Circulation Research

ORIGINAL RESEARCH

Fibroblasts Drive Metabolic Reprogramming in

Pacemaker Cardiomyocytes
Pei-Chun Chou,* Chih-Min Liu ,* Ching-Hui Weng, Kai-Chien Yang , Mei-Ling Cheng, Yuh-Charn Lin, Ruey-Bing Yang ,
Bai-Chuang Shyu , Song-Kun Shyue, Jin-Dian Liu, Shih-Pin Chen , Michael Hsiao, Yu-Feng Hu

BACKGROUND: The sinoatrial node (SAN) is characterized by the microenvironment of pacemaker cardiomyocytes (PCs) encased
with fibroblasts. An altered microenvironment leads to rhythm failure. Operable cell or tissue models are either generally
lacking or difficult to handle. The biological process behind the milieu of SANs to evoke pacemaker rhythm is unknown. We
explored how fibroblasts interact with PCs and regulate metabolic reprogramming and rhythmic activity in the SAN.
METHODS: Tbx18 (T-box transcription factor 18)-induced PCs and fibroblasts were used for cocultures and engineered tissues,
which were used as the in vitro models to explore how fibroblasts regulate the functional integrity of SANs. RNA-sequencing,
metabolomics, and cellular and molecular techniques were applied to characterize the molecular signals underlying metabolic
reprogramming and identify its critical regulators. These pathways were further validated in vivo in rodents and induced
human pluripotent stem cell-derived cardiomyocytes.
RESULTS: We observed that rhythmicity in Tbx18-induced PCs was regulated by aerobic glycolysis. Fibroblasts critically
activated metabolic reprogramming and aerobic glycolysis within PCs, and, therefore, regulated pacemaker activity in PCs.
The metabolic reprogramming was attributed to the exclusive induction of Aldoc (aldolase c) within PCs after fibroblast-
PC integration. Fibroblasts activated the integrin-dependent mitogen-activated protein kinase-E2F1 signal through cell-
cell contact and turned on Aldoc expression in PCs. Interruption of fibroblast-PC interaction or Aldoc knockdown nullified
electrical activity. Engineered Tbx18-PC tissue sheets were generated to recapitulate the microenvironment within SANs.
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Aldoc-driven rhythmic machinery could be replicated within tissue sheets. Similar machinery was faithfully validated in de
novo PCs of adult mice and rats, and in human PCs derived from induced pluripotent stem cells.
CONCLUSIONS: Fibroblasts drive Aldoc-mediated metabolic reprogramming and rhythmic regulation in SANs. This work details
the cellular machinery behind the complex milieu of vertebrate SANs and opens a new direction for future therapy.
GRAPHIC ABSTRACT: A graphic abstract is available for this article.

Key Words: glycolysis ◼ fibroblasts ◼ integrins ◼ metabolomics ◼ vertebrates

In This Issue, see p 1 | Meet the First Author, see p 2 | Editorial, see p 21

T
he sinoatrial node (SAN) initiates electric impulses
for every heartbeat to maintain life. Its dysfunction
causes a slow heart rate, insufficient blood supply,
and detrimental consequences such as cardiac arrest.1 In
contrast to atrial or ventricular cardiac tissue, the SAN
consists of a network of pacemaker cardiomyocytes
(PCs) encased with abundant fibroblasts and a heteroge-
neous connective tissue microenvironment.2–5 This unique
structure of SANs is well conserved across vertebrate
species.5–7 The microenvironment, through the integration
of PCs, mesenchymal lineages (including fibroblasts),
and extracellular matrix (ECM) organization, is required
for the rhythmic activity of SANs during embryogenesis.4
Failure of ECM organization and likely fibroblast inte-
gration results in electrical dysfunction of SANs.4 At the
other extreme, extensive fibrosis of the SAN also leads to
pacemaker failure.8,9 Alteration of the microenvironment
underlies the pathogenesis of SAN disorders. Although

Correspondence to: Yu-Feng Hu, MD, Division of Cardiology, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road, Taipei, 11217, Taiwan. Email yfhu@vghtpe.gov.tw
*P.-C. Chou and C.-M. Liu contributed equally.
Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/CIRCRESAHA.121.320301.
For Sources of Funding and Disclosures, see page 18.
© 2022 American Heart Association, Inc.
Circulation Research is available at www.ahajournals.org/journal/res

6   June 24, 2022 Circulation Research. 2022;131:6–20. DOI: 10.1161/CIRCRESAHA.121.320301

 Chou et al Fibroblast-Pacemaker Cardiomyocyte Integration

Novelty and Significance

Original Research
What Is Known? From the induced rat PCs and engineered tissue sheet
• The sinoatrial node (SAN) initiates electric impulses models to human PCs and in vivo animal experiments,
for every heartbeat, and its dysfunction causes a slow we observed fibroblasts induce aerobic glycolysis and
heart rate or cardiac arrest. regulate rhythmicity by upregulating Aldoc in PCs. In
• SAN is characterized by the microenvironment of pace- addition to explaining the mechanisms of SAN failure,
maker cardiomyocytes (PCs) encased with fibroblasts. Aldoc-driven energy replenishment may also be used
• The disintegration of PCs with fibroblasts results in as a future device-free therapy to restore SAN dys-
SAN dysfunction. function. Establishment of engineered tissue sheets
can be used as an in vitro experimental model for the
What New Information Does This Article study of SAN diseases and preclinical tests.
Contribute?
• Fibroblasts induce metabolic reprogramming and acti-
vate the PC-specific expression of Aldoc (aldolase c)
within SANs through integrin-dependent cell contact.
• The activation of aldolase c critically maintains intrin-
sic aerobic glycolysis in PCs and regulates pacemaker
activities.
• The engineered tissue sheets are established as an in
vitro model of the SAN microenvironment.

The SAN is tiny with a paucity of PCs.3,12 Operable cell

Nonstandard Abbreviations and Acronyms or tissue models are either generally lacking or difficult to
handle.10 To study functional interactions within the micro-
Aldoc aldolase c environment, recapitulation of the complex structure of the
Cx45 connexin 45 PCs and fibroblasts within the SAN is necessary, but such
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DHAP dihydroxyacetone phosphate models have yet to be developed.13 This presents a bar-
ECM extracellular matrix rier to studying the biological function of this critical tissue
G3P glyceraldehyde 3-phosphate and obtaining models of SAN diseases.10 Recently, induced
PCs, generated by Tbx18 (T-box transcription factor 18)
Hcn4 potassium/sodium hyperpolarization-acti-
vated cyclic nucleotide-gated channel 4 transduction or biomaterial, recapitulated not only electri-
cal and morphological phenotypes, but also the metabolic
IPS-CMs induced pluripotent stem cell-derived
cardiomyocytes properties of native SAN cardiomyocytes.14–16 It is pos-
sible that Tbx18-induced PCs (Tbx18-PCs), considered
Itgb1 integrin subunit β1
a replacement for native PCs, might be used to establish
MAPK mitogen-activated protein kinase
engineered models to study unknown SAN biological
PCs pacemaker cardiomyocytes
processes, especially intercellular functional interactions
Pde phosphodiesterase within the microenvironment.17
Pde4a phosphodiesterase In this study, we used Tbx18-PCs and engineered
p-ERK phosphorylated ERK tissue to explore how fibroblasts regulate the functional
PI3K phosphoinositide 3-kinase integrity of SANs. Fibroblasts drove PC-specific expres-
SAN sinoatrial node sion of Aldoc (aldolase c, an enzyme involved in glycolysis
Tbx18 T-box transcription factor 18 metabolism) through integrin-dependent cell contact. This
Tbx18-PC Tbx18-induced PC machinery critically maintained intrinsic aerobic glycolysis
VMs ventricular cardiomyocytes in PCs and regulated pacemaker activities. Aldoc-medi-
ated rhythmic activity was faithfully validated in an in vitro
engineered model, in vivo in mice, and in human-induced
the molecular mechanisms underlying the ability of indi- pluripotent stem cell-derived cardiomyocytes. These find-
vidual PCs to generate rhythmic electrical impulses have ings highlight the importance of the SAN microenviron-
been well studied,10,11 the biological process behind the ment in determining its energy metabolism and rhythmicity.
microenvironmental niche in SANs, especially fibroblast- Moreover, tissue engineered with Tbx18-PCs could be a
PC interactions, remains poorly understood. feasible in vitro platform to study SAN physiology.

