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Isolation and Characterization of Atrioventricular Nodal
Cells From Neonate Rabbit Heart
Xiao Ye Sheng, MD, MSc*; Yang Qu, MD, PhD*; Pauline Dan, PhD; Eric Lin, PhD;
Linda Korthout, MSc; Aaron Bradford, BSc; Leif Hove-Madsen, PhD;
Shubhayan Sanatani, MD, FRCPC; Glen F. Tibbits, PhD
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Background—The properties of the atrioventricular (AV) node in the neonate heart and its role in unique pediatric cardiac
arrhythmias such as junctional ectopic tachycardia (JET) are poorly understood. This is due in large part to the dearth
of information on the structure and physiology of the AV node in the immature myocardium.
Methods and Results—Sinoatrial nodal cells (SANCs), AV nodal tissues, and myocytes (AVNCs) were obtained from
neonatal (10-day-old) rabbits, and the histological, immunohistological, and electrophysiological properties were
characterized in detail. Masson’s trichrome histological staining clearly delineated AV nodal structures including the
inferior nodal extension, compact node, and the bundle of His region. AV tissue sections and AVNCs were
immunolabeled against neurofilament 160 (NF160), connexin 43 (Cx43), hyperpolarization-activated, cyclic nucleotide
modulated channel (HCN4), sodium/calcium exchanger, ryanodine receptor, sarcoplasmic/endoplasmic reticulum Ca2⫹
pump (SERCA), and phospholamban (PLB). In AVNCs with triple-positive NF160, SERCA, and PLB labeling, SERCA
and PLB were found with high degrees of colocalization. The majority (59%) of NF160-positive AVNCs were found
to coexpress HCN4. NF160 and HCN4 expression was found to be even higher in SANCs, where 88% of SANCs
exhibited coexpression. Spontaneous action potentials recorded from isolated neonatal AVNCs were uniformly of the
atrionodal type, showing none of the action potential heterogeneities found in the mature heart. Current recordings found
the hyperpolarization-activated funny current (If) in 55% (11 of 21 cells) of AVNCs, consistent with the immunocytochemistry results.
Conclusions—This represents the first detailed electrophysiological and immunohistological report of the neonatal AV
node and lays the groundwork for a better understanding of heart rate regulation and unique arrhythmias in the neonate
heart. (Circ Arrhythm Electrophysiol. 2011;4:936-946.)
Key Words: cardiac arrhythmogenesis 䡲 pacemaking cells 䡲 HCN4 䡲 NCX 䡲 NF160 䡲 compact node
䡲 inferior nodal extension
T
junction proteins and fast-acting voltage-gated ion channels
impart specific electric characteristics of each region.7–9
he atrioventricular (AV) node represents the sole conduction pathway between the atrial and ventricular compartments of the normal adult heart. The AV node is localized
within the triangle of Koch, bordered by the coronary sinus,
tendon of Todaro, and tricuspid valve.1 Within this diminutive area lie considerable structural and functional heterogeneities that determine the complex physiological functions of
the AV node. Functionally and histologically, the AV node
can be further subdivided into the inferior nodal extension
(INE), compact node (CN), and lower nodal bundle.2– 4 This
intricate structure forms the slowly conducting electric pathway that provides the critical AV delay that ensures adequate
diastolic ventricular filling.5,6 Differential expression of gap
Clinical Perspective on p 946
In addition to the crucial role of the AV node in providing
the required AV delay, the AV node also plays a critical role
as a secondary pacemaking site, preserving cardiac function
in the absence of sinoatrial (SA) pacemaker activity.10 Despite the ability of AV node to act as a fail-safe mechanism,
pathology within the AV node can be a significant source of
life-threatening cardiac arrhythmias. Cardiac morphology and
function continues to develop after birth and specific arrhythmias occur more frequently in certain developmental stages.
Received April 5, 2011; accepted September 12, 2011.
From Cardiovascular Sciences, Child and Family Research Institute, Vancouver, British Columbia, Canada (X.Y.S., Y.Q., P.D., E.L., L.K., A.B., S.S.,
G.F.T.); Molecular Cardiac Physiology Group, Simon Fraser University, Burnaby, British Columbia, Canada (X.Y.S., Y.Q., P.D., E.L., A.B., G.F.T.);
Maastricht University, Maastricht, The Netherlands (L.K.); Cardiovascular Research Center, Hospital de Sant Pau, Barcelona, Spain (L.H.-M.); and
Pediatric Cardiology, BC Children’s and Women’s Hospital, Vancouver, British Columbia, Canada (S.S.).
*Drs Sheng and Qu contributed equally to this work.
The online-only Data Supplement is available at http://circep.ahajournals.org/lookup/suppl/doi:10.1161/CIRCEP.111.964056/-/DC1.
Correspondence to Glen F. Tibbits, PhD, Molecular Cardiac Physiology Group, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada. E-mail
[email protected]
© 2011 American Heart Association, Inc.
Circ Arrhythm Electrophysiol is available at http://circep.ahajournals.org
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DOI: 10.1161/CIRCEP.111.964056
Sheng et al
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Figure 1. Neonatal rabbit atrioventricular (AV) nodal histology. A, Ten-day rabbit endocardium with anatomic landmarks as indicated:
superior vena cava (SVC); inferior vena cava (IVC); tendon of Todaro (tT); coronary sinus (CS); fossa ovalis (FO); aorta (Ao); and tricuspid valve (TV). The approximate location of the inferior nodal extension (INE) and the compact node (CN) of the AV nodal area are
shown in the boxed area. The orientation is also indicated by the arrows in the upper left-hand corner. Masson’s trichrome staining of
3 sections taken from the neonate AV nodal area are shown in B (S125), C (S165), and D (S302), in which red indicates muscular tissue, blue indicates collagen/connective tissue, black indicates nuclei, and white indicates fibrous tissue. B through D illustrate atrial
muscle (AM), aortic valve (AoV), central fibrous body (CFB), ventricular muscle (VM), and a specific regions of the AV nodal tract; INE
(B), compact node (CN) (C), and bundle of His (HB) (D).
