draft · 2018. 8. 3. · draft cjpp-2018-0115_r1 - 2 abstract the hyperpolarization-activated...

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HCN channels: pathophysiological, developmental and

pharmacological insights into their function in cellular excitability

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2018-0115.R1

Manuscript Type: Review

Date Submitted by the Author: 02-May-2018

Complete List of Authors: Spinelli, Valentina; Universita degli Studi di Firenze Scuola di Scienze della

Salute Umana, Department of Neurosciences, Psychology, Drug Research and Child Health Sartiani, Laura; Universita degli Studi di Firenze Scuola di Scienze della Salute Umana, Department of Neurosciences, Psychology, Drug Research and Child Health Mugelli, Alessandro; Universita degli Studi di Firenze Scuola di Scienze della Salute Umana, Department of Neurosciences, Psychology, Drug Research and Child Health Romanelli, Maria Novella; Universita degli Studi di Firenze Scuola di Scienze della Salute Umana, Department of Neurosciences, Psychology, Drug Research and Child Health Cerbai, Elisabetta; Universita degli Studi di Firenze Scuola di Scienze della

Salute Umana, Department of Neurosciences, Psychology, Drug Research and Child Health

Is the invited manuscript for consideration in a Special

Issue: IACS EU Section

Keyword: HCN channels, arrhythmias, cardiomyopathies, HCN blockers, β<sub>3</sub>-adrenergic receptors

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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TITLE

HCN channels: pathophysiological, developmental and pharmacological insights into their

function in cellular excitability.

Valentina Spinelli1, Laura Sartiani

1, Alessandro Mugelli

1, Maria Novella Romanelli

1 and Elisabetta

Cerbai**1

1Department of Neurosciences, Psychology, Drug Research and Child Health (NeuroFarBa),

University of Florence, Florence, Italy;

** Corresponding author: Elisabetta Cerbai, PhD - Department of Neurosciences, Psychology,

Drug Research and Child Health (NeuroFarBa), University of Florence, Viale G. Pieraccini 6,

50139 Florence, Italy, phone: +390552758207 email: [email protected]

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ABSTRACT

The hyperpolarization-activated cyclic-nucleotide-gated (HCN) proteins are voltage-dependent ion

channels, conducting both Na+ and K

+, blocked by millimolar concentrations of extracellular Cs

+

and modulated by cyclic nucleotides (mainly cAMP) that contribute crucially to the pacemaker

activity in cardiac nodal cells and subsidiary pacemakers. Over the last decades, much attention has

focused on HCN current, If, in non-pacemaker cardiac cells and its potential role in triggering

arrhythmias. In fact, in addition to pacemakers, HCN current is constitutively present in the human

atria and has since long been proposed to sustain atrial arrhythmias associated to different cardiac

pathologies or triggered by various modulatory signals (catecholamines, serotonin, natriuretic

peptides). An atypical If occurs in diseased ventricular cardiomyocytes, its amplitude being linearly

relate to the severity of cardiac hypertrophy. The properties of atrial and ventricular If and its

modulation by pharmacological interventions has been object of intense study, including the

synthesis and characterization of new compounds able to block preferentially HCN1, HCN2 or

HCN4 isoforms. Altogether, clues emerge for opportunities of future pharmacological strategies

exploiting the unique properties of this channel family: the prevalence of different HCN subtypes in

organs and tissues, the possibility to target HCN gain- or loss-of-function associated with disease,

the feasibility of novel isoform-selective drugs as well as the discovery of HCN-mediated effects

for old medicines.

Key words: Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels; arrhythmias;

cardiomyopathies; HCN blockers; β3-adrenoceptors.

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INTRODUCTION

The hyperpolarization-activated cyclic-nucleotide-gated (HCN) proteins are voltage-dependent ion

channels, conducting both Na+ and K

+ ions, blocked by millimolar concentrations of extracellular

Cs+ and modulated by cyclic nucleotides (mainly cAMP) (see (Sartiani et al. 2017) for a

comprehensive review). HCN-current was first described in cardiac nodal cells and subsidiary

pacemakers and termed If (funny current) by Dario Di Francesco and coworkers (Brown et al. 1979;

DiFrancesco 1981a, b). Similar recordings were obtained in neurons possessing rhythmic activity

(Yanagihara and Irisawa 1980; Yanagihara et al. 1980), and altogether these findings led to the

classification of If as a pacemaker current playing a relevant yet limited role in cells able to set the

pace. However, during the last decades and mainly after two groups succeeded sequencing the

genes coding for the four HCN isoforms (Ludwig et al. 1998; Santoro et al. 1998), it became more

and more evident that these channels are expressed almost ubiquitously. The expression of the four

different isoforms and the resulting HCN-current varies depending on tissue and cell subtype, age

and developmental stage, disease and genotype. Besides the canonical expression in cardiac

sinoatrial and atrioventricular nodes and in the conducting system, HCN current is constitutively

present in the human atria and has since long been proposed to sustain atrial arrhythmias associated

to different cardiac pathologies or triggered by various modulatory signals (catecholamines,

serotonin, natriuretic peptides) (Sartiani et al. 2017). An atypical If occurs in diseased human and

rodent ventricular cardiomyocytes, its amplitude being linearly related to the severity of cardiac

hypertrophy. This overexpression has been interpreted as the consequence of remodeling toward a

fetal phenotype, hence attention has been devoted to the fate of HCN channels during cardiac

differentiation of embryonic stem cells and in fetal cardiomyocytes. This review will recapitulate

available information concerning the pathophysiological and developmental features of HCN

channel expression in cardiomyocytes, with recent original data from our lab on modulation by β3-

adrenergic stimulation. Finally, we will remark on the progress toward HCN-isoform selective

blockers, and the opportunities for future pharmacological strategies.

MATERIAL AND METHODS

Methods described thereafter refers to original data described in the following sections;

experimental conditions and appropriate ethical statements related to previously published results

have been extensively described in corresponding literature.

