP (which activate NPR-A/B along with NPR-C) increases BR in

the presence of submaximal doses of isoproterentol (10 nM), although effects are smaller than in basal

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conditions due to competition effects of NPR-A/B (increasing BR) and NPR-C (decreasing BR)

stimulation. In fact, in the presence of larger doses of isoproterentol (1 µM), BNP and CNP instead

decrease BR. In agreement with this observation, CNP and cANF can also decrease ICa,L in SAN

myocytes in the presence of high doses of isoproterentol (Rose et al., 2004). Collectively, these studies

demonstrate that NPs can modulate SAN function via NPR-A/B receptors (stimulatory) and NPR-C

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(inhibitory) and that these receptors elicit opposing effects. Because of this, NPs increase BR in some

conditions, but decrease BR in others, which is dependent on the extent of β-AR activation and the

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relative contribution of each NPR in varying physiological conditions (Azer et al., 2014; Azer et al.,

2012; Moghtadaei et al., 2016).

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While much of the focus regarding peptidic SAN regulation has been placed on the effect of NPs,

BR may also be affected by a host of other locally released factors (Beaulieu and Lambert, 1998). For

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instance, angiotensin II, which is locally synthesised by the cardiac renin-angiotensin system and is
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highly concentrated at the level of the SAN artery (Saito et al., 1987), has been shown to affect BR in

isolated SAN cells (Sheng et al., 2011), atrial preparations (Kobayashi et al., 1978), and intact animals
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(Lambert et al., 1991) via type 1 angiotensin II receptors (At1) (Lambert, 1995). Similarly, endothelin-
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1 (as well as -3), produced and secreted by endothelial cells and acting through ETA receptors, alters
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BR of isolated SAN cells (Ono et al., 2001; Tanaka et al., 1997) and atrial preparations (Ishikawa et

al., 1988; Ju et al., 2011; Saito et al., 1987), in a concentration and species dependent manner.
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2.2.3 Tissue Stretch

Throughout the heart, function is intrinsically regulated by feedback from the myocardium’s
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mechanical state to its electrical activity (mechano-electric feedback or coupling) (Quinn and Kohl,
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2016; Quinn et al., 2014). This intracardiac phenomenon is essential for adaptation to constantly

changing physiological conditions, and is important for both normal heart function (Quinn, 2015) and

as a contributor to arrhythmias in various cardiac diseases (Quinn, 2014; Quinn and Kohl, 2011a).

The SAN specifically responds on a beat-by-beat basis to changes in hemodynamic load (Quinn et

al., 2011a; Quinn and Kohl, 2012). First recognised in 1915 by Francis Bainbridge as an increase in

BR with atrial distension by intravenous fluid injection in anaesthetised dogs (Bainbridge, 1915),

similar effects have since been observed in a wide variety of vertebrates (Pathak, 1973), including

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human (Donald and Shepherd, 1978). Stretch-induced increases in BR occur also in the isolated

(decentralised) heart (Blinks, 1956), SAN tissue (Blinks, 1956; Deck, 1964), and SAN cells (Cooper et

al., 2000), and are insensitive to ablation of intracardiac neurons (Wilson and Bolter, 2002), block of

neuronal sodium channels (Chiba, 1977; Wilson and Bolter, 2002), and adrenergic and cholinergic

blockade (Blinks, 1956; Brooks et al., 1966; Chiba, 1977; Wilson and Bolter, 2002), indicating that

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non-neuronal, intracardiac mechanisms must be involved.

Microelectrode recordings in SAN tissue have demonstrated that stretch increases maximum

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diastolic and decreases maximum systolic Vm, and increases DD rate (Deck, 1964). Patch-clamp

recordings in axially stretched isolated SAN cells, combined with computational modelling, have

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demonstrated that stretch responses can be explained by a mechano-sensitive whole-cell current with a

reversal potential of -11 mV (Cooper et al., 2000), presumably carried by cation non-selective stretch-

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activated channels (SACNS). While the molecular identity of SACNS is still not known (Peyronnet et
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al., 2016), it is clear that they are rapidly-activating, with a reversal potential of 0 to -20 mV in cardiac

cells (Craelius et al., 1988; Guharay and Sachs, 1984), and that their block causes a reversible
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reduction of stretch-induced changes in BR (Cooper and Kohl, 2005). Interestingly, even if located
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outside of SAN cells in electrically-coupled fibroblasts (Quinn et al., 2016), SACNS can account for
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stretch-effects (Kohl et al., 1994), as fibroblasts possess SACNS (Stockbridge and French, 1988), are

coupled to SAN cells (Camelliti et al., 2004), and are depolarised by stretch (Kohl et al., 1994; Kohl
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and Noble, 1996).

Of course quantitative plausibility is no substitute for validation (Quinn and Kohl, 2011b), and
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stretch responses may also result from direct effects on mechano-sensitive components of the Vm/Ca2+-
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system. When expressed in oocytes, activation and deactivation rate of HCN channels are increased

with mechanical stimulation (Lin et al., 2007), along with current amplitude (Calloe et al., 2005).