However, the specific differences between the immature and
mature heart are poorly understood. In the present report, we
further a developmental AV nodal model using electrophysiological and immunohistological techniques to investigate
the essential components of the funny current and calcium
clock hypotheses of pacemaker activity,11 including the
hyperpolarization activated, cyclic nucleotide-gated channel
(HCN4), sodium/calcium exchanger (NCX), ryanodine receptor (RyR), phospholamban (PLB) and sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). AV nodal
dysfunctions produce arrhythmias with distinct characteristics at different stages of development. For instance, the
presentation of atrioventricular nodal reentrant tachycardia
(AVNRT) is distinct in pediatric patients12 due to changes in
the electrophysiological properties of the AV node as the
heart matures with development.13 The ontogenic differences
in AVNRT are believed to be due to a higher incidence of
anterograde dual AV nodal pathways in adult patients.
Junctional ectopic tachycardia (JET), another arrhythmia
consistent with an AV nodal dysfunction, is uniquely associated with open heart surgery for congenital heart defects in
infants and young children.14 Nevertheless, for virtually all
arrhythmias associated with AV node dysfunction, the agerelated etiology has not been clearly understood. Intensive
immunohistochemical, electrophysiological and computational modeling studies have been performed on the intact
adult AV node for more than 40 years,2,15,16 whereas the
electrophysiological characteristics of single AV nodal cells
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Figure 2. Immunohistochemistry of the neonate atrioventricular (AV) nodal area. A, B, and C are dual-labeled (NF160 and NCX) images
of sections taken from approximately S125, S165, and S302 as shown in Figure 1A, respectively. Locations of anatomic landmarks are
indicated: atrial muscle (AM), aortic valve (AoV), inferior nodal extension (INE), central fibrous bundle (CFB), ventricular muscle (VM),
and compact node (CN). High-magnification (63⫻) images are provided of the INE (D, I, II, and III), CN (E, I, II, and III), and bundle of
His (HB) (F, I, II, and III) for NF160, NCX, Cx43, and HCN4 Abs, respectively.
Sheng et al
Table 1.
AV Nodal Cells in Neonate Heart
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Immunohistochemical Summary
NF160
NCX
Cx43
HCN4
Atrial muscle
⫺
⫹
⫹⫹
⫺
Inferior nodal extension
⫹
⫹⫹
⫺
⫹
Compact node
⫹
⫹⫹
⫺
⫹
Bundle of His
⫹
⫹⫹
⫹
⫹
Ventricular muscle
⫺
⫹
⫹⫹
⫺
the neonatal AV node at the histological and cellular levels
that provide information that furthers the understanding of
ontogenic changes of the cardiac conduction pathway.
Methods
Heart Excision and Cannulation
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The experimental protocols used in this study were in accordance
with the Canadian Council on Animal Care (CCAC) regulations. The
Animal Care Committee of the University of British Columbia
approved the use of animals for this study. A total of 15, 10-day New
Zealand rabbits were used. Briefly, hearts isolated from 10-day
rabbits were cannulated and retrogradely perfused with Solution A.
Detailed descriptions of the procedures and solution compositions
are provided in the online-only Data Supplement.
Histology and Immunohistochemistry
Figure 3. Outlines of the structures shown in the Masson’s
trichrome and IHC images. Sixteen different sections spanning
the region from S105 to S352 are shown in cartoon fashion to
highlight areas of the atrioventricular (AV) nodal area vis-à-vis
other cardiac structures in the sections. The areas outlined in
red and mustard-colored lines include the atrial and ventricular
tissues, respectively. The areas colored in blue include the aorta
and aortic valves (cyan) and tricuspid valves (dark blue). The
fuchsia-colored area is the central fibrous bundle (CFB) and the
green-colored region represents the nodal track. Each section is
10 ␮m thick and thus the distance, for example between sections 105 and 126 is ⬇210 ␮m. The 16 images span the region
between the 2 long vertical red bars shown in Figure 1A.
(AVNCs) were not explored until a method for the isolation
of single AVNCs in adult hearts was developed by Hancox
and Levi in 1993.17 The extension of these techniques to the
investigation of AV conducting mechanisms found in neonates has so far been extremely limited, due primarily to the
technical difficulties associated with isolation and characterization of cells from this nodal area in the immature heart.
For the present study, we developed an AVNC isolation
technique for the 10-day-old (10-day) rabbit heart, which
consistently produced a high-yield of spontaneously beating
AVNCs. We also performed immunohistochemical and electrophysiological experiments on intact neonatal AV nodal
sections and isolated beating AVNCs. The development of
these techniques has allowed for detailed characterizations of
A rectangular section of right atrial tissue (3⫻5.5 mm) including not
only the triangle of Koch but also the aorta and the bundle of His
(HB) (see Figure 1A) was excised immediately after Solution A
perfusion. The preparation was imbedded in O.C.T Tissue-Tek (No.
4583, Sakura) and frozen in isopentane precooled to ⫺140°C in
liquid N2 and then stored at ⫺80°C; 10-␮m serial cryosections were
sliced perpendicular to the long axis of the tricuspid valve (Figure
1A). Sections were mounted onto poly-L-lysine– coated glass slides,
fixed with 4% paraformaldehyde for 10 minutes, and quenched with
glycine for 10 minutes. Detailed descriptions of the histology and
immunohistochemistry procedures are provided in the online-only
Data Supplement.