Circulation Research. 2022;131:6–20. DOI: 10.1161/CIRCRESAHA.121.320301 June 24, 2022   7

 Chou et al
METHODS
Original Research
A detailed, expanded methods section is provided in the
Supplemental Material. The Institutional Animal Care and Use
Committee at Taipei Veterans General Hospital approved all
animal experiments.
Data Availability
All data supporting the findings of this study are available within
the article and its Supplemental Material. The whole transcrip-
tome analysis data set (Figure 1A and Figure 3A) is provided
in the Sequence Read Archive data at NCBI (PRJNA743181
and PRJNA743409).
Statistical Analysis
Statistical analysis was performed with GraphPad Prism ver-
sion 8.3.0 (GraphPad Software, CA). Data are expressed as
the mean±SD. The Shapiro-Wilks normality test was used to
determine the use of Student t test, 1-way ANOVA, or Mann
Whitney test as appropriate. Repeated measures ANOVA with
the least significant difference (LSD) post hoc test was used
when repeated measures were necessary. Multiple comparison
correction was performed using the false discovery rate control
approach and the Benjamini–Hochberg method when multiple
comparisons of different variants were needed.18 A P value
<0.05 was considered statistically significant.
RESULTS
Glycolysis Metabolism Regulated Rhythmicity
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in Tbx18-PCs
We hypothesized that a cell-specific biological process of
PCs, which is different from that of quiescent ventricular
cardiomyocytes (VMs), would underlie the tailored inte-
gration of fibroblasts and PCs for functional integrity.4,19
Therefore, Tbx18-PCs were selected as the cell model.14,20
The differential biological processes between PCs and
VMs were explored. Whole-transcriptome expression was
compared between Tbx18-PCs and control-VMs (Fig-
ure 1A, Supplemental Data S1).14,20 Glucose metabolism
and glycolysis accounted for the top canonical pathways
(Figure 1B, Table S1, and Supplemental Data S2) and
were predominant in gene ontology analysis (Table S2 and
Supplemental Data S3). Glucose is metabolized to pyru-
vate via a complex enzyme network (Figure 1C). The met-
abolic genes involved in glycolysis were mostly increased
in Tbx18-PCs. Aldoc was an exception, as its transcripts
significantly decreased (Figure 1C). The genes in other
metabolic pathways, including the tricarboxylic acid cycle
cycle, pentose phosphate pathway, pyruvate oxidation, and
fatty acid metabolism were either not different or slightly
increased between Tbx18-PCs and control-VMs (Figure
S1). The differential change in Aldoc transcripts between
Tbx18-PCs and control-VMs was confirmed via real-time
PCR (Figure 1D).
To correlate functional changes in transcriptome expres-
sion, glycolysis and mitochondrial function in Tbx18-PCs
8   June 24, 2022 Circulation Research. 2022;131:6–20. DOI: 10.1161/CIRCRESAHA.121.320301
Fibroblast-Pacemaker Cardiomyocyte Integration
were further analyzed using Seahorse functional assays.
Glycolysis activity, including basal and compensatory glycol-
ysis, and proton efflux rate in Tbx18-PCs were lower than in
control-VMs (Figure 1E). Mitochondrial function, including
basal respiration, proton leak, and ATP production, did not
differ between the 2 groups (Figure S2). The downregula-
tion of glycolysis was correlated with the reduced levels of
Aldoc and suggested that the increased expression of met-
abolic genes other than Aldoc was likely compensatory. To
confirm this idea, we performed metabolomics analysis via
liquid chromatography–mass spectrometry to comprehen-
sively delineate the metabolite levels of the aforementioned
pathways in Tbx18-PCs and control-VMs.
The levels of tricarboxylic acid cycle cycle metabo-
lites, energy molecules (eg, ATP and NADH), pyruvate
conversion metabolites, and pentose phosphate path-
way metabolites were mostly not statistically different
between Tbx18-PCs and control-VMs (Figure S3). Only
within the glycolysis process, almost all metabolites
decreased. A maximal decrease was observed in the lev-
els of glyceraldehyde-3-phosphate (G3P) and dihydroxy-
acetone phosphate (DHAP, Figure 1F), which reached
the nadir of all glycolysis metabolites (Figure 1G). Aldoc
catalyses reversible aldolase cleavage of fructose
1,6-bisphosphate to DHAP and G3P (Figure 1C).21 The
critical reduction in DHAP and G3P levels was consistent
with the reduction in Aldoc expression. The reduction in
lactate levels (end-product of glycolysis) was further con-
firmed by a colorimetric assay (Figure 1H). These results
reflected low glycolysis activity in the single culture of
PCs and demonstrated the key role of reduced Aldoc
expression in determining glycolysis status.
Whether glycolysis activity in PCs has functional impli-
cations must be determined. Supplementation with sodium
pyruvate to replenish the energy supply from glycolysis in
Tbx18-PCs increased the beating rates of PCs and potas-
sium/sodium hyperpolarization-activated cyclic nucleotide-
gated channel 4 (Hcn4) expression (distinct ion channels
that initiate electrical impulses in PCs, Figure 1I). Treatment
with 2-deoxy-D-glucose to inhibit glycolysis decreased
both beating rates and Hcn4 expression in Tbx18-PCs
(Figure 1I). Considered a critical hub in the glycolysis pro-
cess, adenoviral vector-mediated overexpression of Aldoc
was performed to reverse Aldoc expression in Tbx18-PCs
(Figure 1J). The increased expression of Aldoc improved
beating rates and Hcn4 expression (Figure 1K). These
results indicate that reduced Aldoc levels and glycolysis
status dysregulated PC rhythmicity.
Fibroblasts Drive Glycolysis Adaptation
Through Aldoc in Tbx18-PCs
Next, we determined whether fibroblasts regulate pace-
maker rhythm through intrinsic glycolysis within Tbx18-
PCs. Coculture with fibroblasts improved global glycolysis
function, including basal and compensatory glycolysis, as
 Chou et al Fibroblast-Pacemaker Cardiomyocyte Integration

Original Research
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Figure 1. Aldoc (aldolase c)-driven glycolysis regulation and pacemaker rhythmicity.

A, Heatmap of differential gene expression between Tbx18 (T-box transcription factor 18) pacemaker cardiomyocytes (PCs) and control
ventricular cardiomyocytes (VMs). B, Canonical pathways. C, The expression levels of the key metabolic genes in glycolysis between Tbx18-
PCs and control-VMs. Upper: The key metabolic genes (red color) and metabolites in the glycolysis process. In the lower part, the enzymes
involved in glycolysis mostly increased, and only Aldoc was downregulated. A–C, n=4 for each group. *P<5.0×10−2 (Data 1). D, The expression
of metabolic genes in glycolysis determined by real-time PCR (Hk1 [hexokinase-1], control-VMs, n=7; Tbx18-PCs, n=8; Hk2 [hexokinase-2],
control-VMs, n=5; Tbx18-PCs, n=7; Aldolase a and Aldoc, control-VMs, n=7; Tbx18-PCs, n=12). (Continued )

Circulation Research. 2022;131:6–20. DOI: 10.1161/CIRCRESAHA.121.320301 June 24, 2022   9

 Chou et al Fibroblast-Pacemaker Cardiomyocyte Integration

well as the proton efflux rate, compared with Tbx18-PCs function after coculture with fibroblasts (Figure 2H).
alone (Figure 2A). Mitochondrial function (oxidative phos- However, if we performed a separate coculture (cocul-
Original Research

phorylation), including basal respiration, spare respiratory
capacity, proton leak, and ATP production did not differ
between the 2 groups (Figure 2B). Based on metabolo-
mics analysis, compared with the drastic decrease in G3P
and DHAP in the single culture of Tbx18-PCs, cocul-
ture with fibroblasts significantly increased G3P and
DHAP, as well as fructose 1,6-bisphosphate, through the
reverse reaction of aldolase from DHAP (Figure 2C).22
This led to the upregulation of downstream metabolites,
such as 2-phosphoglyceric acid and phosphoenolpyru-
vate. The levels of tricarboxylic acid cycle cycle metabo-
lites, energy molecules (eg, ATP and NADH), pyruvate
conversion metabolites, and pentose phosphate pathway
metabolites were mostly marginally increased or not sta-
tistically different between Tbx18-PCs and cocultures
(Figure S4). The increased levels of DHAP were vali-
dated by an ELISA (Figure 2D).
Aldoc expression in the cocultures was higher than
that in the single PC cultures (Figure 2E), thus sup-
porting the notion that Aldoc expression underlies the
increase in G3P and DHAP levels. In addition, coculture
with fibroblasts was associated with better pacemaker
phenotypes such as beating rate (Figure 2F), as well as
with the expression of PC-specific genes (Hcn4 and
Cx45 [connexin 45], Figure 2G). Spontaneous local
Ca2+ release events are a hallmark of automaticity in
PCs.23,24 Spontaneous local Ca2+ release events could
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ture of fibroblasts and PCs, but PCs and fibroblasts were
separated by porous membranes), the levels of lactate
did not increase. Lactate levels in the single culture of
fibroblasts were also low. These findings indicate that
the microenvironment contributed to the improvement
in glycolytic activity through contact between fibroblasts
and PCs. Moreover, we did not observe any Aldoc pro-
tein expression in fibroblasts (Figure 2I and Figure S6).
These results indicate that the improvement in glycolysis
was due to intrinsic regulation of Aldoc within PCs.
The regulatory enzymes of glycolysis in Tbx18-PC
cocultures were different from those in cocultures of
control-VMs and fibroblasts (Figure S7). In contrast to
increased Aldoc expressions within Tbx18-PCs, fibro-
blasts increased the transcripts of aldolase a in con-
trol-VMs after coculture. Therefore, we observed that
glycolysis function and DHAP levels improved in both
Tbx18-PCs and control-VMs after coculture with fibro-
blasts (Figure S7).
Fibroblasts Switched on Aldoc Expression in
PCs Through Integrin-Dependent Signals
The mechanisms by which fibroblasts regulate Aldoc
expression in PCs were further explored. After coculture
with fibroblasts, PCs were isolated via cell sorting (Figure
S8). The whole transcriptome expression in isolated PCs

