Stem Cell Rev and Rep
DOI 10.1007/s12015-017-9745-1

A Singular Role of IK1 Promoting the Development of Cardiac
Automaticity during Cardiomyocyte Differentiation
by IK1–Induced Activation of Pacemaker Current
Yu Sun 1 & Valeriy Timofeyev 2 & Adrienne Dennis 1 & Emre Bektik 1,3 & Xiaoping Wan 1 &Kenneth R. Laurita 1 & Isabelle Deschênes 1 & Ronald A. Li 4,5 & Ji-Dong Fu 1

# Springer Science+Business Media New York 2017

Abstract The inward rectifier potassium current (IK1) is generally thought to suppress cardiac automaticity by hyperpolarizing membrane potential (MP). We recently observed that IK1
could promote the spontaneously-firing automaticity induced
by upregulation of pacemaker funny current (If) in adult ventricular cardiomyocytes (CMs). However, the intriguing ability of IK1 to activate If and thereby promote automaticity has
not been explored. In this study, we combined mathematicaland experimental assays and found that only IK1 and If, at aproper-ratio of densities, were sufficient to generate rhythmic
MP-oscillations even in unexcitable cells (i.e. HEK293T cells
and undifferentiated mouse embryonic stem cells [ESCs]). We
Yu Sun and Valeriy Timofeyev contributed equally to this study.
Electronic supplementary material The online version of this article(doi:10.1007/s12015-017-9745-1) contains supplementary material,
which is available to authorized users.
* Ronald A. Li
ronaldli@hku.hk* Ji-Dong Fu
jidong.fu@case.edu
1

Department of Medicine, Heart and Vascular Research Center,
MetroHealth Campus, Case Western Reserve University, 2500
Metrohealth Drive, Rammelkamp 650, Cleveland, OH 44109, USA

2

Department of Internal Medicine, University of California,
Davis, CA, USA

3

Ph.D. Program in Human Biology, School of Integrative and Global
Majors, University of Tsukuba, Tsukuba, Japan

4

Dr. Li Dak-Sum Center for Regenerative Medicine, University ofHong Kong, The Hong Kong Jockey Club Building forInterdisciplinary Research, LB 5-065 Sassoon Road,
Pokfulam, Hong Kong

5

Ming-Wai Lau Center for Regenerative Medicine, Karolinska
Institutet, Solna, Sweden

termed this effect IK1-induced If activation. Consistent withprevious findings, our electrophysiological recordings observed that around 50% of mouse (m) and human (h) ESCdifferentiated CMs could spontaneously fire action potentials(APs). We found that spontaneously-firing ESC-CMs
displayed more hyperpolarized maximum diastolic potential
and more outward IK1 current than quiescent-yet-excitable
m/hESC-CMs. Rather than classical depolarization pacing,quiescent mESC-CMs were able to fire APs spontaneouslywith an electrode-injected small outward-current that hyperpolarizes MP. The automaticity to spontaneously fire APs was
also promoted in quiescent hESC-CMs by an IK1-specific agonist zacopride. In addition, we found that the number of
spontaneously-firing m/hESC-CMs was significantly decreased when If was acutely upregulated by Ad-CGI-HCN
infection. Our study reveals a novel role of IK1 promoting
the development of cardiac automaticity in m/hESC-CMs
through a mechanism of IK1-induced If activation and demonstrates a synergistic interaction between IK1 and If that regulates cardiac automaticity.
Keywords IK1 . If . Rhythmic oscillation . Automaticity .
Embryonic stem cell . Cardiomyocyte differentiation

Introduction
Cardiac automaticity refers to spontaneously generated electrical impulses (i.e., action potentials). In the adult heart, it ispacemaker cells in the sinoatrial node that are dedicated togenerate impulses to maintain the heart’s rhythmic beating;however, in the fetal heart, all cardiomyocytes (CMs), including ventricular and atrial CMs, have high automaticity and areable to spontaneously fire action potentials (APs) [1]. It is still
poorly understood how cardiac automaticity is developed

