# The funny current in genetically modified mice

Mattia Difrancesco, Pietro Mesirca, Isabelle Bidaud, Dirk Isbrandt, Matteo Mangoni

► To cite this version:

Mattia Difrancesco, Pietro Mesirca, Isabelle Bidaud, Dirk Isbrandt, Matteo Mangoni. The funny current in genetically modified mice. Progress in Biophysics and Molecular Biology, 2021, 166, pp.39-50. 10.1016/j.pbiomolbio.2021.06.003. hal-03437498

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The funny current in genetically modified mice

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¹⁻⁴Mattia L. DiFrancesco*, ³,⁴Pietro Mesirca, ³,⁴Isabelle Bidaud, ⁵,⁶Dirk Isbrandt ³,⁴Matteo E. Mangoni*

* corresponding authors

1) Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, Genoa, Italy

2) IRCCS Ospedale Policlinico San Martino, Genoa, Italy

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3) Institut de Génomique Fonctionnelle, Université de Montpellier, CNRS, INSERM, Montpellier, France

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4) LabEx Ion Channels Science and Therapeutics (ICST)

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5) Deutsches Zentrum für Neurodegenerative Erktankungen (DZNE), Bonn, Germany

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6) University of Cologne, Institute for Molecular and Behavioral Neuroscience, Cologne, Germany

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@ Authors for correspondence: matteo.mangoni@igf.cnrs.fr; mattia.difrancesco@iit.it,

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**Keywords:**

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Funny current, cardiac pacemaker activity, conduction system, sinoatrial node, genetically modified mice, rhythmogenesis

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Since its first description in 1979, the hyperpolarization-activated *funny* current ($I_f$) has been the object of intensive research aimed at understanding its role in cardiac pacemaker activity and its modulation by the sympathetic and parasympathetic branches of the autonomic nervous system. $I_f$ was described in isolated tissue strips of the rabbit sinoatrial node using the double-electrode voltage-clamp technique. Since then, the rabbit has been the principal animal model for studying pacemaker activity and $I_f$ for more than 20 years. In 2001, the first study describing the electrophysiological properties of mouse sinoatrial pacemaker myocytes and those of $I_f$ was published. It was soon followed by the description of murine myocytes of the atrioventricular node and the Purkinje fibres. The sinoatrial node of genetically modified mice has become a very popular model for studying the mechanisms of cardiac pacemaker activity. This field of research benefits from the impressive advancement of *in-vivo* exploration techniques of physiological parameters, imaging, genetics, and large-scale genomic approaches. The present review discusses the influence of mouse genetics based on the most recent knowledge of the funny current's role in the physiology and pathophysiology of cardiac pacemaker activity. Genetically modified mice have provided important insights into the role of $I_f$ in determining intrinsic automaticity *in vivo* and in myocytes of the conduction system. In addition, gene targeting of f-(HCN) channel isoforms have contributed to elucidating the current's role in the regulation of heart rate by the parasympathetic nervous system. This review is dedicated to Dario DiFrancesco on his retirement.
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# 1. Introduction

## *Historical perspective*

The *funny* ($I_f$) current was first described in 1979 (Brown et al., 1979) in a study in which tiny specimens of sinoatrial node (SAN) were extracted by hand from a rabbit heart using a delicate procedure that was based on cutting and silk-ligaturing 300-µM-thick tissue strips and voltage clamping under a stereoscopic microscope using the two-microelectrode technique (Noma and Irisawa, 1976).

The size of the rabbit heart was appropriate for this demanding procedure. Sinoatrial node strips were just large enough for manual ligaturing under a microscope, and ligatured specimens were just small enough for satisfactory voltage control during two-electrode voltage clamp. These combined features made the rabbit SAN the first, and, for several years, the only mammalian pacemaker tissue to be available for voltage clamp. It was only in the mid-eighties, when methods for enzymatic cell isolation were developed, that the Nobel Prize-winning patch-clamp technique allowed single pacemaker cells to be studied (DiFrancesco, 1986).

Even after the advent of enzymatic cell isolation techniques, the isolation of single pacemaker cells from rabbit nodal tissue remained the method of choice for investigating the *funny* current for several years before the first recordings of $I_f$ in mouse pacemaker myocytes were performed (Mangoni and Nargeot, 2001). Many relevant basic $I_f$ properties originally described in multicellular SAN preparations and used in numerical modelling (see Noble 2021 in this issue) (Noble, 2020), were then confirmed in single-cell studies in rabbit SAN myocytes (DiFrancesco, 1986).

In parallel with the rapidly advancing investigation of cardiac electrical activity thanks to the exploitation of the new cell isolation and patch-clamp techniques, including single ion-channel recordings, methods designed to investigate molecular properties of ion channels became available. DNA sequencing, recombinant DNA, and heterologous expression techniques allowed much deeper insights to be gained into ion channel properties than was previously possible. In 1986, the first cloning, sequencing, and functional expression of sodium channels (Noda et al., 1986) opened new perspectives with respect to the acquisition of a more detailed knowledge of the molecular features of ion channels and their structure-function relationships.

Many subsequent studies benefited from the research efforts to unravel the molecular features of ion channels resulting in the cloning and sequencing of several voltage-dependent $Na^+$, $Ca^{2+}$, and $K^+$ potassium channels. The interested reader may want to read the book by Frances M. Ashcroft, *Ion*
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*Channels and Disease* (Ashcroft, 2001) on this historical topic. Funny channels lagged behind in this race, probably because of a more limited general interest, or simply because of their intrinsic complexity, and were not cloned until the late nineties from mouse brain, sea urchin sperm, and heart tissues (Gauss et al., 1998; Ludwig et al., 1998; Santoro et al., 1998; Vaccari et al., 1999).

Cloning of HCN1-4, the four molecular correlates of the pore-forming subunits of *funny* channels, was the missing step in the advancement of molecular investigations and genetic manipulation of these channels. The time was ripe for the development of a new animal model that was more suitable for the genetic investigation of funny/HCN channels than the rabbit model.

*The funny current in mouse pacemaker myocytes*

Since its first description, the role of the funny current in the genesis and regulation of cardiac automaticity has been under constant debate. A part of this debate is rooted in the methodological approaches used to study SAN pacemaking. Indeed, until the late nineties, the importance of ionic currents in cardiac pacemaking was generally assessed using ionic or organic blockers. Although this approach was appropriate, the limited selectivity of these blockers or agonists, under physiological conditions of cyclical variation of membrane voltage, rendered the interpretation of the results questionable. Thus, it was important to provide genetic evidence of the relevance of ion channels *in vivo* and *ex vivo* using a mammalian model of cardiac pacemaking.