be observed in Tbx18-PC cocultures. The inhibition of
Aldoc by the Aldoc siRNAs decreased both spontaneous
local Ca2+ release events and oscillating calcium tran-
sients (Figure S5). The spontaneous local Ca2+ release
event period, as observed in the Tbx18-PC cocultures,
was linearly correlated with the cycle length of oscillat-
ing calcium transients (Figure S5). Overall, these find-
ings suggest that Aldoc regulates both membrane and
calcium clocks within PCs.
The improvement in glycolysis was related to the
intrinsic regulation of PCs but not to contamination of
fibroblasts. First, lactate levels increased in contact cocul-
tures, which supported the improvement of glycolysis
from PC-fibroblast cocultures was compared with that
from PCs in single PC cultures (Figure 3A, Supplemen-
tal Data S4). The analysis of glycolysis-related genes
revealed that the highest transcriptional changes (4.3-
fold increment) were observed at Aldoc levels compared
with the other glycolysis enzymes (Figure 3B). Other
metabolic genes related to pyruvate oxidation, tricarbox-
ylic acid cycle, the pentose phosphate pathway, and fatty
acid metabolism were either minimally or not statistically
different (Figure S9). Again, Aldoc was the key enzyme
critically regulated by fibroblasts. The relevant pathways
related to metabolic/energetic regulation of automatic-
ity, mainly those within the calcium clock, were analyzed

Figure 1 Continued. E, Seahorse glycolysis stress test. Left: Representative curves from the experiments. Glycolysis was persistently
lower in Tbx18-PCs than in control-VMs. The right part shows the measurements including basal glycolysis, % proton efflux rate (PER) and
compensatory glycolysis. control-VMs, n=18; Tbx18-PCs, n=14. F, The levels of glycolysis metabolites determined by mass spectrometry. The
highest decrease was observed in the relative levels of glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to those
of control-VMs. G, The reduction ratio of metabolites in Tbx18-PCs with reference to control-VMs. The reduction ratio (%)=100×(Tbx18-PC
metabolites−control-VM metabolites/control-VM metabolites). The reduction in DHAP and G3P levels reached a nadir in comparison to the
other metabolites. F and G, control-VMs, n=3; Tbx18-PCs, n=4. H, Lactate levels in PCs determined by a colorimetric assay. n=7 for each group.
I, Modulation of glycolysis changed pacemaker phenotypes, including potassium/sodium hyperpolarization-activated cyclic nucleotide-gated
channel 4 (Hcn4) expression and beating rate. Supplementation of active glycolysis metabolites with sodium pyruvate (1 mmol/L) increased
Hcn4 expression (Tbx18-PCs, n=10; Tbx18-PCs+sodium pyruvate, n=7; Tbx18-PCs+2-DG, n=11,) and beating rates (Tbx18-PCs, n=7; Tbx18-
PCs+sodium pyruvate, n=8; Tbx18-PCs+2-DG, n=7). 2-DG inhibits glycolysis by competitively inhibiting the production of glucose-6-phosphate
from glucose at the phosphoglucoisomerase level. Treatment with 2-DG (5 mmol/L) decreased Hcn4 levels and beating rate. J, Adenoviral
vector-mediated overexpression of Aldoc in Tbx18-PCs (Tbx18-PCs-control, n=8; Tbx18-PCs-Aldoc, n=8). K, Aldoc overexpression increased the
electrical firing rate (Tbx18-PCs-control, n=17; Tbx18-PCs-Aldoc, n=19) and Hcn4 expression (n=8 for each group). Representative tracing of
the electrical firing rate in the Aldoc-overexpression and control vector groups is shown in the left. P value determined by a 2-tailed t test for all
except I, determined by 1-way ANOVA.

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 Chou et al Fibroblast-Pacemaker Cardiomyocyte Integration

Original Research
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Figure 2. Fibroblasts drive Aldoc (aldolase c)-mediated glycolysis adaptation in pacemaker cardiomyocytes.
A, Glycolysis stress test in Tbx18 (T-box transcription factor 18) pacemaker cardiomyocytes (PCs) and cocultures. Coculture with fibroblasts
improved glycolysis, in comparison to the single culture of Tbx18-PCs. The left shows representative curves from the Seahorse glycolysis test
for Tbx18-PCs and cocultures (Tbx18-PCs, n=10; coculture, n=12). B, Mitochondrial stress test in Tbx18-PCs and cocultures. The left shows
the representative curves from the mitochondrial stress test. Measurements of basal respiration, spare respiratory capacity, proton leak and ATP
production did not differ between the 2 groups. Tbx18-PCs, n=11; coculture, n=12. C, The levels in glycolysis metabolites determined by mass
spectrometry were compared between Tbx18-PCs and cocultures (relative expression to control-VMs). After cocultures, most of the metabolites
increased. However, G3P and dihydroxyacetone phosphate (DHAP) levels exhibited the greatest changes (>2-fold, red arrow). (Continued )

Circulation Research. 2022;131:6–20. DOI: 10.1161/CIRCRESAHA.121.320301 June 24, 2022   11

 Chou et al Fibroblast-Pacemaker Cardiomyocyte Integration

(Figure S10).24,25 The spontaneous electrical activity of Figure S11C and S14). Treatment of cocultures with
PCs, especially calcium clock, is driven by a cAMP-medi- a p38-MAPK inhibitor (SB203580) decreased Aldoc
Original Research

ated phosphorylation, which critically relies on the bal-
ance between the cAMP production by adenylyl cyclases
and degradation by cyclic nucleotide Pde (phosphodies-
terases).24,25 The coculture with fibroblasts decreased
the expression of Pde4a (phosphodiesterase) and might
regulate cAMP-mediated protein phosphorylation and
the calcium clock in PCs.26
We performed Ingenuity Pathway Analysis to explore
the pathways to potentially regulate Aldoc expression,
including canonical pathways and gene ontology (Fig-
ure 3C, Tables S3 and S4, Supplemental Data S5 and
S6). Within canonical pathways (Table S3), those related
to ECM (eg, collagen and laminin), relevant surface
receptors (integrins) and their downstream signals (PI3K
[phosphoinositide 3-kinases]/Akt and MAPK [mitogen-
activated protein kinase]) were repeatedly observed.
These results were compatible with the finding that direct
contact between fibroblast-PCs and the ECM is critical
to drive metabolic reprogramming in PCs. Therefore,
we comprehensively analyzed integrins, which are the
predominant ECM-binding receptors in cardiomyocytes
(Figure 3D).27 The Itgb1 (integrin subunit β1) transcripts
were the most abundant and increased significantly. We
further treated Tbx18-PC coculture with an Itgb1 inhibi-
tory antibody, which decreased integrin activation, Aldoc
expression (Figure 3E), and the beating rate (control
versus Itgb-1 antibody: 140.4±92.0 bpm, n=23 versus
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expression, suggesting that p38-MAPK activation is a
downstream signal of integrins (Figure 3I) to increase
Aldoc expression. The p38-MAPK activation induces
the dissociation of Rb and E2F1, and increases E2F1-
driven transcriptional activity.29 Increased expression of
Rb, phosphorylated Rb, and E2F1 was observed in cocul-
tures (Figure 3J, Figure S11D and S11E, S15, and S16).
There are multiple E2F1 binding sites on the promotor
region of Aldoc, suggesting that E2F1 is a transcriptional
regulator of Aldoc (Figure 3K). Inhibition of p38-MAPK,
Rb, and E2F1 via siRNA decreased Aldoc expression
(Figure 3L and Figure S17). We further performed an
in vivo experiment to clarify the link between Aldoc
expression and integrin-dependent signaling. Mice that
received an intraperitoneal injection of the Itgb1 inhibi-
tory antibody (a functional blocking antibody) had lower
Aldoc expression in SANs than control mice (Figure 3M).
Overall, these results suggest that fibroblasts induce
Aldoc expression in PCs through β1-integrin activation
and downstream p38-MAPK/E2F1 signaling.
Engineered Tbx18-PC Tissue Sheets
Recapitulated Aldoc-Driven Rhythmic
Machinery
The microenvironment with fibroblasts is essential for
the functional integrity of pacemaker tissue. We further