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Culture of mouse embryonic stem cells

Mouse embryonic stem cells (mESC) of the line CGR8 commercially available, were used

(Nichols et al. 1990). Cells were propagated at 37 °C and 5 % CO2 in gelatin coated dishes, using

Glasgow Minimum Essential Medium BHK-21 (G-MEM BHK-21), supplemented with 10% fetal

bovine serum (FBS) (GIBCO Invitrogen, Milan, Italy), 2 mM glutamine, 0.1 mM 2-

mercaptoethanol, 1 mM sodium pyruvate, 1% non-essential amino acids, 1%

penicillin/streptomycin (Sigma Aldrich, Milan, Italy) and 1000 units/ml of Leukemia Inhibiting

Factor (LIF, 10 µg/ml – MILLIPORE, Milan, Italy). All other products for cell cultures were

purchased from GIBCO Invitrogen. Prior to splitting, cultures were treated with trypsin/EDTA

(Sigma Aldrich) at room temperature for 30-60 sec in order to eliminate differentiated cells. These

culture conditions allowed CGR8 mESCs to maintain an undifferentiated phenotype characterized

by round-growing, tightly-packed, refrangent colonies.

Cardiac differentiation of CGR8 embryonic stem cells

CGR8 mESC were differentiated using the hanging drop method (Sartiani et al. 2007). Drops of

differentiating medium containing 600 cells were seeded on the lid of bacteriological dishes. When

the dish was closed, this allowed cells to aggregate and grow in suspension (for 2 days) and

eventually form mouse embryoid bodies (mEBs). Differentiation medium, in control condition,

consisted of medium G-MEM BHK21 supplemented with 10% FBS, 2 mM glutamine, 0.1 mM 2-

mercaptoethanol, 1 mM sodium pyruvate, 1% non-essential amino acids, 1%

penicillin/streptomycin. Thereafter, mEBs were re-suspended in differentiation medium and grown

in suspension in bacteriological dishes for 4 days. On the 6th day, mEBs were plated on gelatin-

coated dishes, where they attached. After 24-36 h from plating, cardiac differentiation was

recognizable from the occurrence of spontaneous contracting areas. mEBs were monitored daily by

inverted microscopy, the beating mEBs were counted from day 7th to 21th (D7-D21). mEBs

maturation was followed for no longer than 21 days according to experimental protocols.

To evaluate the modulating effect of β3-adrenergic receptor (β3-AR) signaling during cardiac

maturation, CGR8 mESCs were differentiated in the presence of the selective β3-AR agonist

BRL37344 (BRL, 7 µM) (Sigma Aldrich) and antagonist SR59230A (SR, 10µM) (Sigma Aldrich).

Drugs were administered from day zero (hanging drop formation) to the end of experiments,

renewing media every 2 days.

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Gene expression of pluripotency and cardiac development markers

Samples of mEBs were collected at different time points of differentiation for molecular assessment

by RNA extraction, reverse transcription and gene expression analysis using quantitative Real-Time

PCR (Q-PCR). Total RNA was isolated from mEBs using NucleoSpin® RNA kit (Macherey-Nagel,

Düren, Germany). Subsequently, complementary DNA (cDNA) was synthesized from 1 �g total

RNA using iScript ™ cDNA Synthesis kit (Bio-Rad, Milan, Italy). All these steps were performed

according to the manufacturer’s instructions. Real-time quantitative PCR (Q-PCR) was performed

using the default thermocycler program for all genes: 1 minutes of pre-incubation at 95°C followed

by 40 cycles for 15 seconds at 95°C, 1minute at 60°C and 15 seconds at 95°C, 1minute at 60°C and

1minute 95 °C. Individual real-time PCR reactions were carried out in 10 µl volumes in a 96-well

plate (Applied Biosystems™, London, UK) containing 2 µl RNAse free water, 1 µl of sense and

antisense primers (Bio-Rad) and 5 µl iTaq™ SYBR® Green Universal Supermix (Bio-Rad) plus 2

µl of cDNA sample (5 ng/µl). Each experiment was repeated in triplicate, and quantitative PCR

analysis was performed in triplicate and analyzed with Delta Ct-method. Mouse Glyceraldehyde-3-

Phosphate Dehydrogenase (mGapdh) was used for internal normalization. Results were analyzed by

assuming as 100% the maximal value of gene expression in each experiment.

RESULTS AND DISCUSSION

If current in ventricular cardiomyocytes: a hallmark of cardiac maladaption

The occurrence of a non-canonical pacemaker current in ventricular cardiomyocytes was first

inferred by Yu et al (Yu et al. 1993), based on original and somehow surprising observation of a

cesium-sensitive inward current, activated upon hyperpolarization, in guinea-pig cells. At that time,

some of us were trying to clarify an unexpected finding in papillary muscles from hypertrophied

(but not in normal) rat hearts. Ventricular samples from spontaneously hypertensive rats (SHR)

showed a sort of diastolic depolarization whose steepness was increased by isoproterenol,

eventually giving rise to spontaneous activity (Barbieri et al. 1994). In the following years, we

proved that the mechanism underlying abnormal depolarization and spontaneous activity was the

occurrence of an If -like current (Cerbai et al. 1994), whose amplitude correlated with the severity

of hypertrophy and was therefore very large in SHR with signs of heart failure (Cerbai et al. 1996).

The translational impact of this observation grew up with the demonstration the same held true for

human cardiomyocytes from failing explanted hearts (Cerbai et al. 1997).

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In line with other changes occurring during the progression to cardiac hypertrophy and failure

(Swynghedauw 1999), we inferred that If overexpression was a hallmark of a “regression” to a fetal

phenotype. Supporting this interpretation, If with similar properties (density, occurrence) was

present in immature rat cardiomyocytes from 2-day-old rats and declined thereafter during

maturation (Cerbai et al. 1999).

At that time, two groups succeeded to identify genes coding for the f-channel (HCN1-3) (Ludwig et

al. 1998; Santoro et al. 1998); soon after, a fourth isoform (HCN4) was detected (Ludwig et al.

1999). This advancement made it possible to widen our knowledge concerning gene and protein

expression in different species and tissues and, at a cardiac level, in different sites and experimental

or clinical settings. In parallel, the 2000’s were characterized by an exponential growth of studies

aimed at clarify the ontogenetic and phylogenetic features of this unique family of channels

(Christoffels et al. 2010; Jackson et al. 2007; Mommersteeg et al. 2007).