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during CM differentiation. Cardiac automaticity is the resultof an ensemble of ion currents through sarcolemmal ion channels [2, 3]. Among those ion currents, a pacemaker Bfunny^current (If), encoded by the hyperpolarization-activated,
cyclic-nucleotide gated (HCN)-family of genes [4], is responsible for initiating the diastolic depolarization and determines
the rate of spontaneous pacemaker activity [5–7].
Overexpression of HCN channels in neonatal ventricular
myocytes increases the beating frequency [8], and injectionof adenovirus-HCN into the left atrium in vivo induces ectopic
pacemaking foci [9, 10]. In contrast, the inward rectifier potassium current (IK1) has been generally thought to play aninhibitory role in the induction of cardiac automaticity [2, 3].
IK1, encoded by Kir2.x genes [11], contains both inward(depolarizing) and outward (hyperpolarizing) current components; the outward component stabilizes the resting membranepotential (RMP) and contributes to the phase-3 repolarizationof APs [12–14]. IK1 is abundant in the working myocardiumof the atrium and ventricle but is low [15, 16] or nonmeasurable [17] in pacemaker cells of the sinoatrial node.
As a stabilizer, IK1 is generally recognized to suppress cardiac
automaticity. In a transgenic mouse model, Kir2.1 knockout
prolongs the AP duration (APD) and increases the number ofventricular CMs that can spontaneously fire APs [18].
Andersen-Tawil syndrome in humans with episodic cardiac
arrhythmias and developmental dysmorphic features are also
caused by mutations in Kir2.1 [14, 19]. It has been observedthat the low amount of IK1 and high amount of If contribute to
the high automaticity of fetal CMs [1, 20, 21] as well as CMs
differentiated from mouse (m) and human (h) embryonic stem
cells (ESCs) [22–24], which have been broadly used to understand mouse and human developmental biology and embryogenesis, including cardiogenesis of pacemaker cells and atrial
and ventricular CMs [22–28]. Our previous study found thatoverexpression of Kir2.1 facilitates the electrophysiologicalmaturation of m/hESC-differentiated CMs by converting
spontaneously AP-firing cells into quiescent-yet-excitableelectrophysiological-matured CMs [23]. Meanwhile, the strategy of suppressing IK1 has also been exploited to develop
bioengineered pacemakers. Knocking-down Kir2.1 channels
in adult ventricular CMs resulted in a depolarization of RMP
and induced endogenous pacemaker activity [14, 29], a similar result of developing bio-pacemaker cells from ventricular
CMs by overexpression of HCN channels [9, 10].
Interestingly, our studies observed that co-overexpression of
Kir2.1 with engineered-HCN1 could further enhance the induced automaticity of ventricular CMs to spontaneously fireAPs at a faster rate [30]. Given that If is activated by hyperpolarization, however, the intriguing ability of IK1 to activate If
and thereby promote automaticity has not been explored.
In the present study, we first applied a computational
mathematical-modeling assay to investigate the synergistic
interaction between IK1 and If and found that a combinationof IK1 and If was sufficient to induce rhythmic oscillation of
membrane potential (MP), which was experimentally replicated in unexcitable HEK293T cells and undifferentiated mouse
ESCs. Our electrophysiological study revealed a novel unexpected role of IK1 in promoting the induction of cardiac automaticity in m/hESC-CMs during cardiac differentiation. We
also found that acute upregulation of If induced by infection of
Ad-CGI-HCN virus inhibited the automaticity of m/hESCCMs, which is surprisingly different from an increased automaticity of CMs differentiated from a stable HCN4-transgenic
mESCs [31], but is consistent with our mathematicalmodeling assay.

Materials and Methods
Differentiation and Isolation of Mouse and Human
ESC-CMs
D3 cell line of mouse ESCs (ATCC) were cultivated and differentiated into spontaneously-beating CMs by embryoid
body (EB)-mediated differentiation [32]. The hES2 human
ESCs (ES Cell International [ESI], Singapore) were maintained as previously reported [33]. The hES2 cells were differentiated into CMs by co-culturing with the immortalized
endoderm-like END2 cells [26]. To isolate single CMs, beating outgrowths were microsurgically dissected from differentiated hESCs (Day 16 ~ 20) and mouse EBs (Day 7 + 4) by aglass knife, followed by incubating in collagenase solution(1 mg/ml) at 37 °C for 30 min [22, 27, 32]. The isolated cells
were incubated with KB solution containing (mmol/L): 85
KCl, 30 K2HPO4, 5 MgSO4, 1 EGTA, 2 Na2-ATP, 5 pyruvic
acid, 5 creatine, 20 taurine, and 20 D-glucose, at room temperature for 30 min. After the cells were plated on laminin-coatedglass coverslips for 1 h at 37 °C, the culture media was added
carefully and refreshed the next day.
Lentivirus- and Adenovirus-Mediated Gene Transfer
Fusion protein of human HCN4GFP or Kir2.1dsRed wasinserted into the pLV-EF1α-GFP plasmid to replace GFP at
the Pac I and Nde I restriction sites; these two lentiviral plasmids were used to mediate the upregulation of IK1 and If,
respectively. The recombinant lentiviruses were produced bytransient transfection of HEK293T cells as previously described [22, 24]. Briefly, the lentiviral plasmids pΔ8.91,
pMD.G, and pLV-EF1α-gene vector (2:1:3 mass ratio) wereco-transfected into HEK293T cells. The supernatants that contain lentiviral particles were harvested at 24- and 48-h posttransfection and stored at -80 °C before use. In the presence of
polybrene (6 μg/ml), lentiviral particles with a > 106 titerinfected cells with a multiplicity of infection of 3. The