20.6±55.7 bpm, n=16, P=1.2×10−5). This indicates that
fibroblasts activate Aldoc and glycolysis activity through
integrin-dependent signals.
The common downstream signals of integrins within
cardiomyocytes include PI3K/Akt and MAPK (p38 and
ERK).27,28 The expression of phosphorylated and total Akt
did not change between cocultures and single cultures
of PCs (Figure 3F and Figure S11A and S12). Treat-
ment with a PI3K inhibitor (wortmannin) in PC cocultures
increased Aldoc expression (Figure 3G). These results
indicate that PI3K/Akt activity did not activate Aldoc
expressions after coculture. Considering MAPK path-
ways, ERK activation was suppressed after coculture as
p-ERK (phosphorylated ERK) was lower in cocultures
than in the single culture of PCs (Figure 3H and Fig-
ure S11B and S13). Instead, phosphorylated and total
p38-MAPK increased after coculture (Figure 3H and
constructed an in vitro Tbx18-PC tissue sheet to mimic
the 3-dimensional microenvironment of in vivo SANs to
study the regulatory role of Aldoc in pacemaker rhythmic-
ity. The PC tissue sheet was induced by re-expression of
Tbx18 in an engineered tissue, which was constructed
by mixed culture of VMs and fibroblasts with Matrigel
(Figure 4A). Compared with control tissue sheets (GFP
expression), re-expression of Tbx18 induced sponta-
neous electrical firing that was recorded with a micro-
electrode array (Figure 4B). A sympathomimetic drug
(epinephrine, α- and β-receptor sympathetic agonist)
increased the beating rate of Tbx18-PC tissue sheets
(Figure 4C). Through immunofluorescence staining, PCs
with distinct pacemaker ion channels (HCN4, Figure 4D
and Figure S18) and Cx45 (Figure S19) were observed
in Tbx18-PC tissue sheets but not in controls. PC tissue
sheets also had higher expression of PC-specific genes

Figure 2 Continued. *P<5.0×10−2 Tbx18-PCs vs cocultures; Tbx18-PCs, n=4; coculture, n=3. D, The level of DHAP determined by ELISA in
Tbx18-cocultures was higher than that in Tbx18-PCs (Tbx18-PCs, n=10; coculture, n=11). E, Aldoc transcripts increased in the cocultures with
fibroblasts compared with the level in Tbx18-PCs alone (Tbx18-PCs, n=12; coculture, n=13). F, Coculture increased beating rates compared
with those of single Tbx18-PC cultures (Tbx18-PCs, n=26; coculture, n=37). G, Coculture also increased the expression of distinct pacemaker
genes (Hcn4 [potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4], Cx45 [connexin 45]) compared with Tbx18-
PCs alone (Tbx18-PCs, n=9; coculture, n=10). H, The level of the end-product of glycolysis (lactate) represents the active status of glycolysis.
The separate-coculture of Tbx18-PCs and Tbx18-transduced fibroblasts was not associated with an increased level of lactate. Only contact-
coculture containing both Tbx18-PCs and fibroblasts was associated with increased lactate levels. P value determined by 1-way ANOVA
with an least significant difference (LSD) post hoc test. I, Representative western blot images of Aldoc expression. No Aldoc expression was
noted in fibroblasts with GAPDH as a protein control (n=4). However, Aldoc expression was observed in neonatal cardiomyocytes (ventricular
cardiomyocytes [VMs], n=4). P value determined by Mann-Whitney test (A) or a 2-tailed t test (B–G). OCR indicates oxygen consumption rate.

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Figure 3. Fibroblast-pacemaker interaction to regulate intrinsic expression of Aldoc (aldolase c) in pacemaker cardiomyocytes.
A, Heatmap of differential gene expression in cardiomyocytes isolated from single Tbx18 (T-box transcription factor 18) pacemaker
cardiomyocyte (PC) cultures and fibroblast cocultures (Tbx18-PCs, single culture, n=4; coculture, n=3). B, Transcript levels of the key enzymes
involved in glycolysis between cocultures and single cultures of Tbx18-PCs. *P<5.0×10−2 (Supplemental Data S4). The expression of Aldoc was
increased. C, The canonical pathways determined by IPA analysis. The detailed genes within these pathways, as shown in Table S3, revealed
critical regulation of ECM receptors and their downstream signals after coculture. (Continued )

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 Chou et al Fibroblast-Pacemaker Cardiomyocyte Integration

(Hcn4 and Cx45) than control tissue sheets (Figure 4E). Aldoc Regulated Pacemaker Activity in Human
These findings suggest that engineered Tbx18-PC tis- Induced Pluripotent Stem Cell-Derived
Original Research

sue sheets recapitulated the phenotypes of a native

SAN. Aldoc-driven rhythmic machinery within a 3-dimen-
sional microenvironment was further explored. Aldoc
expression in PC tissue sheets was higher than controls
(Figure 4F). Treatment of PC tissue sheets with Aldoc
siRNAs reduced Aldoc expression (Figure S20) and
spontaneous electrical activity (Figure 4G). Engineered
Tbx18-PC tissue sheets recapitulated the microenviron-
ment of de novo SAN tissues and suggested that the
Aldoc-driven glycolysis machinery regulates PC rhyth-
micity in a 3-dimensional microenvironment.
The Regulation of In Vivo Pacemaker Rhythms
by Aldoc in Vertebrates
The regional distribution of Aldoc and its physiological
function in the hearts of vertebrates were further explored.
In rats and mice, through immunofluorescence staining,
Aldoc expression was observed exclusively within SANs
but not in the atrium or ventricle (Figure 5A). This find-
ing was consistent with the abundance of Aldoc tran-
scripts in rat SANs, while Aldoc expression was almost
undetectable in the atria and ventricles (Figure 5B).30 We
further performed in vivo knockdown of Aldoc in mouse
SANs to determine whether Aldoc regulates the pace-
maker activity of SANs. The efficiency of AAV9 (adeno-
associated virus 9) Aldoc siRNAs in reducing Aldoc
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Cardiomyocytes
Induced pluripotent stem cell-derived cardiomyocytes
(IPS-CMs) were used as a human cell model to study
whether Aldoc regulates pacemaker activity in human
PCs. HCN4 (+) PCs accounted for ≈10% of IPS-CMs31,32
and were responsible for the rhythmic beating of IPS-
CMs.33 Aldoc expression was predominantly observed in
human HCN4 (+) PCs (Figure 6A and 6B). Overexpres-
sion of Aldoc in IPS-CMs via adenoviral vectors signifi-
cantly increased Aldoc levels in IPS-CMs (Figure 6C) and
the electrical firing rate of IPS-CMs (Figure 6D and 6E).
Human PCs share similar machinery in which Aldoc drives
metabolic adaptation to regulate pacemaker rhythm,
highlighting their translational potential for the study of
human SAN physiology or disease prevention.
DISCUSSION
Years after observing the clinical linkage of the micro-
environmental niche in SANs with pacemaker failure,
the exploration of its biological process remains a rocky
terrain.11,34 From the induced rat PCs and engineered
tissue sheet models to human PCs and in vivo animal
experiments, we found that fibroblasts induce metabolic
reprogramming by upregulating Aldoc in PCs through
integrin-dependent MAPK-E2F1 signals. This enhanced

expression was first confirmed via in vitro transduction of
mouse cardiomyocytes (Figure S21). Then, AAV9 Aldoc
siRNAs were delivered into the pericardial recess around
mouse SANs through a mini-thoracotomy and reached
≈80% transduction efficiency (Figure S22). The in vivo
Aldoc expression in the mouse SAN was successfully
reduced (Figure 5C and 5D). Mice that received Aldoc
siRNAs had a lower spontaneous heart rate than those
that received the scrambled controls (Figure 5E and 5F).
In addition, the responses to epinephrine were nullified
in mice that received Aldoc siRNAs (Figure 5F). Accord-
ingly, the expression of Aldoc within the SAN might not
only drive glycolysis machinery but also regulate in vivo
PC rhythmicity.
aerobic glycolysis and regulated the rhythmicity of SANs.
Interruption of the fibroblast-PC Aldoc interaction stag-
nated SAN electrical activity. Aldoc-driven energy replen-
ishment may restore SAN dysfunction and might be a
future device-free therapeutic target. Establishment of
engineered tissue sheets can hopefully be used as an in
vitro experimental model for the study of SAN diseases
and preclinical tests.
Fibroblast and PC interactions in SANs are rarely
addressed. Only a mathematical model suggests that
fibroblasts, as stretch sensors during atrial diastole,
raise the spontaneous depolarization rate of PCs.35
Our findings uncovered functional implications of the
cellular architecture of SANs showing that fibroblasts