Isolation of Single AVNCs and SA Nodal Cells
From the Neonate Rabbit Heart
Isolation procedures specific to AVNCs were adapted from myocyte
isolation protocols used in our laboratory18 for neonatal ventricular
myocytes and from AVNC isolation protocols published for adult
hearts.17 Enzyme concentrations and perfusion rates were refined to
ensure viable, Ca2⫹-tolerant AVNCs and SA nodal cells (SANCs)
from the neonate heart. The complete procedure is described in
online-only Data Supplement Table S2 and related material.
Electrophysiological Recordings
Perforated (amphotericin B, 250 ␮g/mL) whole-cell current-clamp
and voltage-clamp techniques were used to record spontaneous
action potentials and ionic currents (MultiClamp 700A, Axon Instruments, Union City, CA) from isolated single cells as described in the
online-only Data Supplement.
Immunocytochemistry
The excised enzyme-digested rectangular tissue was chopped in a
tissue culture dish containing 2% paraformaldehyde for 10 minutes.
Cells were quenched by 100 mmol/L glycine for 10 minutes,
permeabilized with 0.1% Triton X-100 for 10 minutes, washed with
PBS 2⫻ for 10 minutes each, then allowed to settle onto poly-Llysine– coated (No. P5899, Sigma) coverslips for 1 hour at room
temperature, followed by primary antibodies incubation overnight at
4°C. Detailed descriptions of the immunohistochemistry procedures
are provided in the online-only Data Supplement.
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Figure 4. Action potential characteristics. A shows the length and width of spindle and rod atrioventricular nodal cells (AVNCs) measured using Image J software (publicly accessed at http://rsbweb.nih.gov/ij/). B shows spontaneous action potentials recorded from
current clamped AVNCs and sinoatrial nodal cells (SANCs) as well as an induced action potential from an atrial cell (AC), all of which
are representative for these cell classes. The diastolic depolarization of both AVNCs and SANCs are clearly evident. C indicates the
frequency of depolarization of the 3 cell types. Whereas ACs were stimulated at 0.5 Hz, both AVNC and SANC contracted spontaneously over the period of measurements at an average of ⬇17 and ⬇73 bpm, respectively. The diastolic depolarization rates (DDR) are
indicated and varied in a manner consistent with the spontaneous depolarization rates. Note that the DDR value for ACs was not determined (N.D.), as diastolic depolarization is not observed in these cells. D shows the mean diastolic potential (MDP), action potential
(AP) overshoot, amplitude, and duration (as both time for 50% [AP50] and 75% [AP75] repolarization). These values were not corrected
for differences in cycle length. Statistical analysis of AP parameters demonstrate significant increases in the length (AP50 and AP75) in
the atrial versus both types of pacemakers cells in the neonate heart (*P⬍0.050).
Statistical Analysis
Unless otherwise stated, data are presented as means with 95%
confidence intervals (95% CI), and n represents cell number.
Statistical significance of the results was tested using a 1-way or
2-way ANOVA and a Tukey test was used for post hoc multiple
comparisons between groups with significant differences based on
ANOVA. Student t test for paired samples as indicated or ␹2 test for
categorical frequency data were used. A probability value of ⬍0.050
was considered significant.
Results
Histology and Immunohistochemistry of the
Neonate AV Node
Landmarks demarking the adult AV node are also identifiable
in the neonate heart. The coronary sinus, tendon of Todaro,
and tricuspid valve, which outline the triangle of Koch, can
be clearly observed in Figure 1A. Representative results of
Masson’s trichrome staining are shown in Figure 1B, 1C, and
1D, corresponding to serial sections S125, S165, and S302, as
indicated in Figure 1A.
Protein expression within the AV node was investigated
with immunohistological techniques. Neurofilament 160
(NF160) is widely accepted as a unique cardiac pacemaker
marker in rabbits.19,20 Serial sections neighboring the S125,
S165, and S302 trichrome sections were labeled with NF160
and NCX and are shown as Figure 2A, 2B, and 2C, respectively. Immunolabeling for NF160 demonstrates high expression levels in the INE (Figure 2A and 2C-I), CN (Figure 2B
and 2D-I) and HB (Figure 2C, 2E-I) conductive tissues.
Whereas NF160 expression was extremely low in atrial
tissue, NCX expression was high, providing a clear outline of
the myocardium as well as a counter stain for the conductive
tissues (Figure 2A through 2C).
Connexin isoform expression is a key determinant of the
electric conductivity between cells. Connexin 43 (Cx43) is
the general cardiac isoform which forms high conductance
gap-junctions. However in mature nodal cells, Cx43 is not
expressed and connexin 45 (Cx45) is the predominant isoform, forming relatively low conductance gap-junctions.
Thus, Cx43 expression is associated with low resistance,
fast conducting pathways and Cx45 expression is associated with high resistance, slowly conducting pathways.
Cx43 was found in abundance in atrial and ventricular
muscle as well as within the bundle of His (Figure 2F-II),
all areas associated with rapid conduction speeds. Cx43 in
the atrial and ventricular myocardium was expressed as
highly organized clusters at the intercalated disks. Cx43
expression was only rarely found associated with the INE
(Figure 2D-II) and CN (Figure 2E-II) 2 areas associated
with slow conduction speeds.
Previous studies have suggested that HCN4 is the predominant hyperpolarization activated current (If) expressing channel in adult AVNCs.3,7,19 We used a monoclonal anti-HCN4
antibody on these sections and found that HCN4 labeling was
abundant in the INE (Figure 2D-III), the CN (Figure 2E-III),
and the HB (Figure 2F-III) and rarely seen in working
myocardium.