Similarly, L-type Ca2+ (CaV1.2) (Calabrese et al., 2002; Lyford et al., 2002) and K+ (Kv1, Kv3, Kv7,

KvCa) (Morris, 2011) channels have been shown to be mechanically-modulated in expression

systems. Furthermore, axial stretch of ventricular myocytes causes an acute increase in the frequency

of spontaneous Ca2+ release events from the SR (Ca2+ sparks) via augmented ryanodine receptor open

probability (Gamble et al., 1992; Iribe and Kohl, 2008; Iribe et al., 2009; Prosser et al., 2011; Prosser

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et al., 2013). This increase in Ca2+ sparks may partly involve mechanically-induced mitochondrial Ca2+

release through the mitochondrial Na+-Ca2+ exchanger (Belmonte and Morad, 2008), a channel that

has been shown to alter BR in isolated SAN cells by altering Ca2+ spark frequency (Yaniv et al.,

2012). Thus, if any of the above effects occurs also in SAN cells, they may too contribute to stretch-

induced changes in BR.

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There is also evidence supporting an interaction between mechanical and autonomic BR

modulation. In intact animals (Bolter, 1994; Bolter and Wilson, 1999) and isolated atria (Barrett et al.,

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1998; Bolter, 1996; Wilson and Bolter, 2001), an increase in atrial pressure induces both BR

acceleration and a significant reduction in the percentage response to vagal stimulation. Vice versa,

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when BR is reduced by vagal stimulation the stretch-induced BR response is augmented, an

interaction that may be mediated by muscarinic K+ channels, as they have been shown to close with

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atrial distension (Han et al., 2010). In this way, parasympathetic control of BR may be continuously
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modified by atrial loading, further contributing to the stretch-induced adjustment of BR to fluctuations

in hemodynamic demand, helping prevent excessive slowing and diastolic distension, while
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maintaining cardiac output and adequate circulation (Brooks and Lange, 1977).
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Response of the SAN to stretch, however, differs in some species. While in most medium and large
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mammals the SAN responds to stretch with an increase in BR, in mouse there is instead a decrease in

BR with stretch (Cooper and Kohl, 2005). Thus, the mouse may not represent an appropriate species
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for the investigation of stretch-induced changes in SAN BR, which, as the primary genetically

modifiable experimental model available, has hindered definition of the (still unknown) mechanisms
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responsible for SAN stretch responses. As for integrative studies of the ICNS, the zebrafish may
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represent an alternative model to address current limitations of mammalian models.

3. ZEBRAFISH AS AN ALTERNATIVE MODEL FOR CARDIAC ELECTRO-

PHYSIOLOGICAL STUDIES

The zebrafish is becoming an increasingly powerful experimental model for the study of cardiac

electrophysiological mechanisms, as: (i) it has striking similarities to human, both genetically (almost

every cardiac gene has been identified to have a human ortholog with analogous function) and

functionally (comparable heart rate, AP morphology, and ion channel and Ca2+-handling protein

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expression and function); (ii) its genome has been fully sequenced and can be easily manipulated by

standard techniques; and (iii) its cardiac function can be observed in vivo [relevant studies

demonstrating these important aspects are reviewed in (Brette et al., 2008; Genge et al., 2016; Gut et

al., 2017; Liu et al., 2016; Nemtsas et al., 2010; Poon and Brand, 2013; Vornanen and Hassinen,

2016)].

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3.1 Advantages of the Zebrafish for the Study of SAN Function

In regards to the utility of zebrafish for the study of SAN function, it has been shown that

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pacemaker activity is clearly present, functions similarly (at least in some important respects) to

mammals, and is found in a ring-like structure at the location of the sinoatrial valve (the border

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between the sinus venosus and the atrium) (Arrenberg et al., 2010; Stoyek et al., 2015; Stoyek et al.,

2016; Tessadori et al., 2012). The first indication of the currents involved in generating SAN

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automaticity in the zebrafish came from the discovery of a mutation (slow mo) that caused a reduction
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in heart rate by affecting a hyperpolarization-activated inward current with the properties of If (Baker

et al., 1997; Warren et al., 2001), suggesting that the zebrafish SAN may employ similar pacemaking
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mechanisms to mammals. It has since been shown that SAN development in the zebrafish has
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similarities to mammals, occurring from Isl1- and tbx2b-expressing cells (Tessadori et al., 2012) under
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the control of shox2 (Blaschke et al., 2007; Hoffmann et al., 2013), with a dependence on Popdc1-3

(Schindler et al., 2012) and Fhf2a (Poon et al., 2016).

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Recently, the zebrafish has been used to overcome limitations of larger animal models to explore

both extrinsic autonomic, and intrinsic ICNS, regulation of the SAN (Stoyek et al., 2015). Although
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the relatively small size of the zebrafish heart presents its own technical challenges, it permits
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visualisation of, and accessibility to, the entire ICNS and its external inputs in the whole, intact organ.