Figure 3 Continued. D, The transcript levels of ECM-binding receptors. Compared with the other integrin subunits, Itgb1 levels were the most
abundant and significantly increased after coculture (Tbx18-PCs, single culture, n=4; coculture, n=3). E, Aldoc expression in Tbx18-PC cocultures
decreased after treatment with the Itgb1 inhibitory antibody (IgG control, n=10; Itgb1 antibody, n=11). F, Representative Western blot of total
and phosphorylated AKT showing no difference between Tbx18-PC single cultures and cocultures. G, Aldoc expression after treatment with a
PI3K inhibitor (Wortmannin, n=5 for each group). H, Representative Western blot of total and phosphorylated ERK and p38-MAPK (mitogen-
activated protein kinase) showing increased total and phosphorylated p38-MAPK after coculture with fibroblasts. I, Reduced Aldoc expression
after treatment with a p38-MAPK inhibitor (control, n=9; SB203580, n=9). J, Representative western blot of total and phosphorylated Rb and
E2F1 showing increased total and phosphorylated Rb and E2F1 after coculture with fibroblasts. K, Promoter binding site prediction for Aldoc
determined using AnimalTFDB version 3.0 (http://bioinfo.life.hust.edu.cn/AnimalTFDB/). The prediction showed three E2F1 binding sites within
the promotor of Aldoc. L, Knockdown of p38-MAPK, Rb, and E2F1 downregulated Aldoc expression (nontarget, n=13; Rb siRNA, n=12; E2F1
siRNA, n=13; p38-MAPK siRNA, n=13). Treatment with p38-MAPK, Rb, and E2F1 siRNAs reduced the expression of the corresponding target
genes, as shown in Figure S17. M, Intraperitoneal injection of the Itgb1 inhibitory antibody decreased Aldoc expression in mouse SANs (IgG
control, n=6; Itgb1 antibody, n=8). All P values determined by a 2-tailed t test. Statistical analyses of Western blotting results (F, H, and J) and
uncut blot data are provided in Figure S11 through S16.

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 Chou et al Fibroblast-Pacemaker Cardiomyocyte Integration

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Figure 4. Engineered Tbx18 (T-box transcription factor 18)-pacemaker tissue sheets recapitulate Aldoc (aldolase c)-driven
rhythmic machinery.
A, Immunofluorescence staining of a Tbx18-pacemaker cardiomyocyte (PC) tissue sheet. Cardiomyocytes are interspersed with fibroblasts
(n=4, thickness of 28.3±10.9 μm). The surface of the engineered Tbx18-PC tissue sheets was mostly covered by fibroblasts. DAPI (nucleus):
blue; vimentin (a filament protein in fibroblasts): green; ACTN2 (α-sarcomeric actinin, cardiomyocyte marker): red. B, Electrical recordings of
spontaneous firing in Tbx18-PC and control tissue sheets determined by microelectrode array (MEA; control, n=8; Tbx18-PC tissue sheet,
n=7). C, The autonomic response of Tbx18-PC tissue sheets. Treatment with the sympathomimetic drug (epinephrine, 0.15 µg/mL, agonist of
alpha and beta receptors) increased beating rates in Tbx18-PC tissue sheets compared with rates in controls. There were minimal changes
in the beating rate in the control tissue sheets (control, n=6; Tbx18-PC tissue sheet, n=6). D, Fluorescence staining of HCN4 (potassium/
sodium hyperpolarization-activated cyclic nucleotide-gated channel 4) in Tbx18-PC tissue sheets. No HCN4 expression was observed in
controls (Figure S18). DAPI: blue; HCN4: green; ACTN2: red. E, Hcn4 and Cx45 (connexin 45) transcripts increased in Tbx18-PC tissue
sheets compared with levels in controls. Hcn4, control, n=8; Tbx18-PC tissue sheets, n=5; Cx45, control, n=8; Tbx18-PC tissue sheets,
n=7. F, Aldoc expression was higher in Tbx18-PC tissue sheets than in controls (n=4 for both groups). G, Knockdown of Aldoc by siRNAs in
Tbx18-PC tissue sheets downregulated Aldoc transcripts (Figure S20) and decreased the electrical firing rate of PC tissue sheets (nontarget
siRNA, n=11; Aldoc siRNA, n=10). The left shows a representative MEA tracing of nontarget siRNA and Aldoc siRNAs on Tbx18-PC tissue
sheets. P value determined by a 2-tailed t test (B–G).

drive PC-specific Aldoc expression to modulate cardiac
rhythm. Constitutive Aldoc expression has mainly been
observed in cancer cells or neuronal cells in the brain
and causes cancer progression and neurodegenera-
tion.21,36,37 The physiological roles of Aldoc in the heart
are far from clear. The constitutive expression of Aldoc
in the atria and ventricles is seen in amphibians but not
in mammals.36,38 Aldoc seems inducible only in quiescent
cardiomyocytes during hypoxia, which activates glycoly-
sis (anaerobic glycolysis) to compensate for energy defi-
ciency from impaired mitochondrial function.39–41
In SANs, the presence of fibroblast-PC interactions
activated integrin-dependent pathways and maintained
constitutive Aldoc expression within PCs. Even while the

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 Chou et al Fibroblast-Pacemaker Cardiomyocyte Integration
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Figure 5. Regulation of in vivo pacemaker rhythms by Aldoc (aldolase c) in vertebrates.

A, Aldoc expression in adult rat sinoatrial nodes (SANs) determined by immunofluorescence staining. Aldoc expression was observed in
adult rat SANs (animal=3) but not in adult rat ventricles (animal=2). Aldoc: red; HCN4 [potassium/sodium hyperpolarization-activated
cyclic nucleotide-gated channel 4]: green; DAPI: blue. B, Aldoc transcripts determined by real-time PCR, suggesting dominant expression
of Aldoc in the SAN but not in atrial or ventricular tissues. SAN, n=9; atrium, n=4; ventricle, n=8. P value determined by one-way ANOVA
with an least significant difference (LSD) post hoc test. C and D, In vivo Aldoc expression in the mouse SAN after transduction with AAV9-
Aldoc siRNAs. Regional Aldoc expression within the mouse SAN was best evaluated by immunofluorescence staining, as the SAN could be
precisely localized by the presence of HCN4 channels. A representative image is shown in C. Aldoc: gray; HCN4: red; DAPI: blue. D, The
fluorescence intensity of Aldoc was significantly reduced after transduction with AAV9 siRNA (AU: arbitrary unit). P value by a 2-tailed t
test. Three animals were used for both groups. E, Representative ECG tracings of mice receiving AAV9-Aldoc or scramble siRNAs. In vivo
Aldoc knockdown within SANs led to a slower heart rate compared with that in control mice. The intraperitoneal injection of epinephrine
(2.5 μg, Epi) increased the heart rate in control mice but not in Aldoc knockdown mice. The results are provided in F. Scramble, n=8; Aldoc
knockdown, n=10. P value by a repeated-measures ANOVA followed by an LSD post hoc test.