The first hint from genetics for the field of pacemaking and $I_f$ was published in 1997 and came from a large scale-based screening of mutations affecting the development of the zebrafish cardiovascular system (Stainier et al., 1996). Zebrafish is particularly amenable for studying the cardiovascular development because early embryos and larvae are transparent, and exchanges in oxygen are ensured by simple diffusion. Consequently, the developmental defects that will result in lethality in mammalian embryos can be studied in zebrafish larvae. Among 58 mutations identified in this study, the recessive *slo mo* mutation was characterized and shown to affect the heart rate of the embryo in the absence of other defects in cardiovascular development. The *slo mo* zebrafish mutant presented with slower basal heart rate compared to wild-type counterparts (Baker et al., 1997). Patch-clamp recordings of ionic currents from isolated embryonic cardiomyocytes showed a reduction in the density of the fast component of $I_f$ activation (referred to as $I_h$ in this study). No other ionic current known at the time to be involved in pacemaking in the rabbit sinoatrial node was found to be affected in the hearts of *slo mo* embryos, leading to the conclusion that diminished $I_f$ was responsible for bradycardia in this strain. When homozygous *slo mo* mutants were raised to adulthood, they presented with bradycardia and reduced $I_f$ density in atria and ventricles (Warren et
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al., 2001). Unfortunately, this intriguing fish mutant was not investigated further to determine whether it was the funny channel itself, or a protein involved in channel regulation, that carried the *slo mo* mutation. Yet, the *slo mo* zebrafish mutant provided the first genetic evidence of funny current's relevance in determining the heart rate *in vivo* in vertebrates.

In 1998, Wickman and coworkers published the first paper in which an ion channel known to be involved in the regulation of sinoatrial pacemaker activity and heart was ablated by genetic knockout in mice (Wickman et al., 1998). These authors showed that genetic ablation of G protein-activated K⁺ channel 4 (Girk4) channels underlying *IKACh* significantly reduced the degree of cholinergic regulation of heart rate under resting condition by 50% and inhibited the negative chronotropic response to pharmacological activation of muscarinic or adenosine receptors (Wickman et al., 1998). This study provided *in-vivo* evidence of the relevance of *IKACh* in controlling heart rate. Since preparations of isolated mouse sinoatrial node pacemaker cells were not available in the nineties, Wickman and coworkers underscored in the Discussion section of their article that complete dissection of the role of other ion channels involved in cholinergic regulation of heart automaticity would have required the characterization of the mouse sinoatrial node. The last author of the present review article was largely convinced by this assertion and readily began working in this direction. Indeed, the identification of mouse SAN and the isolation of pacemaker cells were considered as hardly feasible, chiefly because of the tiny size of this region (1mm²). However, in 2001, the journal *Cardiovascular Research* published a couple of articles on recordings of intracellular action potentials of the mouse SAN using sharp electrodes (Verheijck et al., 2001) and the first characterization of the funny current in pacemaker cells of mouse SAN (Mangoni and Nargeot, 2001). Since these first studies, the mouse SAN has become a popular model for studying the complex and partially elusive core of the pacemaker mechanisms.

The first study of ionic currents and $I_f$ in mouse SAN (Figure 1) confirmed that the current properties in mouse pacemaker tissue were similar to those previously described in the rabbit. Notably, none of the cloned HCN-channel kinetics known at the time matched perfectly those of mouse $I_f$, the current activation kinetics being slower than that recorded in cells expressing HCN1 or HCN2 alone, yet much faster than that of HCN4-mediated currents (Mangoni and Nargeot, 2001). These first data opened up the possibility of investigating the relevance of specific ionic currents, such as $I_f$, in the regulation of heart rate using genetically modified mice. Recordings of mouse SAN myocytes were then followed by a study on $I_f$ in Purkinje myocytes (Miquerol et al., 2004) and on pacemaker myocytes from the mouse atrioventricular node (Mangoni et al., 2006; Marger et al., 2011a; Marger et al., 2011b).
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## 2. Contribution of different HCN isoforms to the funny current and consequences for genetically modified mice

Early experiments by Shi and co-workers in rabbit and rat heart showed that rodent cardiac tissues express transcripts of HCN1, HCN2, and HCN4 channels (Shi et al., 1999). In addition, they found that in rabbit SAN, expression of HCN4 is predominant (>81%) relative to HCN1 (>18%) and HCN2 (0.6%) (Akbarali et al., 1999; Shi et al., 1999). In neonatal rat ventricles, known to express $I_f$, HCN2 mRNA was, instead, the dominant transcript with a 5:1 ratio of HCN2 to HCN4. A study by Ishi and co-workers (Ishii et al., 1999) confirmed that expression of the HCN4 isoform was prevalent in rabbit SAN and that the properties of the current resulting from heterologous expression of HCN4 were more similar to those of the native current as compared to the currents generated by HCN1 or HCN2 channels.

According to (Moosmang et al., 2001), also in the murine SAN, the most highly expressed HCN isoform is HCN4. However, *in-situ* hybridization experiments showed that, contrary to the findings of Shi and co-workers (Shi et al., 1999), HCN2 mRNA to be expressed more densely than HCN1 mRNA.

A first genome-scale regional ion channel expression profile in the mouse heart was obtained in 2005 (Marionneau et al., 2005). This study confirmed high expression of HCN1 and HCN4 in the SAN, and high expression in the atrioventricular node and atria. Significant HCN2 transcript levels were also detected in the SAN and ventricle. In the latter, HCN4 expression levels were found as well, while HCN3 expression was very low in all compartments. In conclusion, the expression profile found for the HCN gene family was consistent with previous studies in both rabbit and rat heart.

Whereas in early studies no significant expression of HCN3 mRNA was found in the cardiac conduction system or in the working myocardium, in later studies, the HCN3 isoform was shown to be expressed and functional in ventricular cells of murine heart (Fenske et al., 2011).

In conclusion, studies on the distribution of HCN transcripts in the heart provided important information that helped identify the potential composition of native funny channels, but they did not provide insights into the functional properties of native $I_f$, or its importance in mammalian pacemaking. Genetic targeting of *Hcn* gene family members and functional exploration of transgenic mice were the next steps aimed at advancing the knowledge of $I_f$ function. Since $I_f$ is encoded by different HCN isoforms showing different distributions and prevalence in the SAN, in the conduction system and in the working myocardium, it can be assumed that the generation of several mouse models based on the use of different gene targeting techniques will be required to address the role of
funny current in the generation and regulation of heart automaticity. Additional complexity is to be
expected when considering that native f-channels are heteromeric structures that are likely to be
formed by more than one HCN isoform. Compensatory effects exerted by an isoform different from
the deleted one are to be considered, too. Furthermore, f-channels may have different physiological
roles and effects depending on the developmental stage of the organism, and on other organs in
addition to the heart. Conditional and tissue-specific targeting is, thus, important. Finally, the
generation of mouse models in which a function of If is investigated without deletion of the channel
protein is desirable. This last objective has been achieved by studying models carrying modified
HCN4 devoid of cAMP-dependent regulation, or with silenced channel conductance (see below). In
the next two sections, we will summarize insights obtained from the knockout of Hcn genes and from
mice expressing modified HCN4 channels.