Specifically, it has been shown that approximately 90% of all intracardiac neurons (mean total = 197 ±

23) present in the zebrafish heart are located within the SAN region. This neuronal plexus is

innervated by extrinsic axons from the left and right vagal nerves, with terminals concentrated near

intracardiac ganglia, adjacent to cells expressing HCN4 (the principal HCN isoform contributing to

DD in zebrafish and mammals). As would be predicted by the traditional view of the ICNS, it was

found that the vast majority of intracardiac neurons in the SAN region are cholinergic; however some

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neurons contain tyrosine hydroxylase, indicating that they are capable of synthesizing norepinephrine,

so could instead be adrenergic. This finding does not conform to the general understanding of

peripheral autonomic nervous system organisation, in which postganglionic sympathetic cell bodies

are located outside of the organs that they innervate (Nilsson, 1983; Nilsson and Holmgren, 1994). In

addition to cholinergic and adrenergic neurotransmitters, other ‘non-classical’ autonomic transmitters

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have been detected within the zebrafish ICNS. Neuronal nitric oxide synthase exists in a small

percentage of SAN intracardiac neurons, as well as vasoactive intestinal polypeptide in nerve

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terminals. The role of neurons secreting these transmitters in the zebrafish heart is not clear, but it has

been suggested that intracardiac release of nitric oxide is involved in modulating cardiac effector cells

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(Tota et al., 2005).

A particularly intriguing finding from studies in zebrafish is the detection of what appears to be

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neuronal cell bodies in the ICNS, with axons projecting back to the central nervous system. These
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intracardiac neurons may represent afferent neurons located within the heart. Anatomical evidence for

such an arrangement has been shown previously in the rat heart (Cheng et al., 1997) and
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electrophysiological recordings have been made from presumptive intracardiac afferent neurons in the
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canine heart (Ardell et al., 1991). If these labelled neurons in the zebrafish heart are in fact afferent
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neurons, they may provide cardio-sensory inputs to local, intracardiac control circuits, along with

centrally-mediated reflex control (Stoyek et al., 2016).

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Subsequent to these anatomical findings, the zebrafish isolated heart with intact extrinsic

innervation has been established as a novel model for studies of autonomic control of SAN function
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(Stoyek et al., 2016). By combining electrocardiogram (ECG) and voltage optical mapping recordings
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with extrinsic nerve stimulation and pharmacological interventions, it has been demonstrated that

discrete neural pathways modulate SAN function through adrenergic and cholinergic mechanisms in a

similar manner to mammals [effects which are present as early as 4·days post-fertilisation (Schwerte et

al., 2006)]. Using immunohistochemistry, it was further shown that SAN cells express adrenergic and

cholinergic receptors, so are under direct influence of the ICNS.

Combined, these studies using zebrafish as an experimental model represent a first step towards a

more comprehensive understanding of integrated autonomic control of SAN function. This model may

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now be utilised to identify specific relationships between subpopulations of intracardiac neurons and

their targeted effector cells, which have been elusive in other experimental models, to discover basic

mechanisms of intracardiac neural SAN regulation.

There is also evidence to suggest that the zebrafish may be a useful experimental model for

studying the influence of NPs or stretch on SAN function. Intracardiac release of NPs in response to

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physiological stimuli, and their respective intracardiac receptors in zebrafish are comparable to

mammals (Becker et al., 2014; Bendig et al., 2006), and preliminary experiments by our group

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indicate that SAN BR responses are similar to that seen in mammals. Exposure to NPs (ANP 1-28:

Cat. # H-2100; BNP-32: Cat. # H5968; CNP 32-53: Cat. # H-1296; Bachem, Torrance, USA) acutely

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(time to peak change: 21 ± 3 s) increases BR in zebrafish isolated SAN (ANP: 24.3 ± 15.8%; BNP:

16.9 ± 10.2%; CNP: 23.1 ± 6.2%; n = 3 and p < 0.05 by two-tailed, paired Student’s t-test, for each).

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Stretch-induced electrophysiological responses seen in the atria and ventricles of human are also
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observed in the zebrafish, and of particular note for the SAN, it has been (anecdotally) noted that

stretch acutely alters SAN BR (Werdich et al., 2012).

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3.2 Limitations of the Zebrafish for the Study of SAN Function

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While clear similarities in SAN function and its regulation exist between mammals and zebrafish,
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many fundamental mechanisms remain to be explored, and as with any experimental model, known

limitations must be considered.

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There are clear morphological and systemic differences between zebrafish and mammalian hearts

[e.g., 2 vs. 4 chambers, ring- vs. sheet-like SAN structure, low vs. high blood pressure (Hu et al.,
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2001)], but more importantly for the study of SAN function, much of what is known about basic
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mechanisms from mammalian studies has yet to be studied in the zebrafish. While studies of mutant

zebrafish have shown that If is involved in SAN automaticity (Baker et al., 1997; Warren et al., 2001),

the involvement of other membrane-clock mechanisms (i.e., CaV3.1 ICa,T and CaV1.3 ICa,L) and the

Ca2+-clock, as well as the relative importance of these two systems, has not been explored. It may be

that pacemaking in zebrafish is primarily driven by If and that other membrane- and Ca2+-clock

mechanisms play a minimal (or non-existent) role. It is likely, however, that CaV3.1 ICa,T is involved,

as both atrial and ventricular zebrafish cardiomyocytes display a robust current (Nemtsas et al., 2010),

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and possibly also CaV1.3 ICa,L, which is expressed in zebrafish inner ear hair cells (Sidi et al., 2004)

(although its presence in the zebrafish heart has not yet been established). The sparsity of SR in

zebrafish myocardium (Hu et al., 2001), on the other hand, points to the potential absence of a Ca2+-

clock contribution to SAN automaticity. This is further supported by the limited Ca2+ release with

excitation in zebrafish ventricular cells (Bovo et al., 2013) [along with a lack of transverse tubules

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(Brette et al., 2008)], although this remains controversial, as others have shown a strong dependence

of contractile force on SR Ca2+ release (Haustein et al., 2015) and the existence of Ca2+ sparks with

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characteristics similar to mammals (Llach et al., 2011). At the same time, however, the zebrafish

expresses a variety of unique Na+-Ca2+ exchanger paralogs (Marshall et al., 2005), with a higher INCX

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than in mammals (Zhang et al., 2011), which could compensate for a lower spontaneous SR Ca2+

release, allowing a significant contribution of the Ca2+-clock to SAN automaticity.