A schematic representation, combining the trichrome staining and immunohistological results, is presented in Figure 3.
Sheng et al
50 mV
-120 mV
2s
10d AVNC
50 pA
1s
10d AC
AV Nodal Cells in Neonate Heart
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containing 1.8 mmol/L calcium, 10% of cells began beating
spontaneously. These spontaneously beating cells had mostly
spindle-shaped morphologies and sustained their spontaneous
rhythms for several hours at room temperature at 15– 60 beats
per minute. Isolated AVNCs are identifiable by their absence
of strong striations and 2 common cellular morphologies
were identified: rod-shaped and spindle-shaped. Rod-shaped
cells maintained a uniform cell width along the cell body,
whereas spindle-shaped cells had significant tapering at the
cell ends. Isolated atrial cells (ACs) contained obvious
striations and are generally rod-shaped. Some spindle-shaped
ACs with clear striations were also occasionally observed.
General cellular dimensions and membrane capacitance of
both neonate AVNCs (both spindle and rod-shaped) and ACs
are shown in Figure 4A.
Spontaneously Occurring Action Potentials in
Pacemaking Cells
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50 pA
1s
10d SANC
50 pA
1s
Figure 5. Input resistance of atrioventricular nodal cells
(AVNCs), sinoatrial nodal cells (SANCs), and atrial cells (ACs).
The applied voltage protocol is shown. Representative current
traces from ACs but not AVNCs or SANCs exhibited a large
inward rectifier current.
In these drawings, the atria and ventricle are outlined in red
and mustard and the tricuspid value, central fibrous bundle,
aorta, and aortic valve are highlighted in dark blue, fuchsia,
and cyan, respectively. The nodal track, areas expressing
NF160, is demarked in green. In sections S105 and S126, the
nodal track lies on the atrial side of the central fibrous body
(CFB), consistent with the INE propagating toward the
penetrating bundle. The penetrating bundle, where the nodal
track passes through the CFB, appears in S140 –S276. The
nodal track emerges from the CFB as the HB, located in
S302–S352.
The intensities of protein labeling for different areas of the
heart are presented Table 1. As expected, NF160 was only
expressed within conductive tissues, whereas NCX was
widely expressed. However, NCX intensity in the INE, CN,
and HB was consistently higher than that found in the
musculature. Atrial and ventricular cardiomyocytes were
found to express Cx43 strongly with lesser intensities found
in the HB. Neither the INE nor CN was found to express
Cx43; however, these areas were found to express HCN4.
Characterization of Single Isolated AVNC and
SANC in the Neonate Heart
When cells isolated from the AV nodal region were switched
from a storage solution to a physiological Tyrode solution
Action potentials were recorded from both spindled- and
rod-shaped spontaneously beating AVNCs. Both types of
cells appear to share the same general action potential profile,
containing a pronounced diastolic depolarization during
phase 0 (Figure 4B). The neonatal AVNC action potential
quantitatively resembled the atrionodal (AN⫺) type action
potential recorded on adult rabbit AVNCs, in terms of
maximum diastolic potential (MDP), action potential (AP)
overshoot, AP amplitude, and AP duration at 50% (APD50)
repolarization.21 The previously reported nodal (N⫺) type
and nodal-His (NH⫺) type action potentials were not observed in these neonate cardiac preparations.
Action potentials were also recorded from neonatal ACs by
electric stimulation (10 ms, 2⫻ threshold, 0.5 Hz). This
induced action potential configuration was typical of the
working myocardium, with a more hyperpolarized MDP and
significantly longer APD50 and APD75 (P⬍0.050), as seen in
the representative traces shown in Figure 4B. Temporal and
voltage aspects of all three action potentials profiles are
summarized in Figure 4C and 4D.
Input Resistance
Input resistance, measured from the linear component of the
current recording, was used to quantify the susceptibility of a
cell to changes in membrane voltage. Cells with high input
resistance have low background conductance and only require small current injections to induce a change in membrane voltage. A 10-second voltage ramp from ⫺120 mV to
⫹50 mV was applied and representative currents are shown
in Figure 5. A linear current was observed in AVNCs,
suggesting a small inward rectifier current (IK1). This linear
current had a reversal potential of ⫺53.8 mV (⫺58.7 to
⫺48.9 mV) (n⫽19), substantially more positive than the
predicted potassium equilibrium potential (EK⫹), indicating a
relatively low potassium conductance.
In ACs, a strong inwardly rectified current was consistently
recorded as a large inward current at strongly hyperpolarized
potentials. Current recordings in ACs had a reversal potential
of ⫺77.7 mV (⫺85.0 to ⫺70.4 mV) (n⫽9), almost equal to
EK⫹, indicating a large dominating potassium conductance
under diastolic conditions.
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B
10d AVNC
5 mV
-50 mV
-40 mV
-150 mV
100 pA
500 ms
mV -150 -130 -110 -90
-70
-50
*
*
**
**
200 pA
10d SANC
10d AVNCs (n=8)
10d ACs (n=9)
Figure 6. Hyperpolarization-activated currents (If)
of neonate atrioventricular nodal cells (AVNCs),
sinoatrial nodal cells (SANCs), and atrial cells
(ACs). A, Representative If traces for AVNCs, ACs,
and SANCs, from top to bottom, as the result of
the applied voltage protocol (B). C, Averaged
instantaneous currents from AVNCs and ACs
(*P⬍0.050; **P⬍0.010). D, Averaged If density,
measured from the difference between the steadystate and instantaneous currents, for AVNCs, ACs,
and SANCs. The number of cells is indicated
below each bar in the bar graph (**P⬍0.010).