In our lab, we focused first to changes in cardiac HCN expression occurring in response to different

pathophysiological settings. The left panels of Figure 1 show typical recordings of action potentials

stimulated at 0.2 Hz pacing rate, and If obtained in patch-clamped left ventricular cardiomyocytes

from control and failing human hearts. Current density measured at -120 mV resulted significantly

higher in ischemic cardiomyopathy (2.0±0.2 pA/pF) than in dilated cardiomyopathy (1.2±0.1

pA/pF) or control (1.0±0.1 pA/pF). In the same panels of Figure 1, normalization to maximal

current at -120 mV allows comparing activation kinetics and voltage dependence: If activates at less

negative potentials in cardiomyocytes from failing hearts, the voltage of half maximal activation

being shifted by about 10 mV rightwards (Cerbai et al. 2001; Stillitano et al. 2008). Together with If

gain-of-function, the expression of HCN isoforms, remarkably HCN2 and HCN4 but not HCN1,

increased by 7 to 9-fold (Stillitano et al. 2008).

HCN expression in cardiac remodeling and differentiation: a mirror-image process?

Both transcriptional and post-translational processes contribute to gain- or loss-of-function of If in

adult cardiac cells. The over-expression of If, detected in ventricular myocytes from hypertrophied

or failing rat hearts, was counteracted by drugs interfering with the renin angiotensin system, such

as losartan or irbesartan (Cerbai et al. 2000; Cerbai et al. 2003), as well as by the bradycardic agent

ivabradine (Suffredini et al. 2012). Electrophysiological reverse remodeling was associated with

downregulation of mRNA and protein level for the two major ventricular isoforms, HCN2 and

HCN4. The promoter regions of the two isoforms appear to be trans-activated by the DNA-binding

protein Sp1, belonging to the family of zinc-finger transcription factors: hindering Sp1

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overexpression during stimulation with hypertrophic factors (e.g., angiotensin II) prevents

HCN2/HCN4 re-expression in adult ventricular cardiomyocytes (Lin et al. 2009).

Recent evidence highlighted the role of microRNAs, miR-1 and miR-423-5p, targeting HCN4 in the

SAN of trained rats as well as athletes (D’Souza et al. 2014; D’Souza et al. 2017); of note,

endurance athletes often suffer from bradycardia and supraventricular arrhythmias at old age.

Oppositely, decreased miR-1 level in samples from patients with atrial fibrillation is associated to If

gain-of-function in cardiomyocytes (Li et al. 2015; Stillitano et al. 2013).

As mentioned above, upregulation of HCN protein and, correspondingly, f-current likely represents

a marker of re-expression of fetal genes. Indeed, HCN4 expression occurs ever since the appearance

of the pacemaker activity in the precardiac mesoderm and plays a fundamental role thereafter,

(between E9.5 and E11.5 in mice) (Stieber et al. 2003). Developmental changes of HCN expression

during cardiogenesis in mice has been deeply investigated (Barbuti and Robinson 2015; Christoffels

et al. 2010). Similar studies are obviously lacking for human hearts; however, developmental

changes of cardiomyocytes from embryonic or induced Pluripotent Stem (iPS) cells can provide

interesting insights. The right panels of Figure 1 exemplify this concept. Using human embryonic

stem cells (hESC) committed and differentiated toward the cardiac phenotype, we observed that If

evolves from “less mature” to “more mature” biophysical features (smaller amplitude, more

negative threshold of activation), in parallel with changes in action potential profile, during in vitro

maturation from early to late stages (Sartiani et al. 2007). Again, these changes were accompanied

by parallel decline of expression of all HCN isoforms (Bosman et al. 2013; Sartiani et al. 2007), i.e.,

in the reverse direction tracked by maladaption remodeling. This process goes hand-in-hand with

flattening of diastolic depolarization and decrease of spontaneous rhythmicity.

As recently reviewed by Barbuti and Robinson (2015), several groups have been trying to

counteract the fading of spontaneous activity, that is, to enrich the percentage of pacemaker-like

myocytes. Cell-engineering, pharmacological tools and cell selection processes have been applied,

and invariably the maintenance (or increase) of HCN4 expression has been considered a reliable

marker (Garcia-Frigola et al. 2003; Morikawa et al. 2010; Scavone et al. 2013). Among

pharmacological approaches, the most successful were endothelin-1 (Gassanov et al. 2004), the K-

channel modulator EBIO (1-ethyl-2-benzimidazolinone) (Kleger et al. 2010) and suramin (Wiese et

al. 2011). Interestingly enough, in most cases the enrichment in SAN-like cardiomyocytes was

accompanied by upregulation of transcription factors involved in specification of the conducting

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system such as Isl-1, Tbx3 and Shox2 (Kleger et al. 2010). Although very promising, all these tools

were far from the physiological setting.

Very recently, we approached this issue starting from a different point of view consistent with our

experience with the pathophysiology of cardiac adaption. Beta3-adrenergic receptors (β3-AR),

constitutively expressed in human and rodent cardiomyocytes, undergo upregulation and gain-of-

function in heart failure (Gauthier et al. 1996; Moniotte et al. 2001; Sartiani et al. 2006). At

variance with β1- and β2-AR subtypes, β3-AR couple to nitric oxide synthase through activation of

inhibitory G-proteins (Gauthier et al. 1998). To note, this peculiar signaling pathway apparently

results in a negative shift of If activation in rat (Sartiani et al. 2006) and human (unpublished

observations) ventricular cardiomyocytes, i.e., the reverse effect caused by β1- and β2-AR

stimulation).

We investigated whether β3-AR overexpression in myocardial hypertrophy and failure also

represents a regression toward a fetal phenotype. Therefore, we analyzed changes in expression of

β3-AR in murine embryonic stem cells differentiated into cardiomyocytes (mESC-CM). Figure 2A

shows a typical blot obtained in mESC-CM at different stages. Interestingly, these receptor

subtypes were not only present, but also functional: treatment with a well-known receptor

antagonist (SR) or an agonist (BRL) results in opposite effects on growth of embryonic bodies (data

not shown) and timing/percentage of beating areas (Fig. 2B).