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3. Functional studies using genetic ablation of HCN channels

A first approach exploiting genetically modified mice to investigate HCN function was
developed by the generation of HCN2-deficient mice by systemic knockout of *Hcn2* (Ludwig et al.,
2003). As mentioned above, early measurements of mRNA had shown that, together with the
predominant isoform HCN4, also HCN1 and HCN2 are expressed in the mouse SAN, but no evidence
was available as to the relative importance of these two isoforms at the time. In the study by Ludwig
et al., the functional role of HCN2 was investigated, and it was proposed that this isoform plays an
important role in stabilizing mouse cardiac pacemaker activity (Ludwig et al., 2003). Indeed,
telemetric recordings of ECG in mice globally deficient in HCN2 channels (*Hcn2*/-) presented with
sinus dysrhythmia, evidenced by highly increased inter-beat variability. Pacemaker myocytes isolated
from *Hcn2*/- mice showed a 30% reduction in If density, associated with a negative shift of the
maximum diastolic potential. Mice with a cardiac-specific *Hcn2* knockout showed a similar
phenotype, confirming that dysrhythmia was caused by intrinsic SAN dysfunction.
Electrophysiological analysis of If in cells isolated from the SAN revealed that the current kinetics,
in both wild-type and mutant mice, were faster than HCN4-mediated currents recorded in
heterologous expression experiments. The overall findings were consistent with the hypothesis that
the *funny* current in mouse SAN pacemaker myocytes was generated by co-expression of HCN2 and
HCN4 channels, and that HCN2 contributed to basal excitability of SAN myocytes. However, this
conclusion was later challenged by data showing also a contribution of the HCN1 isoform to SAN If
(Fenske et al., 2013). The study by Ludwig et al. (Ludwig et al., 2003) did not only address the cardiac
phenotype generated in *Hcn2*/- mice but also the electrophysiological and functional abnormalities
caused by the *Hcn2* deletion in the nervous system. The authors found that thalamocortical relay
neurons displayed a pronounced hyperpolarization of the resting membrane potential.
Hyperpolarization was due to the deletion of the HCN2-mediated $I_h$ and induced a change in their
firing patterns. This change in intrinsic excitability increased the susceptibility to oscillations and
resulted in spontaneous absence seizures. The authors' study, thus, revealed an important role for
HCN2 in both cardiac automaticity and the control of neuronal and thalamocortical network
excitability.

Further insight into the contribution of HCN isoforms to pacemaker activity in mice was
gained by studying the effects of *Hcn4* knockout in the developing embryonic mouse heart (Stieber
et al., 2003a). This study showed that mice globally deficient in HCN4 (*Hcn4*⁻⁺), or mice with
inducible cardiac-specific deletion of *Hcn4* (*cHcn4*⁻⁺), exhibit embryonic lethality between E9.5 and
E11.5. In embryonic hearts from HCN4-deficient mice, expression of *I*<sub>f</sub> was reduced by 85% relative
to controls. Of note, while embryonic hearts still showed spontaneous contractions at a significantly
slower rate, their beating rate could not be modulated by cAMP. Mutant cardiomyocytes displayed
primitive low-amplitude spontaneous action potentials, but mature SAN-like action potentials, as
recorded from day 9 *post coitum* (E9) in wild-type, could not be recorded. The authors concluded that
HCN4 channels were essential for the basal activity of embryonic hearts even if, at least until E11.5,
the heart rate was sustained, also in the absence of HCN4, by other mechanisms such as other cardiac
HCN isoforms, or by spontaneous intracellular Ca²⁺ cycling (Viatchenko-Karpinski et al., 1999).

The relevance of the HCN1 isoform to mouse pacemaker activity was evaluated by analyzing
mice globally deficient in HCN1 (*Hcn1*⁻⁺) (Fenske et al., 2013). In this study, HCN1 protein
expression was analyzed in the SAN. HCN1 was found to be highly expressed and co-localized with
HCN4. *In-vivo* exploration of heart rate and rhythm in *Hcn1*⁻⁺ mice showed these channels play an
important role in determining SAN function. *Hcn1*⁻⁺ mice displayed hallmarks of SAN dysfunction
including bradycardia, sinus dysrhythmia, prolonged SAN recovery, and prolonged conduction time,
as well as recurrent sinus pauses. Electrophysiological characterization of *Hcn1*⁻⁺ SAN myocytes
showed a significant reduction in *I*<sub>f</sub> current density and slowing of current activation kinetics in line
with the hypothesis of HCN1 contributing to the fast component of *I*<sub>f</sub> activation, in comparison to the
kinetics of HCN4 currents recorded in heterologous expression systems. Spontaneous action
potentials in SAN pacemaker myocytes of *Hcn1*⁻⁺ mice had slower frequency and displayed an
arrhythmic-like phenotype with pronounced beat-to-beat interval variations. Overall, this work has
provided insight into the role of HCN1 in the SAN and thus laid the foundation for a mechanistic
understanding of potential HCN1-based SAN dysfunction in humans.

Until 2011, *Hcn4* knockout mice were generated using non-conditional deletion strategies,
either global or cardiac-specific (Stieber et al., 2003a), or Cre-loxP techniques allowing systemic
1 inducible knockout in adulthood (Herrmann et al., 2007). While, as we have mentioned above, 2 constitutive global or cardiac-specific *Hcn4*⁻/⁻ mice died *in utero*, inducible knockout mice exhibited 3 mild effects on cardiac automaticity and heart rate control, although some hallmarks of SAN 4 dysfunction were recorded in these mice. These include SAN pauses, or an increase in the beat-to-5 beat variability of heart rate, which is indicative of a reduction in the SAN-diastolic depolarization 6 reserve after the loss of *I*<sub>f</sub> function (Herrmann et al., 2007). The distinction between effects of global 7 cardiac-specific deletion of *Hcn4* and selective knockout in the rhythmogenic centers is of interest, 8 which was attempted by generating a mouse model of selective *Hcn4* knockout in the SAN. In this 9 mouse model, the tamoxifen-inducible Cre recombinase (CreERT2) was knocked into the *Hcn4* gene 10 itself (*Hcn4*<sup>CreERT2</sup>) (Hoesl et al., 2008). Analysis of this line showed reporter gene deletion in the 11 SAN and AVN, but not in Purkinje fibers, allowing a mouse model to be generated in which deletion 12 of *Hcn4* was selectively achieved in the SAN and AVN (*Hcn4*-KiT mouse) (Hoesl et al., 2008). 13 Similarly to observations with respect to the cardiac-specific *Hcn4* knockout, tamoxifen-inducible 14 deletion in the SAN during adulthood resulted in SAN pauses without significant slowing of basal 15 heart rate. Furthermore, neither global cardiac nor SAN-AVN-specific deletion of *Hcn4* altered the 16 heart chronotropic competence, indicating that mechanisms other than *I*<sub>f</sub> were also involved in the 17 autonomic control of the heart (Hoesl et al., 2008). The same authors employed an identical 18 *Hcn4*<sup>CreERT2</sup> line to obtain recombination-dependent expression of diphtheria toxin in SAN cells. This 19 is expected to induce the selective death of HCN4-expressing cells within the SAN. Tamoxifen-20 inducible selective deletion of HCN4-expressing cells in the SAN, intriguingly, resulted in a mild 21 phenotype, which, again, was characterized by SAN pauses and by only a moderate increase in 22 mortality (Herrmann et al., 2011). In line with all results obtained from these *Hcn4* knockout mouse 23 models, these authors proposed that, in adulthood, *I*<sub>f</sub> primarily constitutes a *depolarization reserve* 24 that ensures pacemaking under negative chronotropic challenges and is not required for generating 25 automaticity *per se*.