D
Peak difference current
at -150 mV (pA/pF)
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**
500 ms
10
0
-10
-20
-30
-40
-50
-60
-70
Initial current (pA/pF)
C
10d AC
0
10d AVNCs
10d SANCs 10d ACs
8
**
-2
11
-4
-6
100 pA
500 ms
AVNCs had ⬇7-fold higher input resistance than ACs
(AVNCs: 759.9 M⍀ (636.4 – 883.4 M⍀), n⫽19 versus ACs:
103.4 M⍀ (86.0 –120.8 M⍀), n⫽9; P⬍0.010), indicating a
more readily modified membrane potential in AVNCs. The
high input resistance associated with pacemaker-type cells
allows small inward currents, from the presence of If and or
a strong inward NCX current, to effectively depolarize these
cells.
Hyperpolarization-Activated Current If
If is activated by hyperpolarization and can be assessed by
inducing hyperpolarized membrane potentials. Figure 6A
indicates the induced current in neonate AVNCs, ACs, and
SANCs as the result of 2-second voltage-clamps stepped
from ⫺150 mV to ⫺50 mV at 20 mV intervals, as shown on
Figure 6B. If is measured from the difference between the
instantaneous current evoked at the beginning of the voltage
step and the steady-state current found at the end of the
voltage step (Figure 6A). The current-voltage relationships
for this instantaneous current component in ACs were clearly
associated with large inward currents at these hyperpolarized
potentials, stabilizing the resting membrane potential (Figure
6C). If was seen in 55% (11 of 21 cells) of AVNCs and 100%
of SANCs (11 cells) with spontaneously action potential
activity. If density in SANCs was more than twice that of
AVNCs, and no functional expression of If in any of the ACs
(9 cells) was found (Figure 6D).
11
Immunocytochemistry of AVNCs, SANCs, and
ACs From the Neonate Heart
The higher prevalence of If in SANCs with respect to AVNCs
correlated with HCN4 expression intensities observed in
single cells (Figure 7A through 7D). The distribution of
HCN4 was predominately on the sarcolemma in both neonate
AVNCs (Figure 7A) and SANCs (Figure 7B). In NF160positive cells, 87.6% of cells isolated from the SA node were
found to also contain HCN4 (Figure 7C). In neonatal cells
isolated from the AV node, only 58.8% of cells expressing
NF160 were also found to express HCN4 (Figure 7C). No
significant colocalization between HCN4 and NCX was
observed in either SANCs or AVNCs, as in Figure 7D. Figure
7E shows NF160 (red) has a striated appearance in the
intracellular compartment that corresponds to the sarcomere
pattern of the AVNC sarcomere structures from the xz, xy,
and yz planes. There was very little colocalization of NF160
observed with NCX (green).
The distribution patterns of NF160, RyR and NCX are
shown in neonate AVNC (Figure 8A-I), SANC (Figure
8A-II), and AC (Figure 8A-III). All 3 cell types demonstrated
clear RyR striations with ⬇2 ␮m periodicity but only AVNCs
and SANCs were NF160 positive. NCX expression in all 3
cell types was limited to the cell periphery indicating the
absence of significant t-tubular formation. The NCX to RyR
colocalization was significantly higher in neonatal AVNCs
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AV Nodal Cells in Neonate Heart
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Figure 7. Dual NF160 and HCN4 labeling in neonate atrioventricular nodal cells (AVNCs) and sinoatrial nodal cells (SANCs). A and B
are representative images of single cells with NF160 and HCN4 labeling, in AVNCs and SANCs, respectively. C is a 2⫻2 contingency
table that presents the percentage of cells containing NF160 labeling that also contain HCN4 labeling for AVNCs and SANCs. The difference in HCN4 expression levels between AVNCs and SANCs was highly significant (P⬍0.0001). D presents subcellular colocalization
data for AVNCs and SANCs expressing HCN4 and NCX. HCN4-with-NCX colocalization refers to how often HCN4 pixels are found to
also contain NCX and vice versa. There was no significant difference between AVNCs and SANCs HCN4 and NCX colocalization values. E shows the 3D expression of NF160 (red) and NCX (green) in a neonate AVNC. I, II, and III represent the xz, xy, and yz planes,
respectively, from a 3D confocal Z-stack.
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Figure 8. NF160 triple labeling images of neonate atrioventricular nodal cells (AVNCs), sinoatrial nodal cells (SANCs). A presents representative deconvolved images for NF160, RyR and NCX triple labeling for AVNCs (A-I), SANCs (A-II), and atrial cells (ACs) (A-III) and
the subsequent colocalization results (A-IV). B presents representative deconvolved images for NF160, SERCA, and PLB triple labeling
for AVNCs (B-I), SANCs (B-II), and ACs (B-III) and the subsequent colocalization results (B-IV). NCX-with-RyR colocalization indicates
how often a NCX pixel is found to also contain RyR. The NCX to RyR colocalization is significantly higher in AVNCs compared with
ACs (P⫽0.041). Similar calculations were performed for the SERCA and PLB relationship. SERCA to PLB colocalization is significantly
higher in AVNCs compared with SANCs (P⫽0.019).
compared with ACs (Figure 8A-IV). Substantial levels of
SERCA and PLB colocalization were found in all 3 cell types
(Figure 8B-IV) and AVNCs contain significantly higher
degrees of SERCA to PLB colocalization than do SANCs.