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 Chou et al Fibroblast-Pacemaker Cardiomyocyte Integration

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Figure 6. Aldoc (aldolase c) regulates pacemaker activity in human induced pluripotent stem cell-derived cardiomyocytes.
A, Immunofluorescence staining showing Aldoc expression in HCN4 (potassium/sodium hyperpolarization-activated cyclic nucleotide-gated
channel 4; +) pacemaker cardiomyocytes among human induced pluripotent stem cell-derived cardiomyocytes (IPS-CMs). Aldoc: gray;
HCN4: green; ACTN2: red; DAPI: blue. B, Aldoc levels, calculated by fluorescence intensity, were higher in HCN4 (+) PCs than in HCN4 (−)
cardiomyocytes (IPS-CMs, n=35; IPS-PCs, n=23). C, Aldoc levels after transduction with adenoviral Aldoc vectors (control, n=4; Aldoc, n=6). D,
Aldoc overexpression in IPS-CMs after treatment with the adenoviral vector was associated with higher electrical firing rates in the microelectrode
array (MEA) compared with rates in IPS-CMs transduced with control vectors, as shown in the representative MEA tracing. E, Either at baseline
(without epinephrine) or after epinephrine treatment (150 ng/mL), increased Aldoc expression in IPS-CMs drove a higher electrical firing rate
than observed in controls; n=7 for both groups. P value determined by a 2-tailed t test (B and C), or a repeated-measures ANOVA followed by an
least significant difference (LSD) post hoc test (E).

mitochondrial oxidative phosphorylation function remained
activated and unchanged (as shown in the Seahorse,
whole transcriptome, and metabolomics analyses), Aldoc
in PCs activated glycolysis (aerobic glycolysis, Warburg
effect)42 and regulated beating rates in SANs. This phe-
nomenon was observed either at rest or after treatment
with sympathomimetic drugs to mimic exercise. Aerobic
glycolysis is an important energy machinery for prolifera-
tive cells, including cancer cells, stem cells, and endothelial
cells in the heart.42 The present results extend the physio-
logical implications of aerobic glycolysis from cell prolifer-
ation to relentless electrical firing in PCs. Most intriguingly,
this machinery needs to be maintained by the microenvi-
ronment between fibroblasts and PCs.
The upregulation of Itga5 and Itgb1 after the cocul-
ture with fibroblasts in the present work suggests that
the integrin heterodimer (α5β1) is a critical receptor that
drives fibroblast-induced glycolysis signals. The α5β1 is
predominantly a fibronectin receptor, which corresponds
to the abundant expression of fibronectin in the sinus
node ECM.30 Fibroblast-driven Aldoc-mediated PC regu-
lation is similarly observed across different vertebral PC
cells, including rats, mice, and human cells, suggesting
its physiological importance in SAN function. The critical