26 In 2011, Baruscotti and co-workers generated another tamoxifen-inducible and cardiac-27 specific *Hcn4* knockout mouse model based on the Cre-loxP system (Baruscotti et al., 2011). 28 Telemetric recordings of ECG showed that daily injections of tamoxifen in adult mice carrying the 29 *Hcn4*<sup>lox/lox,Cre</sup> gene to induce *Hcn4* deletion led to progressive slowing of heart rate. Heart rate slowing 30 was accompanied by second-degree atrioventricular blocks, subsequently degenerating into complete 31 heart block. Lethality because of heart block occurred when heart rate was approximately 50% of 32 basal rate before tamoxifen injection, and ECG showed only isolated P waves and no QRS complex 33 could be recorded (Baruscotti et al., 2011). None of the 14 mutant mice receiving tamoxifen treatment 34 survived more than 8.5 days. By contrast, no effect was observed in control mice receiving the same
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treatment. As expected, the time course of spontaneous action potential firing rate in SAN myocytes isolated from either control or mutant mice during tamoxifen treatment paralleled that of the heart rate of free-moving animals. All mutant mice developed complete atrioventricular block leading to heart arrest upon treatment with tamoxifen. These data led to the assumption that HCN4 channels are required for normal rhythm generation and conduction and represent a key molecular mechanism for cardiac pacemaking (Baruscotti et al., 2011). The reasons for these differences in phenotype severity between different inducible cardiac-specific *Hcn4* knockouts are not yet understood.

Of note, all these mice display hallmarks of SAN dysfunction, such as SAN pauses or increased susceptibility to cholinergic challenge. Notably, the phenotype of HCN4 knockout mice reported by Baruscotti and co-workers is qualitatively close to that observed in mice expressing silenced *I*<sub>f</sub> current (Mesirca et al., 2014) (see below).

## 4. Genetic silencing of cAMP-dependent regulation of f-channels

The control of the funny current by direct binding of intracellular cAMP to f-channels and the associated cAMP-dependent modulation of heart rate represent landmark findings of the early investigation of processes involved in the autonomic control of cardiac function (DiFrancesco and Mangoni, 1994; DiFrancesco and Tortora, 1991). Like most findings collected on funny current properties and pacemaker activity in the eighties and nineties, these data were obtained in the rabbit SAN. After the cloning of HCN channels, it became apparent that cAMP-dependent activation can substantially vary among different HCN isoforms. According to data from early heterologous HCN expression experiments, HCN2 and HCN4 isoforms were more sensitive to cAMP than was the HCN1 isoform, with cAMP-induced shifts in the order of 15-16 mV for HCN2 and HCN4, relative to shifts in the order of 2-7 mV for HCN1 (Moroni et al., 2001). Since native *I*<sub>f</sub> current shifts were intermediate between these values (about 10-11 mV (DiFrancesco et al., 1986)), these observations gave rise to the hypothesis that native funny channels could result from dimeric expression of different isoforms (Altomare et al., 2003; Moroni et al., 2001; Stieber et al., 2003b). Genetic manipulation of cAMP-dependent regulation of HCN channels combined with time- and tissue-specific control of HCN expression in mice provided new important insights into the contribution of *I*<sub>f</sub> to the regulation of heart rate.

In 2008, (Harzheim et al., 2008) reported on mice with HCN4 channels deficient in cAMP-dependent binding and regulation. By introducing the knock-in mutation R669Q in the CNBD, binding of cAMP to HCN4 channels was abolished. This mutation was lethal during embryonic development at day E11 in homozygous, but not in heterozygous mutants. Analysis of adult

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heterozygous mutant mice showed an increased number of sinus pauses and sinoatrial blocks after exercise, but the authors did not find significant changes in resting heart rate. Recordings of $I_f$ in isolated SAN myocytes showed that the wild-type current had faster activation and slower deactivation kinetics than the current recorded from heterozygous mutant mice. These results led the authors to suggest that the major function of cAMP-dependent regulation of HCN4 in adult mice is to prevent sinus pauses, and that this function can only be fulfilled when channels are activated by cAMP. In addition, these data indicated that cAMP-dependent regulation of HCN4 is necessary for terminal development of the mouse embryo.

A different approach to assess the role of cAMP-dependent regulation of SAN f-channels was developed by Alig and co-workers in 2009 (Alig et al., 2009). They generated mice with heart-specific and time-controlled expression of dominant negative human HCN4 channel (hHCN4-573X) with truncated C-terminus and cAMP-binding domain deficiency (Figure 2). These mutant HCN4 channels recapitulate the human HCN4-573X mutation that is predicted to abolish the cAMP-dependent control of HCN channels present in a founder individual with primary SAND (Schulze-Bahr et al., 2003). One potential advantage of this approach is the possibility of selectively affecting one of more functional properties of f-channels without deleting the protein itself, possibly sparing functions that depend on channel scaffolding or compartmentalization. In addition, it is possible to keep the expression of the mutant channel at low levels compared to endogenous channels while still obtaining total silencing of, for example, cAMP regulation. Indeed, the probability that a significant number of HCN tetramers contain no mutant dominant-negative HCN subunits is very low (Alig et al., 2009). Immunoblotting experiments showed expression of hHCN4-573X protein predominantly in the SAN and atria (Alig et al., 2009). $I_f$ in SAN myocytes expressing mutant hHCN4-573X channels showed normal current densities but displayed a hyperpolarized half-activation and total insensitivity to the adrenergic agonist isoproterenol. Expression of hHCN4-573X induced quiescence in a significant number of SAN myocytes and decreased the maximal spontaneous rate of pacemaker activity. The in-vivo heart rate of conscious hHCN4-573X mice has been measured at different degree of spontaneous physical activity. The heart rate of hHCN4-573X mice was consistently slower than that of control mice at both low and high degrees of physical activity by ≈80 beats per minute (see Fig. 5 in Alig et al., 2009) (Alig et al., 2009). On the one hand, these data demonstrated that cAMP-mediated control of $I_f$ is important in the regulation of basal and maximal frequency of SAN cells and of heart rate, providing evidence that $I_f$ plays a significant role in setting pacemaker rhythm at rest and during activity. On the other hand, heart rate adaptation to physical activity remained functional in transgenic mice expressing hHCN4-573X, thus indicating that mechanisms other than $I_f$ can regulate heart rate under adrenergic activation. This finding is not entirely surprising because
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abolishment of the $I_f$ contribution to cAMP-dependent modulation of heart rate does not eliminate the
contribution of other ion channels sensitive to cAMP-dependent regulation such as L-type Cav1.3
channels and ryanodine receptor-dependent intracellular $Ca^{2+}$ release (Baudot et al., 2020; Mangoni
et al., 2003; Torrente et al., 2016; Vinogradova et al., 2002).