This acceleration/impairment of cardiac commitment and maturation with BRL or SR, respectively,

was accompanied by opposite up- and down-regulation of genetic markers of cardiac and sinoatrial

differentiation. Results summarized in Figure 2C show the effect of treatment with the β3-AR

agonist and antagonist on the relative expression of selected markers (mesoderm, pre-cardiac, early

and late cardiac markers) in a narrow, sensitive window, around 7-day post differentiation, i.e.,

when EBs start beating. However, our attention pointed to the evidence that not only the number of

beating areas, but also their firing rate at 14 days after differentiation was significantly affected by

β3-AR: increased by the agonist, decreased by the antagonist (Fig 3A). This effect was paralleled by

a remarkable upregulation of SAN markers, HCN1 and Cav1.3, in the presence of the β3-AR

agonist, BRL (Fig 3B). The expression of HCN4, in terms of mRNA level, was only slightly

changed by BRL with respect to CTR. However, in the same time window, immunohistochemistry

assay suggests that protein and especially membrane localization of HCN4 was enhanced by BRL

with respect to control and, vice versa, hindered by SR treatment (Fig. 3C); a similar tendency was

observed with α-actinin. Such a discrepancy between HCN4 mRNA and protein expression has

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been already described in atrial tissue likely due to post-transcriptional processing (Stillitano et al

2013). At variance with previous data from our lab obtained in cardiomyocytes from humans

embryonic stem cells (Bosman et al., 2013), we did not observe a membrane “spotty” staining of

HCN4. This difference might be due to the early stage of cardiac differentiation and membrane

organization (i.e., low caveolin-3 expression); however, similar appearance was reported for HCN4

staining in mESC-CM (Saito et al., 2015) and cardiomyocytes isolated from embryonic mouse

hearts and cultured thereafter (Scavone et al., 2013). Overall, although preliminary, these

observations open a new perspective on the mutual relationship between β3-AR and HCN4

(over)expression, whose mechanistic basis deserves to be further investigated also in view of its

possible role in SAN specification.

Pharmacological insights into HCN function in cellular excitability: the dark site of channels

The role of If in primary and subsidiary cardiac pacemakers has been deeply investigated (and also

debated, see for example (DiFrancesco and Noble 2012; Maltsev and Lakatta 2012); the reader can

refer to several reviews dealing with this matter (Biel et al. 2009; Sartiani et al. 2017; Wahl-Schott

et al. 2014). More uncertainty exists on rhythm disturbances due to ectopic or augmented HCN

expression in other cardiac districts. Upregulation of HCN2 and HCN4 channels in a transgenic

model characterized by severe heart failure was associated with ventricular tachycardia and sudden

cardiac death, sensitive to the If blocker ivabradine (Kuwabara et al. 2013; Yamada et al. 2014).

Electrical instability due to HCN overexpression was also detected in a mouse model of ventricular

hypertrophy (Hofmann et al. 2012). Ivabradine was able to ameliorate cardiac function and revert

HCN overexpression in a rat model of heart failure due to myocardial infarction (Ceconi et al. 2011;

Suffredini et al. 2012). Not only gain of function, but also loss-of-function might underlie the

ventricular arrhythmic burden, as recently suggested by the identification of a novel mutation in the

HCN4 channel in a patient with Brugada syndrome (Biel et al. 2016). Seemingly, HCN channel

dysfunction in ventricular tissue, as well as in other cardiac sites, might be unveiled and emphasized

by concomitant changes of other ion currents in congenital or acquired syndromes. In long-QT

syndrome patients carrying mutations of caveolin-3 (a lipid raft component of myocyte membrane),

simultaneous alterations of HCN4 properties as described in naïve cardiomyocytes and cell lines

(Barbuti et al. 2004; Barbuti et al. 2012; Barbuti et al. 2007; Bosman et al. 2013) may contribute to

the clinical phenotype (Motloch et al. 2017). The hypothesis of “polygenic mechanisms underlying

cardiac disorders” involving HCN4 mutation has been inferred also by the association between

sinus bradycardia and left ventricular non-compaction cardiomyopathy (LVNC) in families with

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heritable HCN4 defects (Milano et al., 2014; Schweizer et al. 2014) and further confirmed by recent

investigation in an infant with LVNC (Yokoyama et al. 2018).

The predominant role of HCN4 at cardiac level prompted us to search for isoform selective blockers

(Romanelli et al. 2016). The feasibility of this approach has been demonstrated by the selectivity of

a series of compounds derived from structural modifications of the non-selective blocker

zatebradine (Del Lungo et al. 2012; Koncz et al. 2011; Melchiorre et al. 2010). This strategy opened

up a somehow unforeseen, promising field: exploitation of blockers targeting HCN1/HCN2

isoforms – instead of the “cardiac” isoform, HCN4– to treat extra-cardiac disorders without

affecting heart rhythm and conduction. In fact, HCN1 blockade by the selective inhibitor MEL57A

counteracted hyperalgesia and allodynia in chemotherapy-induced neuropathy, an ill-treated

condition in patients, at dosage devoid of bradycardic effects (Resta et al. 2018). Recent

unpublished data from our lab suggest that a similar result was achieved by simultaneous blockade

of HCN1/HCN2.

Conclusions

Almost 40 years of intensive research, since the first description of a cardiac funny current (Brown

et al., 1979), have allowed an exponential growth of our knowledge on HCN channels’ properties -

from biophysics to structure and molecular features - making it possible the pharmacological and

therapeutic exploitation of selective f-channel blockers. However, the physiological and

pathophysiological regulation of HCN expression in biologically and clinically relevant settings

unveils new and original fields of investigation. In this light, clues emerge from the observation of a

promoting effect of β3-AR stimulation of HCN functional expression during cardiac differentiation,

whose molecular mechanisms deserve further investigation.

ACKNOWLEDGEMENTS

All authors declare no conflict of interest. This work was funded by grants from the University of

Florence (ex 60%) (M.N.R.); Ente Cassa di 390 Risparmio di Firenze (2013.0683 to L.S.);

Normacor project (contract LSH 391 M/CT/2006/018676 to E.C.).

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REFERENCES

Barbieri M., Varani K., Cerbai E., Guerra L., Li Q., Borea P.A., and Mugelli A. 1994.

Electrophysiological Basis for the Enhanced Cardiac Arrhythmogenic Effect of Isoprenaline in

Aged Spontaneously Hypertensive Rats. J. Mol. Cell. Cardiol. 26(7): 849-860. doi:

https://doi.org/10.1006/jmcc.1994.1102.

Barbuti A., Gravante B., Riolfo M., Milanesi R., Terragni B., and DiFrancesco D. 2004.

Localization of Pacemaker Channels in Lipid Rafts Regulates Channel Kinetics. Circ. Res.