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 Chou et al
role of α5β1 in the fibroblast-SAN interaction, ECM, and
microenvironment is worth further investigation. The pau-
Original Research
city of connexin expression at fibroblast-PC contacts,
including Cx45 and Cx43 (data not shown), might be
associated with low electrical conductivity across hetero-
cellular junctions. It remains unclear whether the low con-
ductivity between fibroblasts and PCs might be related to
the initiation or organization of rhythmic electrical activity.
Aldoc expression are confined to the central nervous
system and is not found in the peripheral nervous system
(which innervates the heart or SAN). Aldoc is only detected
in Purkinje cells, a specific type of cerebellar neuronal
cell within the cerebellum.43,44 In other areas of the cen-
tral nervous system, Aldoc is not detected within neuronal
cells and is only observed in nonneuronal cells (astrocytes,
some cells of the pia mater) within the hippocampus and
thalamus.43,44 Additionally, in our study, Aldoc was found to
be expressed exclusively within the cytoplasm of cardio-
myocytes in SANs by immunofluorescence staining. These
findings suggest that Aldoc is activated within PCs but not
via SAN innervation by nerve terminals.
We could never successfully explore the machinery
behind PC and fibroblast interactions without the engi-
neered Tbx18-PC and tissue sheet models. Tbx18-PCs
and tissue sheets recapitulate the phenotypes of de
novo SANs including distinct genes related to pace-
maker functions (Hcn4, Cx45), spontaneous electrical
firing, the calcium clock, and responses to autonomic
stimulation.14 In addition, metabolic machinery including
Downloaded from http://ahajournals.org by on May 8, 2023
Aldoc-driven glycolysis and PC rhythmicity was similarly
replicated in engineered Tbx18-PCs, native SANs in ani-
mals, and PCs among IPS-CMs. This similarity underlies
the successful translation of fibroblast-driven glycolysis
pathways in induced Tbx18-PCs and their rhythmicity
to de novo PCs. The engineered Tbx18-PCs and tissue
sheets may represent an in vitro platform to study SAN
diseases, at least those related to energy metabolism.
The SAN is a complex 3-dimensional structure compris-
ing different structures, such as pacemaker cells, tran-
sitional cells, fibroblasts, and autonomic nerve terminals.
The present engineered model only partially recapitu-
lated SAN phenotypes. Additionally, it should be noted
that not all VMs were transduced with Tbx18 and suc-
cessfully reprogrammed. The Tbx18 transduction rate
was 87.3±10.9% (n=3). The reprogramming rate (ie, the
ratio of HCN4 [+] cells within Tbx18-transduced cardio-
myocytes) was 25.8±14.7%.
Clinical reports addressing Aldoc in SAN pathologies
are not well established. This is probably not a limitation
but rather offers new hope to understand the mecha-
nisms of SAN diseases and develop future therapy. For
example, age-dependent degeneration is a common
cause of SAN dysfunction,11 and a reduction in blood
Aldoc expression is observed with aging.45 Exploring
the potential role of reduced Aldoc expression in the
aging SAN might give birth to a target strategy involving
18   June 24, 2022 Circulation Research. 2022;131:6–20. DOI: 10.1161/CIRCRESAHA.121.320301
Fibroblast-Pacemaker Cardiomyocyte Integration
Aldoc replenishment and restoration of SAN failure.
This could not only be considered a future device-free
therapy but also part of preventive medicine. Regulation
of glucose-related cardiac metabolism by diet, lifestyle,
exercise, or medication for the primary prevention of
cardiac disease is not new.46
ARTICLE INFORMATION
Received November 28, 2021; revision received April 28, 2022; accepted May
3, 2022.
Affiliations
Division of Cardiology, Department of Medicine, Heart Rhythm Center (P.-C.C., C.-
M.L., C.-H.W., J.-D.L., Y.-F.H.) and Department of Neurology, Neurological Institute
(S.-P.C.), Taipei Veterans General Hospital, Taiwan. Institute of Biomedical Scienc-
es, Academia Sinica, Taipei, Taiwan (P.-C.C., C.-H.W., K.-C.Y., Y.-C.L., R.-B.Y., B.-C.S.,
S.-K.S., J.-D.L., Y.-F.H.). Faculty of Medicine, School of Medicine, National Yang
Ming Chiao Tung University, Taipei, Taiwan (C.-M.L., Y.-F.H.). Department and Grad-
uate Institute of Pharmacology, National Taiwan University College of Medicine,
Taipei (K.-C.Y.). Metabolomics Core Laboratory, Healthy Aging Research Center,
Chang Gung University, Taoyuan City, Taiwan (M.-L.C.). Department of Physiology,
School of Medicine, College of Medicine, Taipei Medical University, Taiwan (Y.-C.L.).
The Genomics Research Center, Academia Sinica, Taipei, Taiwan (M.H.).
Acknowledgments
We thank Wei-Chi Wang for bioinformatics analysis and I-Chien Wu for statistical
analysis consultation.
Author Contributions
P.-C. Chou, C.-H. Weng, and J.-D. Liu performed immunostaining, PCR, in vivo and
in vitro cell experiments, and animal experiments; K.-C. Yang performed whole
transcriptome analysis. M.-L. Cheng performed metabolomics analysis; R.-B. Yang
and Y.-C. Lin performed the integrin and mechanism experiments. B.-C. Shyu as-
sisted in microelectrode array experiments. M. Hsiao and S.-K. Shyue assisted in
the gene interference experiments. S.-P. Chen reviewed the results. P.-C. Chou,
C.-H. Weng, and C.-M. Liu wrote the article. Y.-F. Hu designed the project and
revised the article.
Sources of Funding
This work was supported by Taipei Veterans General Hospital (V110C-039,
V110B-043, V111C-047, VGHUST111-G6-3-2, VTA111-A-1-2), the Ministry of
Science and Technology (110-2628-B-075-015, 110-2314-B-075-063-MY3),
National Health Research Institutes (NHRI-109BCC0-MF-202014-02), and
Academia Sinica (AS-TM-110-01-01).
Disclosures
None.
Supplemental Materials
Expanded Methods and Materials
Data Set 1–6
References 14,20,31,33,47–55
REFERENCES
1. Epstein AE, DiMarco JP, Ellenbogen KA, Estes NA 3rd, Freedman RA,
Gettes LS, Gillinov AM, Gregoratos G, Hammill SC, Hayes DL, et al; Ameri-
can College of Cardiology Foundation; American Heart Association Task
Force on Practice Guidelines; Heart Rhythm Society. 2012 ACCF/AHA/
HRS focused update incorporated into the ACCF/AHA/HRS 2008 guide-
lines for device-based therapy of cardiac rhythm abnormalities: a report of
the american college of cardiology foundation/american heart association
task force on practice guidelines and the heart rhythm society. Circulation.
2013;127:e283–e352. doi: 10.1161/CIR.0b013e318276ce9b
2. Camelliti P, Green CR, LeGrice I, Kohl P. Fibroblast network in rabbit
sinoatrial node: structural and functional identification of homogeneous
and heterogeneous cell coupling. Circ Res. 2004;94:828–835. doi:
10.1161/01.RES.0000122382.19400.14
 Chou et al
3. Perde FV, Atkinson A, Yanni J, Dermengiu D, Dobrzynski H. Morphologi-
cal characteristics of the sinus node on postmortem tissue. Folia Morphol
(Warsz). 2016;75:216–223. doi: 10.5603/FM.a2015.0087
4. Bressan M, Henley T, Louie JD, Liu G, Christodoulou D, Bai X, Taylor
J, Seidman CE, Seidman JG, Mikawa T. Dynamic cellular integration
drives functional assembly of the heart’s pacemaker complex. Cell Rep.
2018;23:2283–2291. doi: 10.1016/j.celrep.2018.04.075
5. Bleeker WK, Mackaay AJ, Masson-Pévet M, Bouman LN, Becker AE. Func-
tional and morphological organization of the rabbit sinus node. Circ Res.
1980;46:11–22. doi: 10.1161/01.res.46.1.11
6. Opthof T. The mammalian sinoatrial node. Cardiovasc Drugs Ther.
1988;1:573–597. doi: 10.1007/BF02125744
7. Kalyanasundaram A, Li N, Hansen BJ, Zhao J, Fedorov VV. Canine and
human sinoatrial node: differences and similarities in the structure, func-
tion, molecular profiles, and arrhythmia. J Vet Cardiol. 2019;22:2–19. doi:
10.1016/j.jvc.2018.10.004
8. Monfredi O, Boyett MR. Sick sinus syndrome and atrial fibrillation in older
persons - A view from the sinoatrial nodal myocyte. J Mol Cell Cardiol.
2015;83:88–100. doi: 10.1016/j.yjmcc.2015.02.003
9. Ferrer MI. The etiology and natural history of sinus node disorders. Arch Intern
Med. 1982;142:371–372. doi: 10.1001/archinte.1982.00340150171028
10. Cingolani E, Goldhaber JI, Marbán E. Next-generation pacemakers: from
small devices to biological pacemakers. Nat Rev Cardiol. 2018;15:139–150.
doi: 10.1038/nrcardio.2017.165
11. Dobrzynski H, Boyett MR, Anderson RH. New insights into pacemaker
activity: promoting understanding of sick sinus syndrome. Circulation.
2007;115:1921–1932. doi: 10.1161/CIRCULATIONAHA.106.616011
12. Opthof T, de Jonge B, Mackaay AJ, Bleeker WK, Masson-Pevet M, Jongsma
HJ, Bouman LN. Functional and morphological organization of the guinea-
pig sinoatrial node compared with the rabbit sinoatrial node. J Mol Cell Car-
diol. 1985;17:549–564. doi: 10.1016/s0022-2828(85)80024-9
13. Weinberger F, Mannhardt I, Eschenhagen T. Engineering cardiac muscle tis-
sue: a maturating field of research. Circ Res. 2017;120:1487–1500. doi:
10.1161/CIRCRESAHA.117.310738
14. Kapoor N, Liang W, Marbán E, Cho HC. Direct conversion of quiescent car-
diomyocytes to pacemaker cells by expression of Tbx18. Nat Biotechnol.
2013;31:54–62. doi: 10.1038/nbt.2465
15. Gu JM, Grijalva SI, Fernandez N, Kim E, Foster DB, Cho HC. Induced car-
Downloaded from http://ahajournals.org by on May 8, 2023
diac pacemaker cells survive metabolic stress owing to their low metabolic
demand. Exp Mol Med. 2019;51:1–12. doi: 10.1038/s12276-019-0303-6
16. Hu YF, Lee AS, Chang SL, Lin SF, Weng CH, Lo HY, Chou PC, Tsai YN, Sung
YL, Chen CC, et al. Biomaterial-induced conversion of quiescent cardiomyo-
cytes into pacemaker cells in rats. Nat Biomed Eng. 2022;6:421–434. doi:
10.1038/s41551-021-00812-y
17. Grijalva SI, Gu JM, Li J, Fernandez N, Fan J, Sung JH, Lee SY, Herndon C,
Buckley EM, Park SJ, et al. Engineered cardiac pacemaker nodes created