**5. Interaction between depolarizing and hyperpolarizing contributions to pacemaker activity and relation to autonomic control of rate in mouse SAN: role of the funny current.**

All the results discussed above clearly indicate that cardiac rhythmogenesis is a complex
mechanism involving several actors that possibly play redundant or complementary roles. This
suggests that an accurate description of the pacemaker mechanism could be achieved by combining
the genetic manipulation of multiple ion channels in the same animal.

In 2013, Mesirca and co-workers (Mesirca et al., 2013) investigated the effects of genetic ablation of G protein-gated $K^+$ channel 4 channels (*Girk4*), which encode the muscarinic-activated $K^+$ current ($I_{KACh}$). The cardiac $K_{ACh}$ channel is a heterotetramer composed of GIRK1 and GIRK4-channel subunits (Wickman et al., 1997). However, because GIRK4 subunits are required for cellular trafficking of GIRK1, knockout of *Girk4* results in the complete loss of cardiac $I_{KACh}$ (Wickman et al., 1998). *Girk4* knockout *Girk4*⁻/⁻ mice exhibit slightly increased basal heart rate (10%) and a reduction of about 50% in the high- and low-frequency domain spectra of heart rate variability (Mesirca et al., 2013; Wickman et al., 1998). The negative chronotropic effect of acetylcholine (ACh) on pacemaker activity of isolated SAN myocytes was similarly reduced by 50%. Genetic ablation of $I_{KACh}$ did not affect other major ionic currents $I_f$ and $I_{CaL}$ (Mesirca et al., 2013). *In vivo*, *Girk4*⁻/⁻ mice displayed prolonged recovery time of basal heart rate after physical exercise, or after β-adrenergic stimulation of heart rate via intraperitoneal injection of isoproterenol. This work revealed that $I_{KACh}$ is an important mechanism in mouse SAN physiology for mediating recovery of resting heart rate after adrenergic activation. This function was previously undetected in the SAN of other species such as the rabbit (DiFrancesco et al., 1989) and illustrates the importance of genetically-modified mice in the field of cardiac automaticity. In addition, these data strongly suggested that $I_{KACh}$ acts as a permanent *background brake* of heart rate, in addition to the previously known regulatory effect on heart rate variability. This view has been confirmed in mice expressing silenced f-channels (see below).
The contribution of $I_{KACH}$ to autonomic-driven control of rhythm raises the question of whether this knowledge can be used to gain an understanding of the role of $I_f$ under physiological or pathological conditions. In general, the diastolic depolarization phase in SAN pacemaker myocytes can be visualized as a sort of *racing car* equipped with both an *accelerator* and a *brake* mechanism that can be controlled by autonomic nervous system stimulation of β-adrenergic and muscarinic receptors, respectively, resulting in a fine tuning of heart rate. In such a complex machinery, $I_f$ acts, among other channels, as an accelerator, while $I_{KACH}$ has the role of a brake (see metaphoric cartoon in Figure 1 of DiFrancesco (DiFrancesco, 2006)). To put these considerations in a concrete experimental context, and to take into account the correct reciprocal equilibrium between accelerating and decelerating elements needed for a proper control of normal physiological functions, Mesirca and co-workers (Mesirca et al., 2014) proposed that SAN dysfunction and atrioventricular block secondary to removal of a diastolic depolarization accelerator mechanism, such as the contribution of $I_f$, could be rescued by inactivating the $I_{KACH}$-mediated brake. To test this hypothesis, a complete conditional and time-controlled silencing of the *funny* current was achieved by expression of another dominant-negative, non-conductive hHCN4-channel subunit (hHCN4-AYA). The advantage of this approach is “silencing” $I_f$ conductance without removal of HCN channel proteins. This point can be important, since the effects of HCN deletion on scaffolding and organization of membrane caveolar microdomains in SAN myocytes (Barbuti et al., 2007) are unknown. This strategy is similar to that applied for creating mice expressing dominant-negative hHCN4-573X channels (Alig et al., 2009). Two lines of transgenic mice carrying the tTA transactivator and the mutant HCN4 are crossed (Figure 3). The resulting double transgenic mice are deprived of doxycycline, and the expression of dominant negative hHCN4-AYA channels is triggered to reach a maximum three weeks after antibiotic removal (Figure 3). This mouse model was characterized by electrophysiological analysis of single pacemaker cells and *in-vivo* telemetric ECG recordings. Complete silencing of $I_f$ in automatic myocytes of the SAN, the atrioventricular node and Purkinje fibers network had profound consequences on automaticity. Expression of mutant channels led to a dramatic drop in the action potential basal firing rate and in the occurrence of delayed afterdepolarizations (DADs) of membrane voltage, as well as in a failure of action potential generation in all three cell types analyzed. Intracellular $Ca^{2+}$ dynamics was also disrupted upon $I_f$ silencing, resulting in the loss of the coordination of local $Ca^{2+}$ release events, which normally lead to the generation of diastolic inward $Na^+/Ca^{2+}$ exchanger current and cell-wide calcium transients in wild-type SAN myocytes (Vinogradova et al., 2002). Confocal microscope line-scan analysis did not only confirm the generation of DAD events in mutant SAN pacemaker cells but also the decrease in the frequency of
spontaneous intracellular Ca²⁺ transients, which was only partially rescued under perfusion of increasing doses of isoproterenol. Following withdrawal of doxycycline from the diet and the resulting onset of hHCN4-AYA protein expression, the mutant mouse experienced progressive slowing of heart rate, accompanied by atrioventricular blocks, ventricular extrasystoles, and ventricular tachycardia (Mesirca et al., 2014). Once the interaction between the components working in opposite directions with respect to the control of cardiac rhythmogenesis was confirmed, the HCN loss-of-function in mutant mice was counterbalanced by crossing them with the *Girk4*⁻/⁻ mouse strain (Mesirca et al., 2013). Indeed, ECG recordings of double-mutant hHCN4-AYA/*Girk4*⁻/⁻ mice revealed improved heart rate in comparison to hHCN4-AYA counterparts and prevention of SAN pauses, atrioventricular dysfunctions and ventricular arrhythmia. These novel findings highlighted the importance of studying cardiac rhythmogenesis as a concerted balance between diverse ion channels with complementary physiological roles. The study by Mesirca and co-workers was the first to provide direct evidence of an interplay between an inward current *I*<sub>f</sub> and an outward current *I*<sub>KACh</sub> in generating heart automaticity, as originally proposed by Noble and co-workers for *I*<sub>f</sub> and background currents using a numerical model of SAN automaticity (Noble et al., 1992). The work by Mesirca et al. led also to the concept of “compensatory” targeting of *I*<sub>KACh</sub> to rescue sinus node dysfunction (Mesirca et al., 2016a; Mesirca et al., 2016b). Indeed, beside hHCN4-AYA mice, we have recently showed that genetic ablation or pharmacologic inhibition of *I*<sub>KACh</sub> can rescue sinus node dysfunction and atrioventricular block in mice lacking L-type Ca<sub>v</sub>1.3 and T-type Ca<sub>v</sub>3.1 channels (Bidaud et al., 2020a; Mesirca et al., 2016a). Finally, have showed also that the *I*<sub>KACh</sub> blocker tertiapin-Q normalizes the heart rate of mice expressing hHCN4-573X (Bidaud et al., 2020a). In conclusion, beside *I*<sub>f</sub>, the concept of *I*<sub>KACh</sub> targeting to restore the balance between inward and outward currents in pacemaking has been now extended to include other *accelerators* of diastolic depolarization such as L-type and T-type Ca²⁺ channels (Bidaud et al., 2020a; Mesirca et al., 2016a). It will be interesting to know if this interplay between inward and outward currents, which determines the cell’s *depolarization reserve*, also applies to the cardiac Na⁺/Ca²⁺ exchanger current (NCX1), which is another important contributor to the SAN depolarization reserve and an *accelerator* of diastolic depolarization (Bogdanov et al., 2001; Gao et al., 2013; Torrente et al., 2015) in cooperation with *I*<sub>f</sub> (Kohajda et al., 2019). In this respect, Mesirca and co-workers showed that automaticity in SAN myocytes isolated from *Girk4*⁻/⁻ mice has reduced sensitivity to application of ryanodine in comparison to automaticity of wild-type counterparts (Mesirca et al., 2016a).