94(10): 1325-1331. doi: 10.1161/01.res.0000127621.54132.ae.

Barbuti A., and Robinson R.B. 2015. Stem Cell–Derived Nodal-Like Cardiomyocytes as a Novel

Pharmacologic Tool: Insights from Sinoatrial Node Development and Function. Pharmacol. Rev.

67(2): 368-388. doi: 10.1124/pr.114.009597.

Barbuti A., Scavone A., Mazzocchi N., Terragni B., Baruscotti M., and DiFrancesco D. 2012. A

caveolin-binding domain in the HCN4 channels mediates functional interaction with caveolin

proteins. J. Mol. Cell. Cardiol. 53(2): 187-195. doi: https://doi.org/10.1016/j.yjmcc.2012.05.013.

Barbuti A., Terragni B., Brioschi C., and DiFrancesco D. 2007. Localization of f-channels to

caveolae mediates specific β2-adrenergic receptor modulation of rate in sinoatrial myocytes. J.

Mol. Cell. Cardiol. 42(1): 71-78. doi: https://doi.org/10.1016/j.yjmcc.2006.09.018.

Biel M., Wahl-Schott C., Michalakis S., and Zong X. 2009. Hyperpolarization-Activated Cation

Channels: From Genes to Function. Physiol. Rev. 89(3): 847-885. doi:

10.1152/physrev.00029.2008.

Biel S., Aquila M., Hertel B., Berthold A., Neumann T., DiFrancesco D., Moroni A., Thiel G.,

and Kauferstein S. 2016. Mutation in S6 domain of HCN4 channel in patient with suspected

Brugada syndrome modifies channel function. Pflügers Archiv – Eur. J. Physiol. 468(10): 1663-

1671. doi: 10.1007/s00424-016-1870-1.

Bosman A., Sartiani L., Spinelli V., Del Lungo M., Stillitano F., and Nosi D., et al. 2013.

Molecular and Functional Evidence of HCN4 and Caveolin-3 Interaction During Cardiomyocyte

Differentiation from Human Embryonic Stem Cells. Stem Cells Dev. 22(11): 1717-1727. doi:

10.1089/scd.2012.0247.

Brown H.F., DiFrancesco D., and Noble S.J. 1979. How does adrenaline accelerate the heart?

Nature 280: 235. doi: 10.1038/280235a0.

Ceconi C., Comini L., Suffredini S., Stillitano F., Bouly M., and Cerbai E., et al. 2011. Heart

rate reduction with ivabradine prevents the global phenotype of left ventricular remodeling. Am.

J. Physiol. Heart Circ. Physiol. 300(1): H366-H373. doi: 10.1152/ajpheart.01117.2009.

Cerbai E., Barbieri M., and Mugelli A. 1994. Characterization of the hyperpolarization-activated

current If in ventricular myocytes isolated from hypertensive rats. J. Physiol. (London) 481:

585-591.

Page 12 of 21



Draft

CJPP-2018-0115_R1 - 13

Cerbai E., Barbieri M., and Mugelli A. 1996. Occurrence and properties of the hyperpolarization-

activated current If in ventricular myocytes from normotensive and hypertensive rats during

aging. Circulation 94: 1674-1681.

Cerbai E., Crucitti A., Sartiani L., De Paoli P., Pino R., and Rodriguez M.L., et al. 2000. Long-

term treatment of spontaneously hypertensive rats with losartan and electrophysiological

remodeling of cardiac myocytes. Cardiovasc. Res. 45(2): 388-396. doi: 10.1016/s0008-

6363(99)00344-2.

Cerbai E., De Paoli P., Sartiani L., Lonardo G., and Mugelli A. 2003. Treatment With Irbesartan

Counteracts the Functional Remodeling of Ventricular Myocytes From Hypertensive Rats. J.

Cardiovasc. Pharmacol. 41(5): 804-812.

Cerbai E., Pino R., Porciatti F., Sani G., Toscano M., and Maccherini Met al. 1997.

Characterization of the hyperpolarization-activated current If in ventricular myocytes from

human failing heart. Circulation 95: 568-571.

Cerbai E., Pino R., Sartiani L., and Mugelli A. 1999. Influence of postnatal-development on I(f)

occurrence and properties in neonatal rat ventricular myocytes. Cardiovasc. Res. 42(2): 416-423.

Cerbai E., Sartiani L., DePaoli P., Pino R., Maccherini M., and Bizzarri F., et al. 2001. The

Properties of the Pacemaker Current IFin Human Ventricular Myocytes are Modulated by

Cardiac Disease. J. Mol. Cell. Cardiol. 33(3): 441-448. doi:

http://dx.doi.org/10.1006/jmcc.2000.1316.

Christoffels V.M., Smits G.J., Kispert A., and Moorman A.F.M. 2010. Development of the

Pacemaker Tissues of the Heart. Circ. Res. 106(2): 240-254. doi:

10.1161/circresaha.109.205419.

D’Souza A., Bucchi A., Johnsen A.B., Logantha S.J.R.J., Monfredi O., and Yanni J., et al. 2014.

Exercise training reduces resting heart rate via downregulation of the funny channel HCN4. Nat.

Commun. 5: 3775. doi: 10.1038/ncomms4775

D’Souza A., Pearman C.M., Wang Y., Nakao S., Logantha S.J.R.J., and Cox C., et al. 2017.

Targeting miR-423-5p Reverses Exercise Training–Induced HCN4 Channel Remodeling and

Sinus Bradycardia. Circ. Res. 121(9): 1058-1068. doi: 10.1161/circresaha.117.311607.

Del Lungo M., Melchiorre M., Guandalini L., Sartiani L., Mugelli A., and Koncz I., et al. 2012.

Novel blockers of hyperpolarization-activated current with isoform selectivity in recombinant

cells and native tissue. Br. J. Pharmacol. 166(2): 602-616. doi: 10.1111/j.1476-

5381.2011.01782.x.

DiFrancesco D. 1981a. A new interpretation of the pace-maker current in calf Purkinje fibres. The

J. Physiol. 314(1): 359-376. doi: 10.1113/jphysiol.1981.sp013713.

DiFrancesco D. 1981b. A study of the ionic nature of the pace-maker current in calf Purkinje

fibres. J. Physiol. 314(1): 377-393. doi: 10.1113/jphysiol.1981.sp013714.