by TBX18 gene transfer overcome source-sink mismatch. Adv Sci (Weinh).
2019;6:1901099. doi: 10.1002/advs.201901099
18. Glickman ME, Rao SR, Schultz MR. False discovery rate control is a recom-
mended alternative to Bonferroni-type adjustments in health studies. J Clin
Epidemiol. 2014;67:850–857. doi: 10.1016/j.jclinepi.2014.03.012
19. Furtado MB, Nim HT, Boyd SE, Rosenthal NA. View from the heart: car-
diac fibroblasts in development, scarring and regeneration. Development.
2016;143:387–397. doi: 10.1242/dev.120576
20. Hu YF, Dawkins JF, Cho HC, Marbán E, Cingolani E. Biological pace-
maker created by minimally invasive somatic reprogramming in pigs with
complete heart block. Sci Transl Med. 2014;6:245ra94. doi: 10.1126/
scitranslmed.3008681
21. Arakaki TL, Pezza JA, Cronin MA, Hopkins CE, Zimmer DB, Tolan DR, Allen
KN. Structure of human brain fructose 1,6-(bis)phosphate aldolase: link-
ing isozyme structure with function. Protein Sci. 2004;13:3077–3084. doi:
10.1110/ps.04915904
22. Choi KH, Shi J, Hopkins CE, Tolan DR, Allen KN. Snapshots of catalysis:
the structure of fructose-1,6-(bis)phosphate aldolase covalently bound to
the substrate dihydroxyacetone phosphate. Biochemistry. 2001;40:13868–
13875. doi: 10.1021/bi0114877
23. Hu YF, Lee AS, Chang SL, Lin SF, Weng CH, Lo HY, Chou PC, Tsai YN, Sung
YL, Chen CC, et al. Biomaterial-induced conversion of quiescent cardiomyo-
cytes into pacemaker cells in rats. Nat Biomed Eng. 2022;6:421–434. doi:
10.1038/s41551-021-00812-y
24. Lakatta EG, Maltsev VA, Vinogradova TM. A coupled system of intracellular
Ca2+ clocks and surface membrane voltage clocks controls the timekeep-
ing mechanism of the heart’s pacemaker. Circ Res. 2010;106:659–673. doi:
10.1161/CIRCRESAHA.109.206078
Circulation Research. 2022;131:6–20. DOI: 10.1161/CIRCRESAHA.121.320301
Fibroblast-Pacemaker Cardiomyocyte Integration
25. Yaniv Y, Juhaszova M, Lyashkov AE, Spurgeon HA, Sollott SJ, Lakatta
EG. Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells
Original Research
links ATP supply to demand. J Mol Cell Cardiol. 2011;51:740–748. doi:
10.1016/j.yjmcc.2011.07.018
26. Vinogradova TM, Sirenko S, Lukyanenko YO, Yang D, Tarasov KV, Lyashkov
AE, Varghese NJ, Li Y, Chakir K, Ziman B, Lakatta EG. Basal spontane-
ous firing of rabbit sinoatrial node cells is regulated by dual activation
of PDEs (phosphodiesterases) 3 and 4. Circ Arrhythm Electrophysiol.
2018;11:e005896. doi: 10.1161/CIRCEP.117.005896
27. Israeli-Rosenberg S, Manso AM, Okada H, Ross RS. Integrins and integrin-
associated proteins in the cardiac myocyte. Circ Res. 2014;114:572–586.
doi: 10.1161/CIRCRESAHA.114.301275
28. Roux PP, Blenis J. ERK and p38 MAPK-activated protein kinases: a family
of protein kinases with diverse biological functions. Microbiol Mol Biol Rev.
2004;68:320–344. doi: 10.1128/MMBR.68.2.320-344.2004
29. Wang S, Nath N, Minden A, Chellappan S. Regulation of Rb and E2F by
signal transduction cascades: divergent effects of JNK1 and p38 kinases.
EMBO J. 1999;18:1559–1570. doi: 10.1093/emboj/18.6.1559
30. Linscheid N, Logantha SJRJ, Poulsen PC, Zhang S, Schrölkamp M, Egerod
KL, Thompson JJ, Kitmitto A, Galli G, Humphries MJ, et al. Quantitative
proteomics and single-nucleus transcriptomics of the sinus node elucidates
the foundation of cardiac pacemaking. Nat Commun. 2019;10:2889. doi:
10.1038/s41467-019-10709-9
31. Tsai MH, Chiu YT, Chan DZ, Wen CH, Syu SH, Lu HE, Huang CF, Lin YJ,
Chang SL, Lo LW, et al. Generation of IBMS-iPSC-021, -022, -023 human
induced pluripotent stem cells (IBMSi016-A, IBMSi017-A, and IBMSi018-
A) derived from patients with the ALDH2 rs671 polymorphism. Stem Cell
Res. 2021;54:102416. doi: 10.1016/j.scr.2021.102416
32. Chang CW, Kao HKJ, Yechikov S, Lieu DK, Chan JW. An intrinsic, label-free
signal for identifying stem cell-derived cardiomyocyte subtype. Stem Cells.
2020;38:390–394. doi: 10.1002/stem.3127
33. Chiu YT, Tsai MH, Chan DZ, Ko HW, Lu HE, Huang CF, Lin YJ, Chang
SL, Lo LW, Huang CY, et al. Generation of IBMS-iPSC-015, -016, -017
human induced pluripotent stem cells (IBMSi013-A, IBMSi014-A, and
IBMSi015-A) derived from patients with atrial fibrillation. Stem Cell Res.
2021;54:102419. doi: 10.1016/j.scr.2021.102419
34. Bañuls MP, Alvarez A, Ferrero I, Zapata A, Ardavin C. Cell-surface marker
analysis of rat thymic dendritic cells. Immunology. 1993;79:298–304.
35. Kohl P, Kamkin AG, Kiseleva IS, Noble D. Mechanosensitive fibroblasts
in the sino-atrial node region of rat heart: interaction with cardiomyo-
cytes and possible role. Exp Physiol. 1994;79:943–956. doi: 10.1113/
expphysiol.1994.sp003819
36. Shiokawa K, Kajita E, Hara H, Yatsuki H, Hori K. A developmental biological
study of aldolase gene expression in xenopus laevis. Cell Res. 2002;12:85–
96. doi: 10.1038/sj.cr.7290114
37. Yu Q, Huang Q, Du X, Xu S, Li M, Ma S. Early activation of Egr-1 promotes
neuroinflammation and dopaminergic neurodegeneration in an experimen-
tal model of Parkinson’s disease. Exp Neurol. 2018;302:145–154. doi:
10.1016/j.expneurol.2018.01.009
38. Langellotti S, Romano M, Guarnaccia C, Granata V, Orrù S, Zagari A,
Baralle FE, Salvatore F. A novel anti-aldolase C antibody specifically inter-
acts with residues 85-102 of the protein. MAbs. 2014;6:708–717. doi:
10.4161/mabs.28191
39. Yang H, Shao N, Holmstrom A, Zhao X, Chour T, Chen H, Itzhaki I, Wu H,
Ameen M, Cunningham NJ, et al. Transcriptome analysis of non-human
primate induced pluripotent stem cell-derived cardiomyocytes in 2D
monolayer culture versus 3D engineered heart tissue. Cardiovasc Res.
2020;117:2125–2136. doi: 10.1093/cvr/cvaa281
40. Jian B, Wang D, Chen D, Voss J, Chaudry I, Raju R. Hypoxia-induced altera-
tion of mitochondrial genes in cardiomyocytes: role of Bnip3 and Pdk1.
Shock. 2010;34:169–175. doi: 10.1097/SHK.0b013e3181cffe7d
41. Bertero E, Maack C. Metabolic remodelling in heart failure. Nat Rev Cardiol.
2018;15:457–470. doi: 10.1038/s41569-018-0044-6
42. Chen Z, Liu M, Li L, Chen L. Involvement of the warburg effect in non-
tumor diseases processes. J Cell Physiol. 2018;233:2839–2849. doi:
10.1002/jcp.25998
43. Thompson RJ, Kynoch PA, Willson VJ. Cellular localization of aldol-
ase C subunits in human brain. Brain Res. 1982;232:489–493. doi:
10.1016/0006-8993(82)90294-3
44. Walther EU, Dichgans M, Maricich SM, Romito RR, Yang F, Dziennis S,
Zackson S, Hawkes R, Herrup K. Genomic sequences of aldolase C
(Zebrin II) direct lacZ expression exclusively in non-neuronal cells of
transgenic mice. Proc Natl Acad Sci U S A. 1998;95:2615–2620. doi:
10.1073/pnas.95.5.2615
June 24, 2022   19
 Chou et al
45. Spinetti G, Sangalli E, Specchia C, Villa F, Spinelli C, Pipolo R, Carrizzo
A, Greco S, Voellenkle C, Vecchione C, et al. The expression of the
Original Research
BPIFB4 and CXCR4 associates with sustained health in long-living
individuals from Cilento-Italy. Aging (Albany NY). 2017;9:370–380. doi:
10.18632/aging.101159
46. Krist AH, Davidson KW, Mangione CM, Barry MJ, Cabana M, Caughey AB,
Donahue K, Doubeni CA, Epling JW, Jr, Kubik M, et al. Behavioral coun-
seling interventions to promote a healthy diet and physical activity for
cardiovascular disease prevention in adults with cardiovascular risk fac-
tors: us preventive services task force recommendation statement. JAMA.
2020;324:2069–2075. doi: 10.1001/jama.2020.21749
47. Kizana E, Chang CY, Cingolani E, Ramirez-Correa GA, Sekar RB, Abraham
MR, Ginn SL, Tung L, Alexander IE, Marbán E. Gene transfer of connexin43
mutants attenuates coupling in cardiomyocytes: novel basis for modulation
of cardiac conduction by gene therapy. Circ Res. 2007;100:1597–1604.
doi: 10.1161/CIRCRESAHA.106.144956
48. Sekar RB, Kizana E, Cho HC, Molitoris JM, Hesketh GG, Eaton BP,
Marbán E, Tung L. IK1 heterogeneity affects genesis and stability of spiral
waves in cardiac myocyte monolayers. Circ Res. 2009;104:355–364. doi:
10.1161/CIRCRESAHA.108.178335
49. Neuss M, Regitz-Zagrosek V, Hildebrandt A, Fleck E. Isolation and char-
acterisation of human cardiac fibroblasts from explanted adult hearts. Cell
Tissue Res. 1996;286:145–153. doi: 10.1007/s004410050683
Downloaded from http://ahajournals.org by on May 8, 2023
20   June 24, 2022 Circulation Research. 2022;131:6–20. DOI: 10.1161/CIRCRESAHA.121.320301
Fibroblast-Pacemaker Cardiomyocyte Integration
50. Tsai YC, Tsai TH, Chang CP, Chen SF, Lee YM, Shyue SK. Linear correla-
tion between average fluorescence intensity of green fluorescent protein
and the multiplicity of infection of recombinant adenovirus. J Biomed Sci.
2015;22:31. doi: 10.1186/s12929-015-0137-z
51. Yang KC, Ku YC, Lovett M, Nerbonne JM. Combined deep microRNA
and mRNA sequencing identifies protective transcriptomal signature
of enhanced PI3Kα signaling in cardiac hypertrophy. J Mol Cell Cardiol.
2012;53:101–112. doi: 10.1016/j.yjmcc.2012.04.012
52. Goodson JM, MacDonald JW, Bammler TK, Chien WM, Chin MT. In utero
exposure to diesel exhaust is associated with alterations in neonatal cardio-
myocyte transcription, DNA methylation and metabolic perturbation. Particle
and Fibre Toxicology. 2019;16:17. doi: 10.1186/s12989-019-0301-9
53. Chen KH, Cheng ML, Jing YH, Chiu DT, Shiao MS, Chen JK. Resveratrol
ameliorates metabolic disorders and muscle wasting in streptozotocin-
induced diabetic rats. Am J Physiol Endocrinol Metab. 2011;301:E853–
E863. doi: 10.1152/ajpendo.00048.2011
54. Pinto AR, Ilinykh A, Ivey MJ, Kuwabara JT, D’Antoni ML, Debuque R, Chandran A,
Wang L, Arora K, Rosenthal NA, Tallquist MD. Revisiting cardiac cellular compo-
sition. Circ Res. 2016;118:400–409. doi: 10.1161/CIRCRESAHA.115.307778
55. Claycomb WC, Lanson NA Jr, Stallworth BS, Egeland DB, Delcarpio JB,
Bahinski A, Izzo NJ Jr. HL-1 cells: a cardiac muscle cell line that contracts
and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl
Acad Sci U S A. 1998;95:2979–2984. doi: 10.1073/pnas.95.6.2979