In the same vein as Mesirca's et al.'s work, a study by Fenske and co-workers (Fenske et al., 2020) provided further insight into the role of HCN4 in cAMP-dependent regulation of pacemaker activity and rate control by the autonomic nervous system using a knock-in mouse model expressing
cAMP-insensitive HCN4 channels (HCN4FEA) (Fenske et al., 2020). While hHCN4-AYA expressing mice enable studying the role of $I_f$ without disrupting channel protein, the main advantage of the HCN4FEA mouse model resides in the possibility of studying the physiological role of cAMP-dependent regulation of $I_f$ without causing direct channel loss-of-function. Similarly to $I_f$ in mice expressing hHCN4-573X-channel subunits, HCN4FEA channels are insensitive to cAMP, yet their half-activation voltage is more positive compared to that of wild-type controls. hHCN4-573X and HCN4FEA mice are thus highly complementary, because activation of $I_f$ in hHCN4-573X-expressing myocytes is set as if intracellular concentration of cAMP was very low, while HCN4FEA channels mimic a slightly increased basal cAMP concentration. Fenske and co-workers proceeded on the assumption that one of the main functions of HCN4 channels is to counteract slowing of heart rate induced by vagal stimulation, that is, to prevent parasympathetic override, detrimental rate slowing, and deep bradycardia (see review article by Hennis et al. 2021, in this issue)(Hennis et al., 2021). As in previous studies in which the reduction or removal of HCN4 contribution to activity was investigated, the HCN4FEA mice exhibited bradycardia within a complex phenotype that was also characterized by sinus pauses, showing the importance of $I_f$ in dynamic regulation of heart rate. The authors proposed that rate slowing caused by removal of cAMP modulation of HCN4 is due to the presence of non-firing cells, which interact with firing cells in a tonic entrainment process. cAMP activation of HCN4 channels increases the fraction of firing vs non-firing cells, thus representing a mechanism that opposes augmented responses to vagal stimulation (Fenske et al., 2020).

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# 6. Notes on the role of the funny current in the atrioventricular node

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The atrioventricular node ensures conduction of the SAN impulse between the atria and ventricles. In addition, the atrioventricular node is able to drive the heartbeat in case of SAN failure. Even if the role of $I_f$ is generally thought to be prominent in pacemaking, genetically modified mice have provided evidence of an important role of the funny current in impulse conduction in the atrioventricular node. In 2011, Baruscotti and co-workers reported that atrioventricular node myocytes isolated from tamoxifen-treated Hcn4$^{lox/lox,Cre}$ mice showed about 50% reduction in $I_f$ density associated with the occurrence of atrioventricular conduction block (Baruscotti et al., 2011). In the same year, Marger et al. showed that, while hHCN4-573X-expressing mice did not show atrioventricular dysfunction, atrioventricular myocytes isolated from these mutants displayed ≈65% slowing of pacemaker activity in comparison to control myocytes (Marger et al., 2011a). The work by Mesirca et al. in 2014, was the first to correlate severe atrioventricular dysfunction with silencing of $I_f$ and reduced pacemaker activity in atrioventricular myocytes expressing hHCN4-AYA channels

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1 (Mesirca et al., 2014). Importantly, it showed also that atrioventricular block in mice expressing
2 hHCN4-AYA subunits was highly correlated with onset of sustained ventricular tachycardia (Mesirca
3 et al., 2014). In further support of a role of *I*<sub>f</sub> in atrioventricular conduction, mouse models in which
4 TRPM7 channels have been genetically ablated, show diminished *I*<sub>f</sub> densities in the atrioventricular
5 node and display atrioventricular blocks (Sah et al., 2013a; Sah et al., 2013b). Clinical studies indicate
6 that athletes are more prone to atrioventricular block than the general population. This phenomenon
7 provided an exciting chance to test the role of *I*<sub>f</sub> in atrioventricular conduction in a very relevant way
8 using mouse models of long-term training and *in vivo* ECG recordings in racing horses (Mesirca et
9 al., 2021b). Mesirca and co-workers have reported that mice trained for 5 months to swimming
10 develop 1st degree atrioventricular block, similarly to racing horses. Consistent with observations in
11 training induced SAN bradycardia, atrioventricular block was due to downregulation of HCN4
12 mRNA and *I*<sub>f</sub>.

13 It may seem counterintuitive that a slow-onset current activated upon hyperpolarization can
14 play an important role in impulse conduction, which is generally considered to be reliant on faster
15 voltage gated currents activated upon depolarization. However, this apparent paradox can be solved
16 by considering the concept of depolarization reserve, as it applies for the SAN diastolic phase. First,
17 spontaneously active mouse atrioventricular node myocytes express lower I<sub>f</sub> densities in comparison
18 to SAN myocytes (Marger et al., 2011b). More generally, previous work in mouse atrioventricular
19 node myocytes has demonstrated lower densities of the principal ionic currents involved in
20 pacemaking including Ca<sub>v</sub>1.3-mediated I<sub>CaL</sub> and Ca<sub>v</sub>3.1 -mediated I<sub>CaT</sub> as compared to SAN myocytes
21 (Marger et al., 2011a; Marger et al., 2011b). This suggests the total depolarization reserve of the
22 atrioventricular node to be lower than that of the SAN. On one hand, this will render the SAN
23 automaticity dominant over that of the atrioventricular node. On the other hand, I<sub>KACh</sub> activated by
24 vagal input will more easily destabilize conduction in the atrioventricular node after genetic silencing
25 of I<sub>f</sub> or genetic ablation of HCN4. In support of this hypothesis, is the observation that knockout of
26 I<sub>KACh</sub> restored atrioventricular conduction and prevented ventricular tachycardia in mice expressing
27 hHCN4-AYA channels (Mesirca et al., 2014). Similarly to I<sub>f</sub> silencing, genetic ablation of I<sub>KACh</sub>
28 restored atrioventricular conduction in Ca<sub>v</sub>1.3<sup>-/-</sup> mice (Mesirca et al., 2016a).