DiFrancesco D., and Noble D. 2012. The funny current has a major pacemaking role in the sinus

node. Heart Rhythm 9(2): 299-301. doi: https://doi.org/10.1016/j.hrthm.2011.09.021.

Page 13 of 21



Draft

CJPP-2018-0115_R1 - 14

Garcia-Frigola C., Shi Y., and Evans S.M. 2003. Expression of the hyperpolarization-activated

cyclic nucleotide-gated cation channel HCN4 during mouse heart development. Gene Expr.

Patterns 3(6): 777-783. doi: https://doi.org/10.1016/S1567-133X(03)00125-X.

Gassanov N., Er F., Zagidullin N., and Hoppe U.C. 2004. Endothelin induces differentiation of

ANP-EGFP expressing embryonic stem cells towards a pacemaker phenotype. FASEB J. 18(14):

1710-1712. doi: 10.1096/fj.04-1619fje.

Gauthier C., Leblais V., Kobzik L., Trochu J.N., Khandoudi N., and Bril A., et al. 1998. The

negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric

oxide synthase pathway in human ventricle. J. Clin. Invest. 102(7): 1377-1384. doi:

10.1172/jci2191.

Gauthier C., Tavernier G., Charpentier F., Langin D., and Le Marec H. 1996. Functional beta3-

adrenoceptor in the human heart. J. Clin. Invest. 98(2): 556-562. doi: 10.1172/jci118823.

Hofmann F., Fabritz L., Stieber J., Schmitt J., Kirchhof P., Ludwig A., and Herrmann S. 2012.

Ventricular HCN channels decrease the repolarization reserve in the hypertrophic heart.

Cardiovasc. Res.h 95(3): 317-326. doi: 10.1093/cvr/cvs184.

Jackson H.A., Marshall C.R., and Accili E.A. 2007. Evolution and structural diversification of

hyperpolarization-activated cyclic nucleotide-gated channel genes. Physiol. Genomics 29(3):

231-245. doi: 10.1152/physiolgenomics.00142.2006.

Kleger A., Seufferlein T., Malan D., Tischendorf M., Storch A., and Wolheim A., et al. 2010.

Modulation of Calcium-Activated Potassium Channels Induces Cardiogenesis of Pluripotent

Stem Cells and Enrichment of Pacemaker-Like Cells. Circulation 122(18): 1823-1836. doi:

10.1161/circulationaha.110.971721.

Koncz I., Szel T., Jaeger I., Baczko I., Cerbai E., Romanelli M.N., Papp J.G., and Varro A.

2011. Selective Pharmacological Inhibition of the Pacemaker Channel Isoforms (HCN1-4) as

New Possible Therapeutical Targets. Curr. Med. Chem. 18: 3662-3674.

Kuwabara Y., Kuwahara K., Takano M., Kinoshita H., Arai Y., and Yasuno S., et al. 2013.

Increased Expression of HCN Channels in the Ventricular Myocardium Contributes to Enhanced

Arrhythmicity in Mouse Failing Hearts. J. Am. Heart Assoc. 2(3). doi: 10.1161/jaha.113.000150.

Li Y., Hong Y., Yusufuaji Y., Tang B., Zhou X., Xu G., Li J., Sun L., Zhang J., Xin Q.,

Xiong J., Ji Y., and Zhang Y. 2015. Altered expression of hyperpolarization-activated cyclic

nucleotide-gated channels and microRNA-1 and -133 in patients with age-associated atrial

fibrillation. Mol. Med. Rep. 12(3): 3243-3248. doi: 10.3892/mmr.2015.3831.

Lin H., Xiao J., Luo X., Chen G., and Wang Z. 2009. Transcriptional Control of Pacemaker

Channel Genes HCN2 and HCN4 by Sp1 and Implications in Re-expression of These Genes in

Hypertrophied Myocytes. Cell. Physiol. Biochem. 23(4-6): 317-326. doi: 10.1159/000218178.

Ludwig A., Zong X., Jeglitsch M., Hofmann F., and Biel M. 1998. A family of

hyperpolarization-activated mammalian cation channels. Nature 393: 587-591.

Ludwig A., Zong X., Stieber J., Hullin R., Hofmann F., and Biel M. 1999. Two pacemaker

channels from human heart with profoundly different activation kinetics. EMBO J. 18(9): 2323-

2329. doi: 10.1093/emboj/18.9.2323.

Page 14 of 21



Draft

CJPP-2018-0115_R1 - 15

Maltsev V.A., and Lakatta E.G. 2012. The funny current in the context of the coupled-clock

pacemaker cell system. Heart Rhythm 9(2): 302-307. doi:

https://doi.org/10.1016/j.hrthm.2011.09.022.

Melchiorre M., Del Lungo M., Guandalini L., Martini E., Dei S., and Manetti D., et al. 2010.

Design Synthesis and Preliminary Biological Evaluation of New Isoform-Selective f-Current

Blockers. J. Med. Chem. 53(18): 6773-6777. doi: 10.1021/jm1006758.

Milano A., Vermeer A.M., Lodder E.M., Barc J., Verkerk A.O., and Postma A.V., et al. 2014.

HCN4 mutations in multiple families with bradycardia and left ventricular noncompaction

cardiomyopathy. J Am Coll Cardiol. 64(8):745-56. doi: 10.1016/j.jacc.2014.05.045.

Mommersteeg M.T.M., Hoogaars W.M.H., Prall O.W.J., de Gier-de Vries C., Wiese C., and

Clout D.E.W., et al. 2007. Molecular Pathway for the Localized Formation of the Sinoatrial

Node. Circ. Res. 100(3): 354-362. doi: 10.1161/01.RES.0000258019.74591.b3.

Moniotte S., Vaerman J.L., Kockx M.M., Larrouy D., Langin D., Noirhomme P., and Balligand

J.L. 2001. Real-time RT-PCR for the Detection of Beta-adrenoceptor Messenger RNAs in Small

Human Endomyocardial Biopsies. J. Mol. Cell. Cardiol. 33(12): 2121-2133. doi:

https://doi.org/10.1006/jmcc.2001.1475.

Morikawa K., Bahrudin U., Miake J., Igawa O., Kurata Y., and Nakayama Y., et al. 2010.

Identification Isolation and Characterization of HCN4-Positive Pacemaking Cells Derived from

Murine Embryonic Stem Cells during Cardiac Differentiation. Pacing Clin. Electrophysiol.