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The presence of a host of paralogous genes in zebrafish [which is a result of the zebrafish having
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undergone two whole genome duplications after splitting from the phylogenetic pathway that includes

humans (Howe et al., 2013)] is another potential limitation of the use of zebrafish for understanding
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mammalian cardiac electrophysiology, as the duplicated genes are often involved in sub-, neo-, or
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unknown functions. Specific to the SAN, HCN4 (the gene primarily responsible for If. in zebrafish and
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mammals) also has numerous paralogs in zebrafish (Jackson et al., 2007). While the functional impact

of these paralogous genes is unclear, they may have important consequences for characteristics of If.
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One possibility is that function of the various HCN4 isoforms in zebrafish vary with temperature,

which could be an important evolutionary adaptation allowing the ectothermic zebrafish to maintain
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SAN function over a broad range of temperatures.

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The effect of temperature itself is another important consideration. It has been shown that cardiac

electrophysiology in the zebrafish is extremely temperature-sensitive. In the SAN, cooling causes a

reduction in the rate of DD, resulting in a 4% decrease in BR per °C drop in temperature (Q10 = 1.67),

and has further direct (i.e., BR-independent) effects on AP duration (Lin et al., 2014), highlighting the

importance of tight temperature control for experimental studies.

3.3 Summary

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Thus, the zebrafish appears to retain all critical components of intrinsic SAN regulation seen in

mammals (keeping in mind known limitations), making it a potentially powerful experimental model

for studies of underlying mechanisms. However, as the SAN response to stretch in the zebrafish has

not yet been systematically investigated, our goal was to examine the effects of controlled stretch on

BR of the zebrafish isolated SAN, in order to evaluate its utility as a novel model for the study of

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intrinsic regulation by mechanical effects.

4. METHODS

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4.1 Ethical Approval

All experimental procedures were approved by the Dalhousie University Committee for Laboratory

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Animals and followed the guidelines of the Canadian Council on Animal Care. Details of

experimental protocols have been reported following the Minimum Information about a Cardiac

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Electrophysiology Experiment (MICEE) reporting standard (Quinn et al., 2011b), see online
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repository (https://www.micee.org/?q=node/00001379).

4.2 SAN Preparation

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Adult (6-12 months post-fertilisation) wild-type (AB) zebrafish were euthanised with tricaine (2
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mM; E10521, Sigma-Aldrich, Oakville, Canada) in Tris-buffered (pH 7.4; BP152, Fisher Scientific,
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Ottawa, Canada) room temperature tank water until there was no response to fin pinch. Fish were then

placed into a Sylgard-lined dish (DC 170, Dow Corning, Midland, USA) filled with Krebs-Henseleit
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solution (containing [in mM]: 120 NaCl, 4.7 KCl, 26 NaHCO3, 1.4 NaH2PO4, 1.0MgCl2, 1.8 CaCl2,

5.0 Glucose; osmolality: 300 ± 5 mOsm/kg; pH: 7.40 ± 0.05) bubbled with carbogen (95% O2, 5%
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CO2) and maintained at 28.0 ± 0.2 °C (physiological zebrafish temperature) with a temperature-
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controlled (TC-344C, Warner Instruments, Hamden, USA) warmed platform (WP-16, Warner

Instruments). A ventral midline incision was made through the body wall and a block of tissue

encompassing the ventral aorta, ventricle, atrium, and sinus venosus was removed and placed in a

separate dish. In the two-chambered zebrafish heart, the SAN is arranged in an oval ring that

surrounds the orifice between the sinus venosus and the atrium. To expose the SAN, the ventricle was

removed and the atrium cut open and pinned flat. A custom suction microelectrode (1.00/0.58 mm

outer/inner diameter; 1B100 and MPH6R10, World Precision Instruments, Sarasota, USA) connected

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to an ECG amplifier (Animal Bio Amp, ADInstruments, Colorado Springs, USA) was positioned with

a three-axis rack and pinion stage (62041, Edmond Optics, Barrington, USA) at the edge of the atrium

and suction applied with a 1 ml syringe to measure the local ECG. Bath temperature was measured

using a thermocouple (T-type pod, ADInstruments). Temperature and ECG signals were recorded at

2000 Hz using a data acquisition (DAQ) device (PowerLab, ADInstruments) controlled by LabChart

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(ADInstruments; Figure 3).