Stem Cell Rev and Rep

fluorescence of GFP or dsRed was used to select positivelytransduced cells.
An adenovirus vector of pAd-CMV-GFP-IRES (Ad-CGI)with bioengineered mouse HCN1 (pAd-CGI-HCN) was used
to mediate the overexpression of If in m/hESC-CMs as in ourprevious study [10, 23, 34]. The empty adenovirus was usedas the control. Adenoviruses were generated by Cre-lox recombination of purified ψ5 viral DNA and shuttle vector
DNA. The recombinant products were plaque-purified, expanded, and concentrated by CsCl gradient, yielding concentrations of 1010 plaque formation unit (PFU)/ml. For transduction, adenoviral particles were added at a concentration of~2 × 109 PFU [10]. Adenoviral-infected cells were used forelectrophysiological characterization within two to four days
after adenoviral infection.
Electrophysiological Characterization
Electrophysiological experiments were performed at the single cell level by whole-cell patch-clamp with an Axopatch
200B amplifier and the pClamp10.2 software (Axon
Instruments Inc., Foster City, CA) [22–24, 35]. Patch pipettes
were prepared from 1.5 mm thin-walled borosilicate glass
tubes using a Sutter micropipette puller P-97 and had typicalresistances of 4–6 MΏ when filled with an internal solution
containing (mmol/L): 110 K-aspartate, 20 KCl, 1 MgCl2, 0.1
Na-GTP, 5 Mg-ATP, 5 Na2-phospocreatine, 1 EGTA, 10
HEPES, pH adjusted to 7.3 with KOH. The external
Tyrode’s bath solution consisted of (mmol/L): 140 NaCl, 5
KCl, 1 CaCl2, 1 MgCl2, 10 D-glucose, 10 HEPES, pH adjusted to 7.4 with NaOH. Voltage- and current-clamp recordings
were performed at 37 °C. The electrode potential was adjusted
to zero after immersion of the pipette tip, which caused apositive voltage bias of measured MPs and was corrected witha junction potential (−14.9 mV).
APs were directly recorded from spontaneously-firingm/hESC-CMs by current-clamp without electrical stimulus
and were elicited and recorded in quiescent cells with a stimulus of 0.1–1 nA for 5 ms. Based on the parameters of APs(Table 1), m/hESC-CMs were categorized into pacemaker,
atrial, or ventricular phenotypes [23, 36]. In brief, ventricularm/hESC-CMs have longer APD, APD50, and APD90 thanatrial cells; a ratio of the duration at different levels of 10%
repolarization, APD30–40/APD70–80 that indicates Bplateau^during phase 2, was used to categorize the cells as atrial(<1.4) or ventricular (≥1.4); the maximum depolarization rate
(dV/dTMax) of AP was applied to distinguish pacemaker cells(<5 mV/ms) from atrial and ventricular cells.
After AP characterization, IK1 and If were measured in thesame cell by switching current-clamp to voltage-clamp recordings. To measure IK1 and If, cells were held at a -30 mV
potential and pulsed for 2 s from 0 mV to -140 mV with
10 mV increments of every sweep, followed by a -100 mV

pulse for 1 s. IK1 was defined as 1 mmol/L Ba2+-sensitive
currents; If was represented by the remaining Ba2+-nonsensitive currents.
Single Cell Real Time RT-PCR
Right after patch-clamp recording of APs, some individual
hESC-CMs were manually picked up by the recording pipetteand transferred into 0.2 ml PCR tubes for reverse transcription(RT) and pre-amplification of cDNA by using CellsDirect
One-Step qRT-PCR kit (Invitrogen) together with pooled
primers (Supplemental Table 1). The amplified cDNA samples were diluted 20 times and stored in −80 °C. To evaluate
the gene expression of individual hESC-CMs, the standard
real time PCR was set up as the product manual of
SsoFast™ EvaGreen® Supermixe with low ROX (Bio-Rad),and data were collected and analyzed on an ABI-7500 realtime PCR system (Applied Biosystems). Ct values were used
to indicate the expression of genes. Supplemental Table 1 includes the information of primers.
Immunocytochemistry
Differentiated hESC-CMs (D21) were digested and plated onlaminin-coated glass coverslips. Cells were fixed for 15 min atroom temperature with methanol-acetone (1:1) mixture. Afterwashing with PBS, cells were incubated with 10% goat serumfor 30 min and then incubated with primary rabbit anti-Kir2.1
or anti-HCN4 antibody (Alomone Labs) together with mouseanti-α-actinin monoclonal antibody (Sigma). Alexa Fluor-488
goat anti-mouse IgG and Alexa Fluor-594 goat anti-rabbit IgG(Invitrogen) were used for fluorescence imaging. Coverslips
were mounted onto glass slides in Prolong Gold antifade
mountant with DAPI (ThermoFisher). Samples were imaged
on a confocal laser-scanning microscope (Leica TCS SP8).
Formulation of a Mathematical Model
A mathematical modeling approach was applied to investigate
the systematic coordination of IK1 and If by a well-establishedmathematical model that was originally established for rabbit
SAN pacemaker cells [37]. Rather than nine membrane currents applied in the original model, only IK1 and If, without
other ion currents, were included in our single cell model with
their typical kinetics. Time-dependent behavior of two currents is described using gating variables obeying first-order
Hodgkin-Huxley kinetics. Our model uses the same initialextracellular and cytosolic ion concentrations as that in theoriginal model [37]. MATLAB 6.5 was used to perform theprogramming assay. Differential equations were solved using
the Euler method. A fixed constant step of integration 0.01 ms
was used.