Discussion
AV Node and AVNC Morphology in
Neonate Heart
The examination of the histological and immunochemical
sections in our study suggests the neonate heart shares similar
AV anatomic complexity as the adult, consistent with the
findings of other groups.4,19 INE, CN, and HB regions are
clearly identifiable in 10-day AV nodal region. The HCN4
immunolabeling pattern in single cells was also reminiscent
of what others have observed in adult SANCs.22,23 The HCN4
fluorescence intensity was found to be consistently higher in
SANCs compared with that found in AVNCs with the
majority of HCN4 staining distributed along the cell membrane. Occasionally, some cells had HCN4 localized intracellularly, which may be attributable to protein trafficking.19,24 Several different AVNC morphologies have been
reported in adult hearts, including spider, spindle, rod, and
ovoid phenotypes. Salient electrophysiological properties
such as the action potential shape and ionic channel composition appear to vary with these different cellular morphologies.17,21,25 Compared with the adult, neonatal AVNCs were
observed to be relatively homogeneous in the phenotypic
features, and only spindle and rod morphologies were found.
Although spontaneous pacemaker activity was observed more
frequently in spindled cells, both cell shapes had similar
action potential configurations and, If, ICa, and INa current
densities (data not shown). Future studies may determine the
developmental relationship between the variety of AVNC
morphologies present in the adult and the more limited
spindle- and rod-shaped morphologies found in the neonate
heart.
In 10-day rabbit hearts, AVNCs and ACs have similar
cellular dimensions and membrane capacitances (Table 2),
yet these 2 cell types are vastly different at ⬇56 days of
development (Munk et al21,26). Combining our observations
with those of Munk et al, ACs can be expected to grow
⬇1.7-fold in length and ⬇1.5-fold in width during this
development period, with a ⬇2.6-fold increase in membrane
capacitance. We have previously observed a ⬇3.5-fold
change in membrane capacitance in rabbit ventricular myocytes over the same time frame,27 indicating that both atrial
and ventricular myocytes undergo significant changes in cell
morphology over this development period. However, over the
same period, AVNCs were shown to undergo negligible
changes in cell length and increased their cellular volume and
membrane capacitance by only 1.3- and 1.5-fold, respectively. Therefore, in morphological terms, AVNCs appear to
Sheng et al
Table 2.
AV Nodal Cells in Neonate Heart
945
Comparison of Neonatal (10-Day) Versus Mature (⬃56-Day) AVNCs and ACs: Cell Dimensions
AVNCs (Rod-Shaped)
10-Day
ACs
56-Day*
10-Day
56-Day*
Mean
95% CI
Mean
95% CI
% Inc
Mean
95% CI
Mean
95% CI
Length, ␮m
64.5
54.9 –74.1
76.2
69.0 – 83.5
118.1
70.3
58.3– 82.3
121.8
102.2–141.4
173.3
Width, ␮m
7.6
7.0–8.2
10.7
9.9–11.5
140.8
8.2
7.6–8.8
12.6
10.6–14.6
153.7
16.0–37.6
40.7
28.7–52.7
151.9
19.1
16.6–21.7
50.4
35.1–65.7
263.9
Cm, pF
26.8
Cells, n
6
8
9
% Inc
5
AVNCs indicates atrioventricular nodal cells; ACs, atrial cells; CI, confidence interval; and Inc, increase.
*Data taken from Munk et al.21 In that study, the adult rabbits weighed 1.5–2.5 kg, which we have calculated to be approximately 8 weeks of age.26
Downloaded from http://circep.ahajournals.org/ by guest on May 13, 2017
be nearly fully developed even at 10 days of age. The voltage
aspects of the action potential from both 10-day and 56-day
AVNCs are similar (Table 3). The functional consequences of
this observation and the electric properties of these cells at
different development stages remain to be determined.
Electrophysiological Properties of Neonate AVNCs
When compared with AVNCs, SANCs (NF160⫹) exhibit
significantly higher expression of HCN4 and larger If, all
consistent with their role as the dominant pacemaker in the
heart. In the adult rabbit heart, the AV nodal rhythm is
thought to originate from the INE, an area defined by a
specific pattern of protein expression (NF160⫹, Cx43⫹/⫺,
HCN4⫹, CaV1.3⫹), where a AN-type action potential predominates.4,19 Thus, HCN4 expression correlates to regions
of strong pacemaker activity.
In the adult heart, AVNCs appear to be electrically diverse
with multiple action potential phenotypes having been reported.16,17,21,25,28 Among these reports, Billette28 subdivided
the nodal cells into 6 groups, namely AN, ANCO, ANL, N,
NH, and H cells, according to their action potential morphology and responses to periodic premature stimulation. Similar
action potential diversity has also been found in the intact
neonatal AV node, where Hewett et al recorded 4 action
potential configurations designated as AN, N, high NH, and
low NH.29
In our hands, only spindle- and rod-shaped AVNC morphologies could be identified and the action potentials recorded from these cells were uniformly of the AN-type.
Table 3.
Comparison of Neonatal (10-Day) Versus Mature
(⬃56-Day) AVNCs and ACs: Action Potential Characteristics
AVNCs (Rod-Shaped)
10-Day
56-Day*
Mean
95% CI
Mean
95% CI
MDP, ⫺mV
68.8
71.4 – 66.3
71.9
77.4 – 65.4
Overshoot, mV
22.0
12.4–31.6
13.9
9.6–18.2
Amplitude, mV
90.8
82.2–99.4
85.8
81.5–90.1
APD50, ms
44.3
36.7–51.9
70.7
35.2–106.2
Cells, n
8
5
AVNCs indicates atrioventricular nodal cells; ACs, atrial cells; CI, confidence
interval; MDP, mean diastolic potential; and APD, action potential duration.