4.3 SAN Stretch

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Two custom-made micro-sized hooks (~100 µm in diameter; Fig. 1B) fashioned from single-barrel

borosilicate glass capillaries (2.00/1.12 mm outer/inner diameter; 1B200F, World Precision

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Instruments) were coupled with microelectrode holders (MPH110, World Precision Instruments) to

independent three-axis hydraulic micromanipulators (for fine positioning; MHW-103, Narishige

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International, East Meadow, USA) mounted on piezoelectric linear translators (for application of sub-
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nanometer resolution stretch; P-621.1CD, Physik Instrumente, Auburn, USA) fixed to stages on a rack

and pinion track (for coarse positioning; 56798, Edmond Optics). The hooks were inserted opposing
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each other into the SAN ring in the long- or short-axis direction (n=13 for each; Fig. 2A), such that the
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tissue rested within the loops of the hooks (i.e., without tissue puncture/damage), using the
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micromanipulators under an upright microscope (BX63, Olympus, Richmond Hill, Canada) with a 5×

objective (Fig. 1A). Piezoelectric position was controlled by a servo controller (E-665.CR, Physik
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Instrumente) driven by a DAQ device (USB-6361, National Instruments, Austin, USA) with custom

routines developed in LabView (National Instruments) to apply precise magnitudes of stretch (10, 25,
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or 50%; Fig. 2B-C) to the SAN.

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4.4 Experimental Protocol

Preparations with hooks in either the long- or short-axis direction were left to equilibrate for 30 min

to ensure a stable baseline BR. The hooks were separated using the micromanipulators to the point at

which each hook made contact with, and just began to stretch the SAN, but did not affect BR. The pre-

stretch distance between the hooks was measured and this distance was increased by 10% at a rate of 1

mm/s by moving both piezoelectric translators. Stretch was maintained for 30 s followed by return to

the pre-stretch distance at the same rate as stretch. After 120 s of rest the stretch protocol was repeated

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three times (Fig. 3), followed by a repetition of the entire stretch procedure with 25 and 50% increase

in inter-hook distance (for a total of 12 stretches). The two piezoelectric translator positions were

recorded at 2000 Hz with the DAQ device.

4.5 Data Analysis

Data analysis was performed using custom routines in Matlab (MathWorks, Natick, USA). BR was

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calculated from the peaks of the ECG signal and stretch percentage from the two piezoelectric

translator positions (Fig. 3). Baseline BR was measured as the average BR during the rest period prior

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to each stretch. With stretch, average BR was measured over the entire 30 s stretch period and peak

BR was averaged over eleven beats centred on the maximum BR. Time to peak response was

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measured as the time from stretch initiation to the maximum BR. Heart rate variability (HRV) was

assessed during the baseline and stretch periods by calculating the standard deviation of BR and the

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root mean square of the successive differences in BR.
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Statistical analysis was performed in SPSS (IBM, Armonk, USA). Values are presented as mean ±

standard error of the mean, with a p-value < 0.05 indicating significance. Three factors related to the
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stretch response were considered: (i) stretch magnitude (10 vs. 25 vs. 50%), (ii) temporal response
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(average vs. peak), and (iii) direction of stretch (long- vs. short-axis). Two-way repeated-measures
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ANOVA was used to assess: (i) the effect of stretch magnitude, by comparing baseline, average, and

peak BR; (ii) the temporal response, by comparing average and peak percentage change in BR; and
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(iii) HRV at baseline and with stretch, at each stretch magnitude in the long- and short-axis direction.

Two-way mixed ANOVA was used to assess the influence of stretch direction by comparing peak
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percentage change in BR for long- and short-axis stretch at each stretch magnitude. One-way repeated
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measures ANOVA was used to compare time to peak BR at each stretch magnitude in the long- and

short-axis direction. Greenhouse-Geisser correction for non-sphericity was applied when appropriate,

significant main effects were analysed by Bonferroni post hoc tests, and when the interaction effect

was significant, simple effects were analysed by Student’s t-test. SAN ring diameter and average time

to peak BR for long- and short-axis stretch were compared by two-tailed, unpaired Student’s t-test and

initial and final temperature were compared by two-tailed, paired Student’s t-test. The relationship

between the peak change in BR and its baseline value was assessed by linear correlation.

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5. RESULTS

Before commencing the stretch protocol, all isolated SAN preparations displayed regular

spontaneous beating (140 ± 5 beats/min, bpm; n = 26). Before long-axis stretch, the diameter of the

SAN ring was 220 ± 11 µm. With stretch in this direction, BR immediately began to increase (Fig. 3),

reaching a peak on average after 16 ± 1 s, which did not vary with stretch magnitude (10%: 14.8 ± 1.5

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s; 25%: 16.5 ± 1.7 s; 50%: 15.6 ± 2.0 s; n = 13, p = 0.68). For short axis stretch, the initial diameter of

the SAN ring was not different than for long-axis stretch (220 ± 13 µm, p = 0.989) and, the time to

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peak BR was also not dependent on stretch magnitude (10%: 17.8 ± 1.5 s; 25%: 19.8 ± 1.6 s; 50%:

18.3 ± 2.0 s; n = 13, p = 0.62), although the average time to peak BR (18.6 ± 1.0 s) was longer than for

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long-axis stretch (p = 0.03). Upon release, BR began to immediately decrease, returning to baseline

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over a similar time course as the increase with stretch (Fig. 3).