108.0 ± 4.7−67.4 ± 1.3

Paced (35)Firing (0)Paced (27)
136.5 ± 30.3

19.1 ± 1.2

103.9 ± 36.1
39.1 ± 14.2

20.8 ± 2.8
74.7 ± 11.8
66.6 ± 11.8

180.5 ± 34.7
116.0 ± 25.2
APD50 (ms)42.5 ± 6.8
27.9 ± 4.6

26.1 ± 5.1

276.1 ± 44.3
259.6 ± 39.8

67.0 ± 13.6
32.4 ± 3.0

30.2 ± 6.0

APD50 (ms)184.3 ± 31.9

46.4 ± 2.7

172.9 ± 55.9
66.3 ± 28.3

51.8 ± 5.2
123.8 ± 20.7
129.2 ± 10.7

230.6 ± 39.9
236.3 ± 34.8
APD90 (ms)127.5 ± 41.8
79.5 ± 9.4

88.3 ± 15.6

341.0 ± 46.7
370.8 ± 49.9

110.2 ± 24.4
113.2 ± 7.1

149.7 ± 7.3

APD90 (ms)2.0 ± 0.3

0.9 ± 0.2

0.9 ± 0.2
1.0 ± 0.2

0.9 ± 0.1
1.8 ± 0.4
1.9 ± 0.3

APD30–40 /APD70–80
1.0 ± 0.1
1.0 ± 0.2

2.8 ± 0.6
2.5 ± 0.6

0.9 ± 0.4

3.0 ± 0.7
2.7 ± 0.6

1.0 ± 0.4
0.9 ± 0.5

1.0 ± 0.2

APD30–40 /APD70–80

20.4 ± 1.6

21.1 ± 1.4

3.3 ± 1.3
27.0 ± 6.2

28.9 ± 3.4
34.7 ± 13.8
34.6 ± 8.6

15.8 ± 6.2
20.2 ± 1.7
dV/dTMax (mV/ms)
3.1 ± 0.8
38.2 ± 11.8

20.7 ± 1.7

15.2 ± 8.4
22.4 ± 5.3

35.7 ± 14.6
20.6 ± 4.4

4.7 ± 1.0

dV/dTMax (mV/ms)

110.5 ± 23.6
207.5 ± 65.4

175.7 ± 40.5

Firing Rate (fpm)
138.2 ± 69.1
273.0 ± 44.1

34.5 ± 7.0

35.7 ± 6.3

222.6 ± 76.0

48.2 ± 2.5

Firing Rate (fpm)

MDP, maximum diastolic potential; APA, action potential amplitude; APD, action potential duration; APD50 and APD 90, AP duration at 50% or 90% repolarization; APD30–40/APD70–80, a ratio of theduration at different levels of 10% repolarization which indicates Bplateau^ during phase 2; dV/dTMax, maximal rate of depolarization; fpm: firing per minute. Values are expressed as mean ± s.e.m.;*p < 0.05 and **p < 0.01, Spontaneously-Firing vs. Quiescent-Paced counterpart.

V
107.6 ± 5.3

44.4 ± 7.5
71.3 ± 10.8

−66.9 ± 3.1−73.9 ± 2.2*

Pacemaker (3)A Firing (4)V−71.1 ± 2.0

108.9 ± 7.7
84.6 ± 6.7
107.5 ± 6.4−64.8 ± 2.1−72.4 ± 3.1*
−67.5 ± 3.2

Paced (19)Firing (12)Paced (13)HCN

59.3 ± 5.6
80.0 ± 7.4
APA (mV)
49.2 ± 1.6
81.7 ± 12.4

V

−62.0 ± 3.0*
−57.5 ± 2.2
MDP (mV)−65.9 ± 0.9−71.5 ± 1.5**

83.7 ± 6.1

68.1 ± 3.1
90.9 ± 4.8−71.1 ± 4.3*
−64.7 ± 2.7

Firing (9)Paced (12)Pacemaker (0)A Firing (0)Paced (12)−55.4 ± 3.5

67.8 ± 15.8
86.4 ± 6.4

V48.3 ± 1.4−56.0 ± 3.9−69.5 ± 5.1*
−57.6 ± 2.6

Firing (4)Paced (6)

Pacemaker (2)A

APA (mV)Firing (8)Paced (8)
Mouse ESC-CMs
Control Pacemaker (3)A Firing (13)HCN

Control

MDP (mV)

Parameters of Action Potential Recorded in Human and Mouse ESC-CMs

Human ESC-CMs

Table 1

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Statistical Analysis
Data are expressed as mean ± S.E.M. Statistical significanceof differences in means was estimated by unpaired Student’s ttest or Chi-square (x2) test. p < 0.05 was considered statistically significant.