*Data taken from Munk et al.21 In that study, the adult rabbits weighed
1.5–2.5 kg, which we have calculated to be approximately 8 weeks of age.26
Neither the N- nor the NH-type action potentials, associated
with compact nodal and nodal-his cell in the adult, were
observed in our preparation. Immunolabeling results found
both the anatomic INE and the anatomic CN to contain
similar protein expression patterns (NF160⫹, Cx43⫹/⫺,
HCN4 ⫹). However, it is not known whether the single cell
recordings represent just the immature INE or other areas of
the nodal track.
Because of the diminutive size of the neonate nodal
preparation, it is unclear whether the AN-type uniformity
stems from an immature nodal action potential phenotype
common among all nodal cells or from the relative prevalence
of AN-type nodal cells in this preparation. We observed the
diameter of CN in neonatal rabbit to be approximately 0.25
times of the adult compact node, and it is possible that
non–AN-type cells are present but not observed due to their
limited number. As the excision area predominantly isolated
the AV node and only included a limited extension of the His
Bundle, it is also conceivable that this cell type may be
limited within the isolated cells. Nevertheless, HCN4 staining
and the lack of Cx43 expression in both the INE and compact
node could suggest that both regions may contain active
pacemaker activity in the neonate heart. Further studies will
be required to determine the functional contribution of HCN4
expression in the compact node.
Although many AVNCs (NF160⫹) exhibited HCN4
and/or If expression, not all AVNCs contain these pacemaker
hallmarks. All AVNCs were, however, found to contain the
major components of the Ca2⫹ clock pacemaker mechanism
(eg, RyR, NCX, SERCA, and PLB). Whether the prevalence
or role of the calcium clock mechanism are the same as in the
adult heart remains to be determined.
In summary, neonatal AV nodal tissue shows similar
histological complexity as the adult AV node. Although
further structural and functional studies using optical mapping and computer reconstruction techniques need to be
performed to delineate the neonatal AV nodal conduction
pathways, this study represents the first characterization of
isolated neonate AV cells. These findings should make a
significant contribution to our understanding of AV nodal
function in the neonate heart and the pathologies therein that
are unique to that stage of ontogeny.
Acknowledgments
We thank Toro Min for contributions to the study.
946
Circ Arrhythm Electrophysiol
December 2011
Sources of Funding
These studies were generously supported by an operating grant from
the Canadian Institutes of Health Research (to Dr Tibbits) and a
junior research fellowship from the Heart and Stroke Foundation of
Canada (to Dr Qu). Dr Tibbits is the recipient of Tier I Canada
Research Chair.
Disclosures
None.
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Downloaded from http://circep.ahajournals.org/ by guest on May 13, 2017
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CLINICAL PERSPECTIVE
The atrioventricular (AV) node represents the sole conduction pathway between the atrial and ventricular compartments
of the normal adult heart. Many properties of the adult AV node have been elucidated, including anatomy, molecular
architecture, and electric activity. However, the properties of the neonatal AV node and its role in unique pediatric cardiac
arrhythmias such as junctional ectopic tachycardia (JET) are poorly understood. For the first time, histological and
immunochemical examination of sections suggests the neonate heart shares similar AV anatomic complexity as the adult.
Compared with the adult, single neonatal AV nodal cells were observed to be relatively mature in size and homogeneous
in phenotypic features, as only spindle and rod morphologies were found. Spontaneous action potentials recorded from
isolated neonatal AV nodal cells were uniformly of the atrionodal-type, showing none of the action potential
heterogeneities found in the mature heart. Our report lays the groundwork for a better understanding of heart rate regulation
and future studies aiming to find mechanisms of unique arrhythmias in the neonate heart such as JET.
Isolation and Characterization of Atrioventricular Nodal Cells From Neonate Rabbit
Heart
Xiao Ye Sheng, Yang Qu, Pauline Dan, Eric Lin, Linda Korthout, Aaron Bradford, Leif
Hove-Madsen, Shubhayan Sanatani and Glen F. Tibbits
Downloaded from http://circep.ahajournals.org/ by guest on May 13, 2017
Circ Arrhythm Electrophysiol. 2011;4:936-946; originally published online October 14, 2011;
doi: 10.1161/CIRCEP.111.964056
Circulation: Arrhythmia and Electrophysiology is published by the American Heart Association, 7272 Greenville
Avenue, Dallas, TX 75231
Copyright © 2011 American Heart Association, Inc. All rights reserved.
Print ISSN: 1941-3149. Online ISSN: 1941-3084
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Supplementary Materials
Methods
Heart Excision and Cannulation
The experimental protocols used in this study were in accordance with the Canadian Council on
Animal Care (CCAC) regulations. The Animal Care Committee of the University of British
Columbia approved the use of animals for this study. Isolation procedures specific to AVNCs were
adapted and modified from isolation protocols used in our laboratory 1 formulated for neonatal
ventricular myocytes and AVNC isolation protocols published for adult hearts 2. 10 day old (10-d)
New Zealand rabbits (150 to 240 g) of either sex were deeply anaesthetized by an intra-peritoneal
injection of thiopental sodium (60 mg/kg body weight) and sodium heparin (102.1 mg/kg body
weight). Upon the disappearance of pinch-toe response the heart was rapidly excised. The aorta
was cannulated 1.5 mm above the coronary orifice on a Langendorff apparatus providing retrograde
perfusion. A 15 gauge stainless needle was used as a cannula to tightly fit into the 10-d aorta.
Successful isolations required this entire procedure, from puncturing the thoracic cavity to perfusion
of the heart, to be completed in less than one minute.