The temporal effect-stretch magnitude interaction was significant for both long- and short-axis
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stretch (p = 0.042 and 0.021, respectively). For long-axis stretch, peak BR after stretch was greater
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than baseline for all stretch magnitudes, while average BR was only greater than baseline for 50%

stretch (Fig. 4). For short-axis stretch, both average and peak BR were greater than baseline for all
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stretch magnitudes, and both were greater for 50% stretch than for 10 or 25%. Across the stretch
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protocol there was no change in baseline BR (Fig. 4) or temperature (initial: 28.4 ± 0.2 °C, final: 27.9

± 0.2 °C¸ p = 0.18), and the peak change in BR was not related to baseline BR (all R2-values < 0.1).
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Figure 5 shows the absolute peak and average change in BR for long- and short-axis stretch,

demonstrating the temporal- (average vs. peak) and stretch direction- (long- vs. short-axis) dependence
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of stretch effects. For comparison, changes in BR were normalised to the pre-stretch value by
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calculating the percentage change in BR. The temporal effect-stretch magnitude interaction was

significant for short-axis stretch only (p = 0.039), with a greater peak than average percentage change

in BR for all stretch magnitudes (Fig. 6), reflecting the continuous increase in rate during the stretch

period. Further, the direction-stretch magnitude interaction was significant (p = 0.013), with a greater

peak percentage change in BR for 25% stretch in the short compared to long-axis direction (Fig. 7).

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For HRV measured by the standard deviation of BR, the stretch-stretch magnitude interaction

effect was significant for both long- and short-axis stretch (p = 0.003 and 0.016, respectively). HRV

decreased for 10% stretch in both the long- and short-axis direction and increased for 50% stretch in

the long-axis direction (Fig. 8). No differences were seen in the root mean square of the successive

differences of BR.

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6. DISCUSSION

We performed a systematic investigation of the response of the zebrafish isolated SAN to varying

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degrees of controlled stretch. Overall, it was found that stretch causes an immediate and continuous

increase in BR, which reaches a maximum part way through a period of sustained stretch, and the

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extent of which is dependent on the magnitude and direction of stretch. This chronotropic response is

comparable to what occurs in isolated SAN from most mammals (although not from mice), suggesting

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that the zebrafish may represent a novel experimental model for the study of mechanisms involved in
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the intrinsic regulation of SAN function by mechanical effects.

6.1 Determinants of the Response to Stretch

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The increase in BR with stretch in our experiments was magnitude-, direction-, and time-
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dependent. The importance of stretch magnitude is well established from previous work (Quinn and
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Kohl, 2012), yet it remains unknown whether: (i) the key mechanical parameter is strain (stretch)

(Kamiyama et al., 1984; Sanders et al., 1979), stress (tension) (Arai et al., 1996; Brooks et al., 1966;
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Chiba, 1977; Cooper and Kohl, 2005), or a combination of both (Lange et al., 1966); (ii) the response

is dependent (Brooks et al., 1966; Lange et al., 1966) or independent (Chiba, 1977) of stretch rate; or
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(iii) there is an effect of the spatial nature of stretch (i.e., linear, biaxial, or concentric) (Deck, 1964).
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The dependence on stretch direction in our experiments may reflect an influence of anisotropic tissue

properties, which could affect the spatial distribution of stress and/or strain across the SAN tissue. To

address this, it is essential that future experiments include the measurement of applied force during

stretch, which will also to provide insight into the dependence of the increase in BR on stress vs.

strain. It may also help reduce the relatively large inter-subject variability of the stretch-induced

change in BR seen in our experiments, as pre-stretch hook separation may be more consistently

determined by pre-stretch force.

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Our experiments demonstrated that while BR immediately began to increase with stretch, there was

a delay to the peak response, and that the return of BR to baseline after release occurred over a similar

timescale. This finding could also be explained by the mechanical properties of the SAN, with visco-

elastic dampening of the translation of applied stress into tissue stretch, as well as its decline after

release. Alternatively, it may point to the involvement of a (more slowly activating) second messenger

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system in the stretch-induced response to BR, which requires time for removal after a return to normal

tissue length. An important consequence of this temporal aspect of the stretch response is that the

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change in BR averaged over the entire stretch period will be lower than the peak response, which

complicates comparison of previous studies, which involved different stretch durations and

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measurements of BR.

The temporal nature of the stretch response also highlights the importance of stretch timing. In

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ventricular myocytes, it has been shown that stretch effects on the AP are phase-dependent (Calkins et
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al., 1991; Franz et al., 1992; Hansen et al., 1990; Nishimura et al., 2008). This means that the timing of

stretch application must be controlled to mimic desired physiological and pathophysiological states. In
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the case of the SAN, under normal conditions stretch will be greatest at the end of atrial filling, which
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is when SAN cells are moving towards AP initiation. In this way, ‘mechanical priming’ of SAN cells
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could adjust function on a beat-by-beat basis to diastolic load, contributing to the matching of cardiac

output (BR × stroke volume) to venous return. In pathophysiological conditions that involve
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dyssynchronous contraction, on the other hand, systolic/early diastolic stretch could occur, which will

affect cell repolarisation, potentially slowing BR and destabilising function. Past studies (including the
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present investigation), however, have utilised sustained (and often excessive) stretch. Studies
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investigating the effects of stretch timed with distinct phases of the cardiac cycle are needed to

understand the importance of the chronotropic response to stretch.