Results
IK1-Induced If Activation Programmed Rhythmic
Oscillations in in Silico Analysis
Since we recently found that I K1 could promote thespontaneously-firing automaticity induced by upregulationof If in adult ventricular CMs [30]; we first investigated how
IK1 and If synergistically interact with each other by mathematical simulation. Without any other ionic currents, only IK1and I f with a typical current-voltage (I-V) relationship(Fig. 1a) were included in a well-established mathematicalmodel that was originally developed for rabbit SAN pacemaker cells [37]. Remarkably, the rhythmic oscillation of MP, apacemaker-like activity, could be successfully programmed
by If and IK1 (Fig. 1b). The MP was oscillating between 80 mV and -35 mV in our simulation assay. In theory, theoutward current of IK1 hyperpolarizes MP, which subsequently activates HCN-channels; the inward current of If depolarizes MP, which then activates the outward current of IK1 tohyperpolarize MP again and to induce rhythmic MPoscillation (Fig. 1c). We termed this mechanistic model IK1–
induced If activation. Noticeably, inducing MP oscillation requires a large amount of If and a small amount of IK1, meaningthat any small-amount of increased IK1 will require much
more If to induce MP oscillation.
We calculated the oscillation rate after a 10-s simulation of
IK1 and If and found that a proper ratio of IK1 and If was critical
for the induction of this MP oscillation and controlled thefrequency of rhythmic oscillation (Fig. 1d). A specific levelof If and IK1 at spot Ba^ in Fig. 1d could induce a 3 Hz oscillation of MP, which was increased to 4.5 Hz with more IK1(spot Bb^); however, a further increasing of IK1 quickly suppressed the rhythmic oscillation (spot Bc^). This result demonstrated that a proper amount of IK1 could help If to accelerate this rhythmic MP oscillation. Interestingly, too much If
without a corresponding increase of IK1 suppressed the induction of MP oscillation (spot Bd^ in Fig. 1d).
Generating Rhythmic Oscillations in Unexcitable Cells
by IK1–Induced If Activation
We next investigated if this computational simulation ofrhythmic oscillation, driven by IK1–induced If activation, happens in a real biological system. We introduced two fusion

proteins––Kir2.1dsRed and HCN4GFP––into unexcitable
HEK293T cells by lentiviral transduction (Fig. 2a) and measured a huge IK1 with a big outward current (8.0 ± 6.1pA/pF at-60 mV, n = 8) and an intermediate inward current of If(−3.7 ± 2.0 pA/pF at -60 mV, n = 8) in the Kir2.1dsRed/
HCN4GFP-cotransduced cells (Fig. 2b-c). With such a huge
IK1, the MP of HEK293T cells that overexpressed Kir2.1 were
hyperpolarized to −92.1 ± 0.5 mV (n = 68) and−91.1 ± 1.9 mV (n = 24) with and without the expression of
HCN4 channels, respectively (Fig. 2d), while the MP wasmuch more positive in wild-type (−28.2 ± 1.1 mV, n = 21)
and HCN4-transduced (−35.3 ± 1.9 mV, n = 19) cells.
Remarkably, the MP of HCN4/Kir2.1-co-transduced
HEK293T cells was rhythmically oscillating between 85 mV and -35 mV when IK1 was partially blocked by
Ba2+ perfusate (40 μmol/L, n = 5 out of 11) or when theblocked IK1 was partially recovered (n = 8 out of 11) by
washing out Ba2+ (Fig. 2e). Neither HEK293T cells thatoverexpressed Kir2.1 alone nor those that overexpressed
HCN4 alone could ignite a rhythmic electrical oscillation(Fig. S1), indicating a unique ying-and-yang interaction
between IK1 and If. Indeed, this pacemaker-like rhythmic
electrical activity driven by IK1 and If could be replicatedin undifferentiated mESCs (another unexcitable cell type)(Fig. S2). When Kir2.1dsRed was introduced into mESCs,
in which we could record I f but not IK1 [35], its MP
became hyperpolarized as anticipated (Fig. S2c).
Similarly, spontaneous MP-oscillations between -70 mV
to -35 mV were observed in Kir2.1-transduced mESCs(n = 3 out of 6) when I K1 was partially blocked by
Ba2+. Collectively, our experiments demonstrated thatthe unique synergistic interaction between IK1 and If issufficient to program a rhythmic electrical activity in unexcitable cells.
In HCN4/Kir2.1-transduced HEK293T cells, IK1 hyperpolarizes MP and If is the major inward current for depolarization. Given that If is insensitive to millimolar concentrations of external Ba2+ [4], If would be expected to remain
largely unchanged while I K 1 is modulated by Ba 2 +(40 μmol/L) perfusion or wash-out (Fig. 2e). When Ba2+
was administered to quiescent HCN4/Kir2.1 HEK293T
cells, IK1 was decreased gradually, yielding rhythmic oscillations of MP (time point Ba^ of Fig. 2e); when IK1 wasfurther decreased as Ba2+ blockade continued, the firing rate
decreased (time point Bb^) until the cells subsequently became quiescent at a MP (< −45 mV) that was no longer
hyperpolarized by insufficient outward current of IK1 for
If-activation. Oscillations were restored during Ba2+ washout when IK1 gradually increased back to the suitable range
where IK1-activated If activation takes place. These experiments successfully replicated our computational assay that asynergistic interaction of IK1 and If is sufficient to induce
rhythmic MP oscillation.