29 Taken together, these studies have demonstrated a previously unexpected role of the funny
30 current in atrioventricular conduction, but also in stabilizing ventricular rhythm. Future studies will
31 be required to fully understand the functional role of the funny current in impulse conduction.
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34 **7. General considerations on mouse models of I<sub>f</sub> function and of other currents involved in**
**pacemaking**
Since the first mouse model of knockout of *I<sub>KACb</sub>* in 1998 (Wickman et al., 1998) and description of mouse SAN automaticity in 2001 (Mangoni and Nargeot, 2001; Verheijck et al., 2001), many genetically modified mice have been created to study the role of *I<sub>f</sub>* and other currents involved in pacemaker activity. For more exhaustive overview of mouse models of ion channels involved in heart automaticity, as well as of models of inherited sinus node dysfunction and arrhythmias, the reader is referred to recent review articles (Mesirca et al., 2021a; Wallace et al., 2021).

As a general outcome, genetically modified mice carrying a *Hcn* gene knockout and mice expressing modified HCN isoforms have confirmed that *I*<sub>f</sub> plays an important role in the generation of SAN function, atrioventricular conduction and basal heart rate. Indeed, while the degree of severity of the phenotype varies among models and gene targeting approaches, SAN dysfunction is a consistent finding (see sections 3-5). Reasons for the variability in phenotype severity between different knockouts of *Hcn4* are poorly understood. Some potential factors may be considered. First, the exact composition in HCN subunits of SAN f- channels has not been fully elucidated, even if we assume HCN4 to be the predominant channel subunit. The possibility that residual HCN1 and HCN2 subunits may compensate for the loss of HCN4 in models, at different developmental stages and adulthood would need further investigation. In addition, different promoter lines driving expression of Cre recombinase in the heart and the SAN may have variable efficiency, leaving significant amounts of residual HCN4 in cell groups within the SAN or the conduction system. Finally, another important point is the capability of the physiological system to compensate for *I*<sub>f</sub> loss-of-function in the heart. Intact HCN4 function is a critical factor for heart automaticity in the embryo, while other currents may compensate for *Hcn4* knockout or pharmacologic inhibition of *I*<sub>f</sub> in adulthood, when the conduction system is fully developed, and the set of ion channels involved in pacemaking is fully expressed.

The mouse SAN has been used also for deciphering the role of *I*<sub>f</sub> in important physiological processes in the heart that are independent from the autonomic nervous system. The role of *I*<sub>f</sub> in setting the basal heart rate has been explored also in a mouse model of SAN bradycardia induced by training (D'Souza et al., 2014). It has been shown that physical training induces slowing of basal heart rate by a mechanism that is independent from the input of parasympathetic nervous system but is due to downregulation of mRNA coding for HCN4 and *I*<sub>f</sub> density, leading to a decrease of the intrinsic SAN beating rate (D'Souza et al., 2014). A recent work by D'Souza and co-workers shows that day-night regulation of HCN4 expression underlies circadian variations of SAN pacemaking and heart rate, again independently from the input of the parasympathetic nervous system (D'Souza et al., 2021). Regulation of the day-night cycle of intrinsic SAN automaticity is possibly one of the most
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important roles of the funny current in cardiac physiology. While these mouse models of $I_f$ function do not involve direct genetic manipulation, they are supportive of the role of this current in determining the heart rate as observed in different genetic models of HCN4 function in the heart (see also article by Boyett et al. in this issue).

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It is a general observation that the heart rate and pacemaker myocytes of adult mice carrying loss-of-function of $I_f$ or cAMP-dependent regulation of HCN4 do not show reduced response to sympathetic agonists, irrespectively of the severity of the phenotype, thereby demonstrating that other currents are involved in sympathetic regulation of cardiac automaticity. The NCX1 current is a core component of the *coupled clock* model of pacemaking (Lyashkov et al., 2018). NCX1 activity has been proposed to couple with spontaneous local $Ca^{2+}$ release from ryanodine receptors of the sarcoplasmic reticulum to initiate diastolic depolarization and mediate its regulation by the sympathetic nervous system (Lakatta et al., 2010). In line with this model, mice carrying knockdown of expression of NCX1 shows normal basal heart rate but a significant reduction of positive chronotropic response to isoproterenol (Gao et al., 2013). This is consistent with other works reporting reduction of sympathetic regulation of heart rate in mouse models of loss-of-function of type 2 ryanodine receptors (Broun et al., 2012; Shan et al., 2010). If the cardiac phenotype of mouse models carrying phosphorylation defective type 2 ryanodine receptors align with the *coupled clock* model of pacemaking, paradoxical results on SAN automaticity have been obtained in mouse models of inherited catecholaminergic polymorphic ventricular tachycardia (CPVT) (Neco et al., 2012; Wang et al., 2017). These mice show SAN bradycardia, despite increased basal and isoproterenol stimulated $Ca^{2+}$ release from type 2 ryanodine receptors. SAN bradycardia has been explained by reduced sarcoplasmic reticulum $Ca^{2+}$ load and concomitant tonic $Ca^{2+}$ dependent inhibition of $I_{CaL}$ (Neco et al., 2012; Wang et al., 2017).

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While we can consider that the activity of NCX1 supports automaticity following loss-of-function of $I_f$, recent data show that the funny current contributes to automaticity in mice with an atrial knockout of NCX1 (Torrente et al., 2015). Indeed, SAN automaticity in these mice is characterized by burst activity, in which automaticity alternates with periods of quiescence. Torrente and co-workers have shown that automaticity in isolated SAN/Atria preparations is slowed by the $I_f$ blocker ivabradine (Torrente et al., 2015). This work also revealed that arrest of pacemaker activity in NCX1 deficient SAN can be rescued by inhibition of $Ca^{2+}$ activated $K^+$ channels, again showing that several classes of ionic channels contribute to set the pacemaker action potential (Yue et al., 2020).

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Accumulating evidence shows that L-type $Ca_v$1.3 channels are a major component of the cardiac pacemaker mechanism. Indeed, $Ca_v$1.3 channel contribute to pacemaking in the mouse SAN
by at least three pathways. First, they drive $Ca^{2+}$ current throughout the range of voltage spanning the diastolic depolarization (Mangoni et al., 2003; Torrente et al., 2016). Second, this $Ca^{2+}$ current controls diastolic ryanodine receptor dependent $Ca^{2+}$ release during pacemaking (Torrente et al., 2016). Third, $Ca_v1.3$ channels constitute a necessary molecular component of the sustained inward $Na^+$ current $I_{st}$ (Toyoda et al., 2017). Consistent with these multiple pathways of contribution to pacemaking automaticity of $Ca_v1.3^/-$ SAN/atria preparations and that of isolated SAN myocytes is reduced by ≈80% after inhibition of $I_f$ by ivabradine (Baudot et al., 2020). These data strongly suggest that $Ca_v1.3$ channels constitute an effective mechanism sustaining automaticity under conditions of loss-of-function of the funny current.