Phase-dependent effects may also explain the observed species-dependence of the response to

sustained stretch [i.e., increased BR in most mammals, but a decrease in mice (Cooper and Kohl,

2005)]. Assuming activation of SACNS is primarily responsible for changes in BR in both cases, in

SAN from animals with APs characterised by a relatively slow upstroke and prominent plateau-like

early repolarisation phase (with Vm moving away from the reversal potential of SACNS [~-10 mV]
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during a larger proportion of each cycle), sustained stretch will more often accelerate changes in Vm

(by pulling Vm in the direction of the reversal potential), thus increasing BR (Fig. 9A). In SAN from

mice (with faster upstrokes, often carried by a mix of Na+ and Ca2+ currents (Lei et al., 2007), and a

swift initial repolarisation), on the other hand, sustained stretch will do the opposite (Fig. 9Bs). This

theory fits with our results found in the zebrafish, as zebrafish AP morphologies are closer to large

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mammals than mice (Nemtsas et al., 2010), so would be expected to have an increase in BR with

sustained stretch (Fig. 9C). One should use caution, however, when generalising the shape of SAN

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APs from different species, as heterogeneity in SAN cell electrophysiology results in spatially-varying

AP morphologies (Boyett et al., 2000; Monfredi et al., 2010), resulting in an overlap between mice

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and other mammals. While SAN cells from larger mammals generally display longer APs, central

SAN cells from mice have very little detectable Na+ current and slower upstrokes, while peripheral

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SAN cells in larger mammals often display rapid upstrokes. It is difficult to predict how SAN cell
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heterogeneity will contribute to the stretch response in the intact SAN; a spatial assessment of APs in

the zebrafish SAN may help determine its important. That said, from the chronotropic response
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measured in the present work, it appears that the zebrafish represents an appropriate experimental
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model for the investigation of the effects and relevance of timed SAN stretch, and to test the species-
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dependence hypothesis.

6.2 Mechanisms of the Response to Stretch

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As SACNS activity in myocytes can account for the electrophysiological changes that occur with

stretch of the SAN, and their pharmacological block causes a reversible reduction of the stretch-
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induced change in BR (Cooper and Kohl, 2005), they have been implicated as the primary mediator of
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stretch effects. Yet, while a long list of candidates exist, their molecular identity remains unknown

(Peyronnet et al., 2016). As stretch responses are present in zebrafish, and powerful tools exist for

their genetic modification, they may represent a useful experimental model for determining the

identity of SACNS. Of course, mechanisms other than SACNS activation in myocytes may be involved

in stretch responses. For instance, SACNS in fibroblasts, if coupled to myocytes, may contribute. Here

again the zebrafish may be a powerful experimental tool, as by cell-specific expression of genetically-

encoded voltage-sensitive fluorescent proteins one can probe the role of different cell types to

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electrophysiological responses (Quinn et al., 2016). Fluorescent-based optical mapping techniques

may also prove useful for investigating the effects of stretch on AP morphology, Ca2+ handling, or the

pattern of SAN excitation.

Stretch effects may also be conferred by mechano-sensitivity of other components of the Vm/Ca2+

pacemaking system, such as HCN channels or internal Ca2+ handling, or other intrinsic regulatory

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pathways, such as interactions with autonomic BR modulation (Barrett et al., 1998; Bolter, 1994;

Bolter, 1996; Bolter and Wilson, 1999; Wilson and Bolter, 2001) or local stretch-induced release of

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NPs (de Bold et al., 1996). In the zebrafish these possibilities may be investigated using genetic or

pharmacological techniques, and may further benefit from computational modelling for quantitative

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assessment of their plausibility, experimentally-testable prediction and hypothesis generation, and

ultimately integration of findings and their projection across relevant species and spatial/temporal

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scales (Muszkiewicz et al., 2016; Quinn and Kohl, 2013).
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6.3 Relevance for Normal and Pathophysiological Function

Intrinsic regulation of SAN function by mechanical factors is an area which deserves greater
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attention, as in vivo the SAN is continuously subjected to cyclic variations in hemodynamic load,
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while isolated cells – the principal experimental model for insight into the molecular mechanisms of
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pacemaker function – have mostly been studied in unloaded conditions. For instance, it appears that

physiological loading may be essential to normal SAN automaticity, as slack tissue often shows no or
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irregular rhythm, while moderate stretch can restore normal activity (Brooks et al., 1966). This

apparent requirement for a minimum mechanical stimulus is also apparent during ontogenetic
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initiation of the first heartbeat, as fluid pressure build-up in the quiescent cardiac tube appears to be
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required for the initiation of its spontaneous activity (Chiou et al., 2016; Rajala et al., 1977).

Moreover, the adjustment of BR to diastolic filling is essential for the matching of cardiac output (BR

× stroke volume) to changes in venous return.

The mechanical regulation of BR works, however, only within a particular range of stretch. After

an initial acceleration in BR, excessive stretch can unbalance pacemaker function, inducing

arrhythmias (Brooks et al., 1966; Hoffman and Cranefield, 1960). This pathophysiological response

may be important in diseases associated with atrial volume overload (Morton et al., 2003; Sanders et

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al., 2003; Sparks et al., 1999), atrial fibrillation (Elvan et al., 1996; Kumagai et al., 1991), advanced

age (Mohler and Anderson, 2008; Rubenstein et al., 1972), or other cardiac pathologies (Akoum et al.,

2012; Bockstall and Link, 2012; Kottkamp, 2012; Nakao et al., 2012) that are accompanied by SAN

dysfunction, especially in cases where increased fibrosis will alter SAN mechanical properties and the

distribution of stretch (interestingly, with similar mechanical load, changes in BR are greater in SAN

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preparations from younger animals (Deck, 1964), in which generally less fibrosis is present).This will

be further exacerbated if fibroblast mechano-sensitivity plays a role in SAN stretch-responses.