33(3): 290-303. doi: 10.1111/j.1540-8159.2009.02614.x.

Motloch L.J., Larbig R., Darabi T., Reda S., Motloch K.A., and Wernly B., et al. 2017. Long-

QT syndrome-associated caveolin-3 mutations differentially regulate the hyperpolarization-

activated cyclic nucleotide gated channel 4. Physiol. Int. 104(2): 130-138. doi:

10.1556/2060.104.2017.2.6.

Nichols J., Evans E.P., and Smith A.G. 1990. Establishment of germ-line-competent embryonic

stem (ES) cells using differentiation inhibiting activity. Development 110(4): 1341-1348.

Resta F., Micheli L., Laurino A., Spinelli V., Mello T., and Sartiani L., et al. 2018. Selective

HCN1 block as a strategy to control oxaliplatin-induced neuropathy. Neuropharmacology 131:

403-413.

Romanelli M.N., Sartiani L., Masi A., Mannaioni G., Manetti D., Mugelli A., and Cerbai E.

2016. HCN channels modulators: the need for selectivity. Curr. Top. Med.Chem. 16: 1764-1791.

Saito Y., Nakamura K., Yoshida M., Sugiyama H., Ohe T., and Kurokawa J., et al. 2015.

Enhancement of Spontaneous Activity by HCN4 Overexpression in Mouse Embryonic Stem

Cell-Derived Cardiomyocytes - A Possible Biological Pacemaker. Plos One Sept 18.

doi.org/10.1371/journal.pone.0138193

Santoro B., Liu D.T., Yao H., Bartsch D., Kandel E.R., and Siegelbaum S.A., et al. 1998.

Identification of a Gene Encoding a Hyperpolarization-Activated Pacemaker Channel of Brain.

Cell 93(5): 717-729. doi: 10.1016/S0092-8674(00)81434-8.

Sartiani L., Bettiol E., Stillitano F., Mugelli A., Cerbai E., and Jaconi M.E. 2007.

Developmental Changes in Cardiomyocytes Differentiated from Human Embryonic Stem Cells:

Page 15 of 21



Draft

CJPP-2018-0115_R1 - 16

A Molecular and Electrophysiological Approach. Stem Cells 25(5): 1136-1144. doi:

10.1634/stemcells.2006-0466.

Sartiani L., De Paoli P., Stillitano F., Aimond F., Vassort G., and Mugelli A., et al. 2006.

Functional remodeling in post-myocardial infarcted rats: focus on beta-adrenoceptor subtypes. J.

Mol. Cell. Cardiol. 40(2): 258-266. doi: http://dx.doi.org/10.1016/j.yjmcc.2005.11.011.

Sartiani L., Mannaioni G., Masi A., Novella Romanelli M., and Cerbai E. 2017. The

Hyperpolarization-Activated Cyclic Nucleotide–Gated Channels: from Biophysics to

Pharmacology of a Unique Family of Ion Channels. Pharmacol. Revi. 69(4): 354-395. doi:

10.1124/pr.117.014035.

Scavone A., Capilupo D., Mazzocchi N., Crespi A., Zoia S., and Campostrini G., et al. 2013.

Embryonic Stem Cell–Derived CD166+ Precursors Develop Into Fully Functional Sinoatrial-

Like Cells. Circ. Res. 113(4): 389-398. doi: 10.1161/circresaha.113.301283.

Schweizer P.A., Schröter J., Greiner S., Haas J., Yampolsky P., and Mereles D., et al. 2014. The

symptom complex of familial sinus node dysfunction and myocardial noncompaction is

associated with mutations in the HCN4 channel. J Am Coll Cardiol. 64(8):757-67. doi:

10.1016/j.jacc.2014.06.1155.

Stieber J., Herrmann S., Feil S., Löster J., Feil R., and Biel M., et al. 2003. The

hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action

potentials in the embryonic heart. Proc. Natl Acad. Sci. USA 100(25): 15235-15240. doi:

10.1073/pnas.2434235100.

Stillitano F., Lonardo G., Giunti G., Del Lungo M., Coppini R., and Spinelli V., et al. 2013.

Chronic Atrial Fibrillation Alters the Functional Properties of If in the Human Atrium. J.

Cardiovasc. Electrophysiol. 24(12): 1391-1400. doi: 10.1111/jce.12212.

Stillitano F., Lonardo G., Zicha S., Varro A., Cerbai E., and Mugelli A., et al. 2008. Molecular

basis of funny current (If) in normal and failing human heart. J. Mol. Cell. Cardiol. 45(2): 289-

299. doi: http://dx.doi.org/10.1016/j.yjmcc.2008.04.013.

Suffredini S., Stillitano F., Comini L., Bouly M., Brogioni S., and Ceconi C., et al. 2012. Long-

term treatment with ivabradine in post-myocardial infarcted rats counteracts f-channel

overexpression. Br. J. Pharmacol. 165(5): 1457-1466. doi: 10.1111/j.1476-5381.2011.01627.x.

Swynghedauw B. 1999. Molecular Mechanisms of Myocardial Remodeling. Physiol. Rev. 79(1):

215-262. doi: 10.1152/physrev.1999.79.1.215.

Wahl-Schott C., Fenske S., and Biel M. 2014. HCN channels: new roles in sinoatrial node

function. Curr. Opin. Pharmacol. 15(0): 83-90. doi:http://dx.doi.org/10.1016/j.coph.2013.12.005.

Wiese C., Nikolova T., Zahanich I., Sulzbacher S., Fuchs J., Yamanaka S., Graf E., Ravens U.,

Boheler K.R., and Wobus A.M. 2011. Differentiation induction of mouse embryonic stem cells

into sinus node-like cells by suramin. Int. J. Cardiol. 147(1): 95-111. doi:

https://doi.org/10.1016/j.ijcard.2009.08.021.

Yamada Y., Kinoshita H., Kuwahara K., Nakagawa Y., Kuwabara Y., and Minami T., et al.

2014. Inhibition of N-type Ca2+

channels ameliorates an imbalance in cardiac autonomic nerve

Page 16 of 21



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CJPP-2018-0115_R1 - 17

activity and prevents lethal arrhythmias in mice with heart failure. Cardiovasc. Res. 104(1): 183-

193. doi: 10.1093/cvr/cvu185.