Stem Cell Rev and Rep
Fig. 1 The in silico simulationshows that IK1–induced If
activation programs rhythmic
oscillations of membranepotential (MP). a Representative
Current-Voltage (I-V) curves of
IK1 (red) and If (green) wereapplied in the mathematic model.
b Only IK1 and If, without otherion currents, were sufficient toprogram rhythmic oscillations of
MP. c A schema illustrates themechanism of how IK1–induced If
activation generates rhythmic
oscillation of MP. d A surface
contour shows that the ratio of IK1and If regulates the frequency ofrhythmic oscillation

IK1 Improves the Induction of Cardiac Automaticity
of m/hESC-CMs during Differentiation
We next investigated if IK1–induced If activation plays a role inregulating cardiac automaticity and studied the EP properties of
m/hESC-differentiated CMs (Table 1). Around half of m/
hESC-CMs could spontaneously fire APs, including ventricular and atrial CMs, with the remaining being quiescent-yetexcitable (Fig. 3a-b). We found that the maximum diastolicpotential (MDP) of spontaneously-firing ESC-CMs was significantly more hyperpolarized than that of quiescent cells, which,upon electrical pacing, could elicit an AP with a phase 4-like
depolarization but failed to elicit subsequent APs. The MDP ofquiescent-yet-excitable atrial and ventricular mESC-CMs were
−64.8 ± 2.1 mV (n = 19) and −67.5 ± 3.2 mV (n = 13), respectively; while the MDP was more hyperpolarized in
spontaneously-firing atrial (−71.5 ± 1.5 mV, n = 13,
p = 0.002) and ventricular (−72.4 ± 3.1 mV n = 12,
p = 0.026) mESC-CMs (Fig. 3c). The same was also true forhESC-CMs (Fig. 3d and Table 1). We also performed single
cell qRT-PCR and confirmed that cells that elicited APs in ourpatch-clamp recording were all CMs with high expression ofcardiac sarcomere genes (i.e. TNNT2, MYH6, MYL7, MYL3)
and cardiac ion channels (Fig. 3e). The mRNA expressions ofion channels varied among individual ESC-CMs and had no
significant difference between quiescent-yet-excitable andspontaneously-firing cells. We found that HCN4 and KCNJ2(Kir2.1) were the major channels mediating IK1 and If in hESCCMs. Consistently, our immunocytochemistry staining confirmed that both HCN4 and Kir2.1 were expressed in hESCCMs, which had formed typical sarcomere structure (Fig. 3f).

We then measured functional currents of IK1 and If andfound that a low amount of IK1 could be measured only inventricular mESC-CMs (Fig. 4a) and hESC-CMs [23], while
If was recorded in all m/hESC-CMs. We found that a bigger If
was measured in quiescent cells than in spontaneously-firing
mESC-CMs (Fig. 4b), but the stable activation curves of If in
both types of cells had no difference (Fig. 4c). However,
spontaneously-firing ventricular mESC-CMs (n = 4) had arelatively bigger outward current of IK1 than quiescent-yetexcitable ventricular cells (n = 5, p = 0.04, Fig. 4d), whichmight be the cause of a relatively positive MDP in quiescentm/hESC-CMs.
Rather than classical depolarization pacing, we applieda modest outward current to mimic IK1 for hyperpolarizing the MP and investigated whether the low automaticity
of quiescent ESC-CMs is caused by the relatively positive
MDP. Upon injection of a 30pA outward current, interestingly, the quiescent-yet-excitable mESC-CMs fired APs
spontaneously (n = 5) (Fig. 4e). We also treated hESCCMs with zacopride, which could specifically enhance IK1
in isolated rat CMs without influence on other ion currents [38], to study if the automaticity can be developed inquiescent-yet-excitable ESC-CMs by an increased IK1, after we validated that zacopride (10 μMol/L) had no influence on If (Fig. S3). We found that zacopride could induce automaticity in quiescent-yet-excitable hESC-CMs(6 out of 8 cells) to spontaneously fire APs (Fig. 4f).
Our results demonstrated that an increased IK1 could enhance the automaticity of m/hESC-CMs and hyperpolarization of MP is important for the development of cardiac
automaticity.

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Fig. 2 Pacemaker-like rhythmic oscillations of MP could be generated
by IK1 and If in bioengineered HEK293T cells. a Two fusion proteins,
HCN4GFP and Kir2.1dsRed, were introduced into HEK293T cells bylentivirus infection. A bar indicates 20 μm. b Representative IK1 and If
were recorded in bioengineered HEK293T cells. c I-V curves of IK1 and If
in bioengineered Kir2.1dsRed/HCN4GFP HEK293T cells. d The RMP
of HEK293T cells with/without infection. **p < 0.01 vs. non-infected

cells. e The oscillation of MP was recorded by patch clamp in abioengineered HEK293T cell co-transduced with LV-HCN4GFP and
LV-Kir2.1dsRed, when IK1 was partially inhibited by Ba2+. The inset
panel compared the oscillations at two different time points (a and b),
showing that an increased IK1 could enhanced the firing rate ofoscillations

Acute Upregulation of If Inhibited the Automaticity
of m/hESC-CMs

adenovirus of Ad-CGI-HCN, which was used to convert
quiescent adult ventricular CMs into spontaneouslyfiring pacemaker-like cells [10, 34]. Ad-CGI-HCN infection significantly increased I f in hESC-CMs(Fig. 5a-b). We found that If upregulation had no significant effect on changing atrial or ventricular cell
types of hESC-CMs (Fig. 5c). However, the percentageof spontaneously-firing hESC-CMs was significantly decreased from 45.5% in the control (n = 33) to 28.5%

Since CMs differentiated from a stable transgenic cellline of mESCs overexpressing HCN4 showed significantly more rapid beating capability [31], we also investigated if bio-engineered pacemaker cells could be
developed from differentiated ESC-CMs by an acute
upregulation of I f . We infected hESC-CMs with