## 8. Conclusions and future directions

Genetic targeting of the funny current in mice has provided new insights in the functional role of HCN channels in heart automaticity in general, but also into the role that other ion channels play in the cardiac pacemaker mechanism *in vivo*. This novel knowledge has confirmed early basic concepts on the relevance of the funny current and introduced new evidence of previously unknown mechanisms that demonstrate the high degree of complexity of the pacemaker process. At the same time, genetic studies on mice models have opened new research avenues for studying the mechanisms controlling pacemaker activity in arrhythmic disease. Genetically modified mice have demonstrated translational relevance also for studying the mechanistic bases of inherited or acquired SAN dysfunction and atrioventricular block in humans. For example, mice expressing hHCN4-573X channels (Alig et al., 2009) have helped elucidate the mechanism of familial $I_f$-mediated SAN bradycardia (Schweizer et al., 2010). Mice expressing hHCN4-AYA channels (Mesirca et al., 2014) present phenotypic traits similar to familial SAN dysfunction associated with ventricular tachycardia due to mutation in the HCN4 channel pore (Milano et al., 2014; Schweizer et al., 2014). hHCN4-AYA as models of HCN4-related rhythm disease, and $Ca_v1.3^/-$ and $Ca_v1.3^/-/Ca_v3.1^/-$ mice, as models of inherited SAN dysfunction (Baig et al., 2011) and congenital heart block (Torrente et al., 2020), respectively, have also demonstrated the relevance of $I_{KACb}$ in establishing SAN dysfunction and atrioventricular block, opening a new avenue of research on GIRK channels as therapeutic targets for managing SAN disease (Mesirca et al., 2016a; Mesirca et al., 2016b). Finally, mouse models of SAN bradycardia and atrioventricular block in athletes (Bidaud et al., 2020b; D'Souza et al., 2014; Mesirca et al., 2021b) are providing and invaluable contribution for understanding the role of $I_f$ in acquired
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bradycardia and will constitute precious models to test innovative therapeutic strategies (See the
article by Boyett et al. in this issue).

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In the future, new insights into the role of the funny current in pacemaking may be obtained
using mice with complex genotype involving targeting of more than one ion channel (see for example
work by Baudot and co-workers in 2020) (Baudot et al., 2020). Such an approach could be combined
with pharmacology and biophysics of ion channels, to overcome some limitations of gene knockout
approaches. It will be also important to further extend the use of mouse models for studying the
mechanisms of congenital disease of SAN pacemaking. This could be achieved by creating mutant
mice in which mutations causative of SAN dysfunction and heart block are inserted in the mouse
genome. This approach could be facilitated by the current availability of patients' databases of
rhythmic disease and Crisper-Cas9 directed mutagenesis. In addition, specific targeting of cellular
subpopulations within the SAN could be achieved using data obtained from single-cell RNA
sequencing of the SAN, the atrioventricular node and Purkinje fibres, which may help identify new
markers of unique pacemaker myocytes. This approach of "intersectional genetics" of specific classes
of pacemaker myocytes, combined with Cre-mediated recombination of construct alleles may
uncover new roles of the funny current, as well as other ion channels and receptors in heart
automaticity. Targeting HCN channels in specific subsets of pacemaker myocytes may also provide
new insights into the role of the funny current in cellular networks within the SAN, as recently
proposed by two recent works on the role of local diastolic Ca²⁺ release (Bychkov et al., 2020) and
the CNBD domain of HCN4 (Fenske et al., 2020).

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3 Acknowledgments

4 Matteo E. Mangoni wishes to acknowledge the paramount importance of the years he spent in Dario
5 DiFrancesco's laboratory working on the *funny* current during his PhD in Physiology (1989-1994).

6 He also wishes to thank Joël Nargeot for his mentorship in studying the biology of voltage-gated
7 calcium channels between 1995 and 1999, and for having supported the first recordings of the funny
current in sinoatrial node pacemaker myocytes in 2000-2001. He thanks also Wayne R. Giles for the
time spent at the University of Calgary (2001-2004) and for important scientific collaboration. Special
thanks go to Jacqueline Alig for characterizing the hHCN4-573X expressing mouse lines. We also
11 thank Laurine Marger for recording the *funny* current in hHCN4-573X expressing mice, and for the
work she provided in studying pacemaker myocytes of the mouse atrioventricular node and Purkinje
fibers. We thank the Réseau d'Animaleries de Montpellier (RAM) of the Biocampus facility for the
mouse lines. We wish to thank all personnel of the PCEA mouse breeding facility in Montpellier, and
of the iExplore platform, for their help in the functional exploration of our collection of genetically
modified mice.
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18 Funding

19 The IGF research group is a member of the Laboratory of Excellence “Ion Channel Science and
Therapeutics”, supported by a grant from ANR (ANR-11-LABX-0015). This work has been
supported by the *Fondation pour la Recherche Medicale "Physiopathologie Cardiovasculaire"*
(DPC20171138970 to M.E.M.), by the Agence Nationale de Recherche (ANR-15-CE14-0004-01 to
M.E.M.), and by the *Fondation Leducq* (TNE FANTASY 19CVD03 to M.E.M.).
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Figure Legends

Figure 1. Representative traces, from the first recordings in 2001, of the principal ionic currents expressed in isolated mouse sinoatrial node myocytes. Adapted from Mangoni et al. Prog. Biophys.

Mol. Biol. 90 (2006) 38-63.

Figure 2. Conditional hHCN4-573X expressing double transgenic (HCN4-CNBD) mice. (A) Schematic representation of single HCN subunits indicating the position of the mutation in the cyclic nucleotide-binding domain (CNBD) linker. (B) Representative scheme of the Tet-Off system. αMHC, alpha-myosin heavy-chain; tTA, Tet-Off system transactivator; Tre, tetracycline-responsive element; DOX, doxycycline. (C) Sample traces of I<sub>f</sub> recorded in isolated sinoatrial node myocytes from control (Cont, left panel) and mutant (HCN4-CNBD, right panel) animals together with current to voltage (I-V) relationships under tyrode (Tyr, black symbols), or isoproterenol (ISO 100 nM, red symbols) perfusion. (D) Representative examples of ECG traces recorded in control (left) and mutant animals on DOX (center), or water (right), at low (top) and high (bottom) levels of spontaneous home cage activity. Adapted from Alig et al. PNAS 106 (2009) 12189-12194.
<figure><img src="image_1.png" /></figure>

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<figure><img src="image_6.png" /><figcaption>Figure 1</figcaption></figure>
A

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B

Cont

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HCN4-AYA

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C

Sinus Pause

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Ventricular Tachycardia

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Atrial Fibrillation

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Atrio-ventricular block

<figure><img src="image_13.png" /></figure>
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B

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C

Cont

<figure><img src="image_16.png" /></figure>

HCN4-CNBD

<figure><img src="image_17.png" /></figure>

D

<figure><img src="image_18.png" /></figure>