Yanagihara K., and Irisawa H. 1980. Inward current activated during hyperpolarization in the

rabbit sinoatrial node cell. Pflugers Arch 385 (1): 11-19.

Yanagihara K., Noma A., and Irisawa H. 1980. Reconstruction of sino-atrial node pacemaker

potential based on the voltage clamp experiments. Jpn J Physiol 30 (6): 841-857.

Yokoyama R., Kinoshita K., Hata Y., Abe M., Matsuoka K., and Hirono K., et al. 2018. A

mutant HCN4 channel in a family with bradycardia left bundle branch block and left ventricular

noncompaction. Heart Vessels. doi: 10.1007/s00380-018-1116-6.

Yu H., Chang F., and Cohen I.S. 1993. Pacemaker current exists in ventricular myocytes. Circ.

Res. 72(1): 232-236. doi: 10.1161/01.res.72.1.232.

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FIGURES AND FIGURE LEGENDS

Figure 1. Representation of membrane potential or action potential, HCN-current and HCN

isoform relative expression in human adult cardiomyocytes (Panel A) and cardiomyocytes

differentiated from human embryonic stem cells (Panel B). Action potentials in normal or

failing hVCM were obtained by patch-clamp recordings at 0.2Hz pacing rate. Abbreviation: hESC,

human embryonic stem cells; hESC-CM early, late: cardiomyocytes differentiated from hESC at

early (20 days) and late (90 days) phase; hVCM Normal, Failing: cardiomyocytes dissociated the

left ventricle of control donor hearts or explanted hearts from patients with ischemic heart failure.

(Modified from Cerbai et al. 2001; Sartiani et al. 2007; Stillitano et al. 2013; Stillitano et al. 2008).

Figure 2. Molecular and functional expression of ββββ3-AR during cardiac differentiation of

mESC. Panel A: Immunoblot and relative quantification of β3-AR protein in mESC at different

stages (from day 2, D2, to day 14, D14) of cardiac differentiation. Panel B: number of beating areas

in mESC embryo bodies differentiating in control medium supplemented or not with a selective β3-

AR antagonist (SR, red) or agonist (BRL, blue). Panel C: Q-PCR analysis of mRNA levels

measured in mESC-CM cultured in the presence of SR or BRL, compared to matched controls

(CTR). Bars represent the relative expression of genes coding for markers of pluripotency (Oct4),

early mesoderm (Brachiury, Bra); pre-cardiac development (Wnt3a; Wnt11; β-catenin, Ctnnb1);

first and second heart field (Mef2c, Hand1, Hand2, Isl1, Fgf10); cardiac differentiation (Gata4,

Nkx2.5, Tbx3, Tbx18, Shox2, cTnI).

Figure 3. Modulation of mESC-CM differentiation and maturation by ββββ3-AR signaling. Panel

A: Frequency of spontaneous contraction of beating areas in embryoid bodies (EBs) differentiating

in control medium supplemented or not with a selective β3-AR antagonist (SR, red) or agonist

(BRL, blue). *** p<0.01 (BRL vs. CTR and BRL vs. SR), ** p<0.01 (SR vs. CTR). Panel B:

expression of genes coding for alpha 1D subunit of L-type calcium channels, CaV1.3, HCN4 and

HCN1 isoforms evaluated by Q-PCR analysis, expressed as ratio versus control (dashed line), in

mESC-CM cultured in the absence (CTR) or presence of SR or BRL. Panel C: Representative

results from immunohistochemistry assays showing expression and localization of HCN4 and α-

actinin in mESC-CM cultured in the absence (CTR) or presence of SR or BRL.

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Figure 1. Representation of membrane potential or action potential, HCN-current and HCN isoform relative expression in human adult cardiomyocytes (Panel A) and cardiomyocytes differentiated from human

embryonic stem cells (Panel B). Abbreviation: hESC, human embryonic stem cells; hESC-CM early, late: cardiomyocytes differentiated from hESC at early (20 days) and late (90 days) phase; hVCM Normal, Failing: cardiomyocytes dissociated the left ventricle of control donor hearts or explanted hearts from

patients with ischemic heart failure. (Modified from Cerbai et al. 2001; Sartiani et al. 2007; Stillitano et al. 2013; Stillitano et al. 2008).

209x157mm (300 x 300 DPI)

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Figure 2: Molecular and functional expression of β3-AR during cardiac differentiation of mESC. Panel A: Immunoblot and relative quantification of β3-AR protein in mESC at different stages (from day 2, D2, to day 14, D14) of cardiac differentiation. Panel B: number of beating areas in mESC embryo bodies differentiating

in control medium supplemented or not with a selective β3-AR antagonist (SR, red) or agonist (BRL, blue). Panel C: Q-PCR analysis of mRNA levels measured in mESC-CM cultured in the presence of SR or BRL,

compared to matched controls (CTR). Bars represent the relative expression of genes coding for markers of pluripotency (Oct4), early mesoderm (Brachiury, Bra); pre-cardiac development (Wnt3a; Wnt11; β-catenin,

Ctnnb1); first and second heart field (Mef2c, Hand1, Hand2, Isl1, Fgf10); cardiac differentiation (Gata4, Nkx2.5, Tbx3, Tbx18, Shox2, cTnI).

209x157mm (300 x 300 DPI)

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Figure 3. Modulation of mESC-CM differentiation and maturation by β3-AR signaling. Panel A: Frequency of spontaneous contraction of beating areas in embryoid bodies (EBs) differentiating in control medium

supplemented or not with a selective β3-AR antagonist (SR, red) or agonist (BRL, blue). *** p<0.01 (BRL vs. CTR and BRL vs. SR), ** p<0.01 (SR vs. CTR). Panel B: expression of genes coding for alpha 1D subunit of L-type calcium channels, CaV1.3, HCN4 and HCN1 isoforms evaluated by Q-PCR analysis, expressed as ratio versus control (dashed line), in mESC-CM cultured in the absence (CTR) or presence of SR or BRL.

Panel C: Representative results from immunohistochemistry assays showing expression and localization of HCN4 and α-actinin in mESC-CM cultured in the absence (CTR) or presence of SR or BRL.

209x155mm (300 x 300 DPI)

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draft · 2018. 8. 3. · draft cjpp-2018-0115_r1 - 2 abstract the hyperpolarization-activated...

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