Stem Cell Rev and Rep

Fig. 3 Spontaneously-firing m/hESC-CMs had a more hyperpolarized
maximum diastolic potential (MDP). a-b Representative APs of quiescent and spontaneously-firing mouse (a) and human (b) ESC-CMs. c-d
The MDP of spontaneously-firing cells were significantly more
hyperpolarized than that of quiescent cells in mouse (c) and human (d)ESC-CMs. *p < 0.05, **p < 0.01. e A heat map of single cell qRT-PCR
shows gene expression in individual atrial (a) and ventricular (v) hESCCMs of our patch-clamp recording. f Immunocytochemistry showed that
hESC-CMs formed typical sarcomere structure and expressed HCN4 andKir2.1. Bars indicate 10 μm(n = 28, p = 0.03) in Ad-CGI-HCN cells (Fig. 5d). The
spontaneously-firing rate of Ad-CGI-HCN-transduced
ventricular hESC-CMs (34.5 ± 7.0 fpm, n = 8) had no
significant difference than that in control cells(35.7 ± 6.3 fpm, n = 9, Table 1). The same result
was also observed in mESC-CMs. There was around
46.7% of control mESC-CMs (n = 60) that could spontaneously fire APs; however, most Ad-CGI-HCN–Stem Cell Rev and Rep

Fig. 4 A hyperpolarized MP enhanced the automaticity of quiescentm/hESC-CMs. a Representative IK1 and If were recorded in ventricular
mESC-CMs. b-c The I-V curve and stable activation curve of If in quiescent (n = 5) and spontaneously-firing (n = 4) ventricular mESC-CMs. d
The I-V curve of IK1 showing that spontaneously-firing ventricular
mESC-CMs (n = 4) displayed a bigger outward current of IK1 thanquiescent-yet-excitable ventricular mESC-MCs (n = 5). e QuiescentmESC-CMs (n = 5) could spontaneously elicit APs when a properamount of electrode-injected outward current hyperpolarized the MP. f
The automaticity of quiescent hESC-CMs (6 out of 8, right) was enhanced to fire APs spontaneously after zacopride treatment. Dash lines
indicate 0 mV, ▲ indicates an electrical stimulationtransduced mESC-CMs became quiescent-yet-excitableand only 10.1% of them (n = 69, p = 0.01) could fireAPs spontaneously (Fig. 5e) without enhancement of thefiring rate (Table 1). These experiments demonstrated
that acute upregulation of If inhibited the automaticity
of m/hESC-CMs.

Discussion
In this study, we investigated a synergistic interaction of IK1and If that regulates cardiac automaticity and found that IK1induced If activation could generate pacemaker-like rhythmic
oscillation of MP even in otherwise unexcitable cells. We

Stem Cell Rev and Rep

Fig. 5 Acute upregulation of If suppressed the automaticity of m/hESCCMs. a Representative traces of If recorded in Ad-CGI- (control) and AdCGI-HCN–transduced (HCN) ventricular hESC-CMs. b The I-V curvesof If in control and HCN-ventricular hESC-CMs. c Acute upregulation of

If didn’t change chamber-specific cell types of hESC-CMs. d-e Acuteupregulated If significantly decreased the number of spontaneouslyfiring cells in human (d) and mouse (e) ESC-CMs. *p < 0.05

found that IK1 and If could influence each other in enhancing
or suppressing cardiac automaticity. A small amount of increased IK1 could improve the development of cardiac automaticity in m/hESC-CMs through a mechanism of IK1-induced If activation, which is a singular role of IK1 in regulating
cardiac automaticity. We also found that acute upregulation ofIf surprisingly suppressed cardiac automaticity of m/hESCCMs in which a low amount of IK1 was measured. Our study
advances the conventional view of IK1 in regulating cardiac
automaticity of spontaneously-firing APs.
As opposed to the dynamic nature of spontaneously-firingcells, the RMP of quiescent cells is highly stable until they get
excited and is mostly determined by the concentrations andconductance of different ions across the membrane.
Undifferentiated cells show highly variable RMP, which isusually significantly more positive than that of differentiatedcells [39]. In our recordings, the RMP was around -30 mV in
unexcitable HEK293T cells (Fig. 3c) and -15 mV in undifferentiated mESCs. The normal RMP of adult ventricular CMs is
hyperpolarized to -90 mV, mostly due to a constant outward
leak of IK1 potassium current through inward rectifier channels [14, 29]. Therefore, the RMP is being gradually
hyperpolarized during the differentiation and maturation ofCMs. The development of sarcolemmal If plays a major role
to initiate a sarcolemmal electrical automaticity of firing APs[5–7], which was found being developed earlier than cell contraction during CM-differentiation in chicken embryos [40,41]. By definition, the activation of I f requires a
hyperpolarized MP; therefore, hyperpolarization of RMP during CM differentiation is indeed critical for the ultimate development of automaticity in differentiated muscle cells. It has
been found that MP hyperpolarization by IK1 is critical forskeletal muscle differentiation from human myoblasts [42].
Consistently, our EP studies found that spontaneously-firingm/hESC-CMs displayed more hyperpolarized MDP than quiescent ESC-CMs (Fig. 4), in which automaticity was enhanced to fire APs spontaneously by an IK1-specific agonist
zacopride or an electrode-injected outward current. Therefore,
we speculate that quiescients m/hESC-CMs might be less differentiated at an intermediate differentiation stage between
spontaneously-firing ESC-CMs and cardiac progenitor/
precursor cells. The presence of a small amount of IK1 hyperpolarizes RMP and plays a critical role in developing cardiac
automaticity in newly-differentiated CMs through a mechanism
IK1-induced If activation. During the later maturation process, afurther increased IK1 together with a decreased If suppresses
cardiac automaticity in adult ventricular and atrial CMs.
Another possibility that could explain less IK1 measured inquiescient m/hESC-CMs is the vulnerability of IK1 to cellisolation methods [43], which could cause a decreased IK1 insome m/hESC-CMs and consequently led to a suppressed
automaticity. However, this explanation also suggests a novelrole of IK1 in enhancing cardiac automaticity. Indeed, in contrast to the increased beating frequency of ventricularStem Cell Rev and Rep