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Another potentially important consequence of changes in SAN mechanical loading is impacts on

HRV. HRV is generally thought to be an index of autonomic function and decreased HRV is an

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independent predictor of cardiac morbidity and mortality in patients suffering from various forms of

heart disease (Task Force of the European Society of Cardiology and the North American Society of

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Pacing and Electrophysiology, 1996). It has been shown previously that in patients with atrial overload
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secondary to pulmonary arterial hypertension (McGowan et al., 2009), or in anesthetised pigs with

targeted right-atrial distension (Horner et al., 1996), HRV is reduced. In agreement with these previous
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findings, we found that 10% stretch was associated with a decrease in HRV. With greater stretch (50%
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in the short-axis direction), however, HRV was increased. The reason for this discrepancy may relate
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to differences in stretch characteristics (for instance stretch duration, which was an order of magnitude

shorter in our experiments than in the pig), chronic vs. acute effects, or preparation- (isolated vs. intact
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heart) / species-related differences. Intriguingly, as we used isolated, and thus decentralised hearts in

our experiments, changes in HRV may have represented changes in intrinsic (rather than centrally-
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mediated) ICNS-based regulation of SAN function.

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7. CONCLUSION

The zebrafish isolated SAN responds to stretch in a similar manner to human (and most other

mammals), with an immediate magnitude-dependent increase in BR. This is an important mechanism

for intrinsic regulation of SAN function, which allows the heart to adapt to changes in hemodynamic

load on a beat-by-beat basis. Yet, the mechanism of this positive chronotropic response is unknown,

due in part to the lack of an appropriate genetically modifiable species for targeted investigations. The

zebrafish, with its functional and genetic similarities to human, and the relative ease of its genetic

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modification, represents an alternative experimental model for the study of intrinsic regulation of SAN

function.

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ACKNOWLEDGEMENTS

This work was supported by the Natural Sciences and Engineering Research Council of Canada

(RGPIN-2015-04759 and RGPIN-2016-04879 to TAQ), the Canada Foundation for Innovation

(project number 32962), and the Dalhousie Medical Research Foundation. TAQ is a National New

Investigator of the Heart and Stroke Foundation of Canada.

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FIGURE LEGENDS

Fig. 1. (A) Experimental setup showing three-axis hydraulic micromanipulators mounted on

piezoelectric linear translators for positioning and controlled manipulation of micro-sized hooks

inserted into the zebrafish isolated sinoatrial node (SAN) ring, with electrocardiogram (ECG)

measured by a suction microelectrode. This was performed in a heated and bubbled bath under an

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upright microscope with a 5× objective. (B) Custom-made micro-sized hooks fashioned from single-

barrel borosilicate glass capillaries.

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Fig. 2. (A) Zebrafish isolated atrium pinned flat to expose the oval sinoatrial node (SAN) ring

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(outlined by green dashed line), in which glass hooks have been inserted opposing each other in the

short-axis direction (indicated by red dashed line, with long axis indicated by blue dashed line) and

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separated to the pre-stretch position, with ECG measured by a suction microelectrode. (B) 25% and
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(C) 50% short-axis SAN stretch.
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Fig. 3. Representative electrocardiogram (ECG), temperature (TEMP), beating rate (BR), and stretch
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magnitude (STRETCH) signals for four repetitions of 25% short-axis stretch.

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Fig. 4. Baseline, average, and peak beating rate for 10, 25, and 50% stretch in the (A) long-axis and
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(B) short-axis direction. *p<0.05 for average vs. baseline and #p<0.05 for 50% vs. 10 or 25%.
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Fig. 5. Absolute average and peak change in beating rate (BR) for 10, 25, and 50% stretch in the long-
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axis and short-axis direction.

Fig. 6. Average and peak percentage change in beating rate (BR) for 10, 25, and 50% stretch in the

(A) long-axis and (B) short-axis direction. *p<0.005 for peak vs. average.

Fig. 7. Peak percentage change in beating rate (BR) for 10, 25, and 50% stretch in the long-axis and

short-axis direction. *p<0.05 for short-axis vs. long-axis.

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Fig. 8. Standard deviation (SD) of beating rate (BR) at baseline and during 10, 25, and 50% stretch in

the (A) long-axis and (B) short-axis direction. *p<0.05 for stretch vs. baseline.

Fig. 9. Theoretical effects of cation non-selective stretch-activated channels (SAC,NS) on the

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sinoatrial node action potential of (A) rabbit, (B) mouse, and (C) zebrafish. Experimental membrane

potential recordings from each species show the relation of electrophysiological parameters

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(maximum diastolic and systolic membrane potential, MDP and MSP, respectively) and SAC,NS

reversal potential (ESAC,NS). The time periods during which SAC,NS activity would either accelerate

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(↑∆V) or slow (↓∆V) changes in membrane potential are indicated. In rabbit and zebrafish, SAC,NS

would accelerate changes in membrane potential during ~70 and 80% of the action potential,

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respectively, while in mouse this would occur over only ~45% of the action potential. Adapted (with
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addition of zebrafish recording) from (Cooper and Ravens, 2011), with permission.
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