myocytes, a consistently slower sinus-paced rhythmic heart
rate in the Kir2.1-knockout mice [18] may also indicate thepossibility that IK1 has a critical role of enhancing cardiac
automaticity.
The mechanism of IK1-induced If activation is consistent
with previous studies of developing biological pacemakers.
The amount and ratio of IK1 and If is critical to eliciting rhythmic electrical activity. Normally, adult cardiac ventricular
muscle cells have a large amount of IK1 and a low level ofIf; therefore, both strategies of down-regulating IK1 [29] andup-regulating If [9, 10] in ventricular CMs could successfully
develop bio-engineered pacemaker cells to spontaneously fireAPs. Saito et al. established a transgenic cell line of mouse
ESCs stably overexpressing HCN4GFP and found that CMs
differentiated from HCN4GFP-transgenic mESCs displayed
an enhanced automaticity [31]. Although they didn’t categorize mESC-CMs into chamber-specific cell types for the comparison of automaticity, this study still inspired us to develop
bio-pacemaker cells from m/hESC-CMs. Unfortunately, acute
upregulation of If actually suppressed cardiac automaticity in
m/hESC-CMs, which was also observed in atrial CMs [44].
Although it is unknown if a constant overexpression of HCN4
in pervious study [31] or acute overexpression of HCN4 in ourcurrent study would influence the expression of immaturity
markers in m/hESC-CMs, our study demonstrates that m/
hESC-CMs couldn’t be directly converted into biopacemaker cells by acute-upregulation of If.
It has been noticed that different ESC lines have distinct
cardiogenic preferences [45] and the culturing and differentiation methods of ESCs have a big impact the efficiency andcardiac lineages of CM-differentiation [46], which might possibly influence the expression of cardiac ion channels andsubsequently affect the development of cardiac automaticity.
This might be an important concern for induced pluripotent
stem cells (iPSCs), which remain an epigenetic memory of thedonor cell source [47]. In our study, we found that IK1-induced
If activation could regulate the automaticity of mouse andhuman ESC-CMs differentiated with two different methods,
EB-differentiation and inductive co-culture, indicating a general role of IK1-induced If activation during the developmentof cardiac automaticity. This is consistent with previous finding that the epigenetic memory of cardiac somatic cell source
does not influence the functional outcome of iPSCdifferentiated CMs, while it improves cardiac differentiation
efficiency [48].
One limitation is that our current study focused on regulating cellular automaticity by the sarcolemmal currents of IK1induced If activation but didn’t include the influence of intracellular Ca2+ transients, which coordinates together with sarcolemmal ion currents to control cellular automaticity [49,50]. Recently, Vaidyanathan et al. found that upregulated IK1
could enhance the amplitude of Ca2+ transients in iPSC-CMs[51]; therefore, the automaticity development promoted by

IK1-induced If activation might be also mediated partiallythrough intracellular Ca2+ transients. In addition, only Kir2.1
was utilized to study I K 1 -induced If activation inbioengineered-HEK293T cells. It has been found that Kir2.1
is the major component of murine IK1, but Kir2.2 also contribute to the native IK1 in mouse ventricular CMs [18].
Therefore, It is possible that both Kir2.1 and Kir2.2, or even
Kir2.3, contribute to the development of cardiac automaticity
in m/hESC-CMs.
In conclusion, our study explored the synergistic interaction between IK1 and If and revealed a singular role of IK1 inthe ultimate development of cardiac automaticity in m/hESCCMs by IK1-induced If activation. Our study provides a morecomprehensive understanding of IK1 regulating cardiac automaticity, which will guide us to investigate the role of IK1 inarrhythmogenesis and develop efficient strategies for antiarrhythmic therapy.
Acknowledgments We are grateful to Dr. Jill Dunham for editorialassistance. This work was supported by the Start-up Fund from The
MetroHealth System (to J.D.F.) and grants from the American Heart
Association-13SDG14580035 (to J.D.F.), the Research Grant Council(TRS T13-706/11 to R.A.L.) and the National Institutes of Health(NIH)-R01HL096962 (to I.D.), NIH-R21HL123012 (to K.R.L.).
Compliance with Ethical Standards
Disclosures No disclosure of conflict interest.

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