Histology and Immunohistochemistry
A rectangular section of right atrial tissue (3 x 5.5 mm) including not only the triangle of Koch but
also the aorta and the bundle of His (see Figure 1A) was excised immediately after Solution A
perfusion. The preparation was imbedded in O.C.T Tissue-Tek (#4583, Sakura) and frozen in
isopentane pre-cooled to -140°C in liquid N2 and then stored at -80°C. 10 µm serial cryosections
were sliced perpendicularly to the long axis of tricuspid valve (Figure 1A). Sections were mounted
onto poly-L-lysine coated glass slides, fixed with 4% paraformaldehyde for 10 minutes and blocked
with glycine for 10 minutes
Masson’s trichrome staining was conducted using the manufacturer’s prescribed protocol (#HT15,
Sigma). After staining, sections were rinsed, dehydrated with alcohol and cleared in xylene
followed by Histomount (#19479, Ted Pella) mounting. Histological images were acquired using
an Olympus BX51 microscope with a 4x objective. Mosaic images were created from ten to fifteen
individual images using Adobe Photoshop CS5.5 (Figure 1B-D).
Supplementary Materials ‐ AV nodal cells in neonate heart Immunohistology was applied to the serial sections adjacent to the trichrome sections. Sections
were permeabilized with 0.1% Triton X-100 for 10 minutes and then washed with PBS 2x for 10
minutes each. Sections were then incubated with a variety of primary antibodies including: chicken
anti neurofilament 160 (NF160) IgY, mouse anti NF160 IgG1, mouse anti sodium/calcium
exchanger (NCX) IgM, mouse anti connexin 43(Cx43) IgG1 and mouse anti HCN4 IgG1) overnight
at 4°C. After being washed with PBS 3x 10 minutes, sections were incubated with the secondary
antibodies (goat anti-chicken IgY conjugated to Alexa 488 and goat anti-mouse IgG1 conjugated to
Alexa 488 or 555) for 1 hour at room temperature. Detailed antibody information and dilutions are
provided in Table S1. After 3 additional washes with PBS, the sections were mounted in Antifade
reagent (#S36936, Sigma) and coverslips sealed with nail polish. Fluorescence images were
acquired using an Olympus BX61 microscope with a 10x objective. On average 100 individual
fluorescence images (tiles) were stitched together (Image Pro, MediaCybernetics) to generate the
mosaic images (Figure 2A-C).
Enzymatic Perfusion
Following cannulation, the heart was retrogradely perfused for 5 minutes with solution A
containing (in mM: 130 NaCl, 4.5 KCl, 3.5 MgCl2, 0.75 CaCl2, 0.4 NaH2PO4, 5 HEPES, 50 taurine,
10 glucose, titrated to pH 7.25 with NaOH) to clear the blood in the coronary circulation (18 ml
min-1); followed with calcium-free solution A for 5 minutes (10 ml min-1). The perfusate was then
switched to an enzyme solution which contained collagenase (0.1 mg/ml, Yakult, Japan), protease
(0.05 mg/ml, P5147, Sigma) and BSA (1 mg/ml, #10775835001, Roche) in solution A. The
perfusion volume of enzyme solution was adjusted from 65 to 95 ml depending on rabbit weight.
The flow rate was maintained at 3.6 ml min-1. Detailed perfusion rates and enzyme concentrations
are provided in Table S2.
Mechanical Dissociation
After digestion, the heart was removed and the AV nodal area exposed in a dissecting dish filled
with cold storage solution (in mM: 100 L-glutamate potassium, 30 KCl, 5 Na-pyruvate, 20 taurine,
5 creatine, 5 succinic acid, 2 Na2ATP, 5 β-OH butyrate, 20 glucose, 5 MgCl2, 1 EGTA, 10 HEPES,
titrated to pH 7.4 with KOH). A ~2 x 4 mm section of tissue was excised around the AV node for
mechanical dissociation. This tissue was then transferred to a separate dish containing cold storage
solution and minced with a surgical blade. Dissociated cells were filtered through a nylon mesh
Supplementary Materials ‐ AV nodal cells in neonate heart (105 µm) and centrifuged at 80 x g for 2.5 minutes. The cell pellet was re-suspended and allowed
to rest in storage solution for 30 minutes at room temperature and then transferred to Ca2+-free
Tyrode’s solution (in mM: 140 NaCl, 5.4 KCl, 1 MgCl2, 10 HEPES,10 glucose, titrated to pH 7.4
with NaOH). The cellular solution was then titrated to 1.8 mM [Ca2+] over 20 minutes. Single
atrial cells (ACs) were isolated from areas adjacent to the excised area from the same heart.
Isolation of sinoatrial nodal cells (SANCs) from 10-d rabbit heart
SANCs were isolated from 10-d rabbit hearts using similar procedures as that for AVNCs. Fifty ml
of enzyme solution containing collagenase (0.2 mg/ml), elastase (0.4 mg/ml, #2292, Worthington),
and protease (0.1 mg/ml) were recirculated at 3.6 ml min-1 for around 30 minutes to digest the
whole heart (see Table S1). The endocardial surface was exposed with a longitudinal incision in the
free wall and a rectangular section of tissue (~2 x 3 mm) including the whole SA node, superior
vena cava and inferior vena cava 3 was excised. Cells were dissociated and dispersed into the
storage solution, then washed and resuspended in Tyrode’s solution.
Determinants for the successful isolation of AVNCs in neonatal heart
We have established a well-defined isolation protocol through which a consistent high-yield of
single ventricular myocytes and AVNCs from the neonatal heart was achieved. For successful
isolation of functional AVNCs there were several critical factors. While some of these are intuitive,
the technical aspects of this process cannot be overstated. The first is the rapidity and precision of
anesthesia administration, heart excision and aortic cannulation. We observed that cannulation at
~1.5 mm above the coronary orifice provided the most efficient perfusion in hearts of this size. The
second is the enzyme concentration and perfusion rate. In contrast to other adult AV nodal isolation
protocols 2 4, it was determined that a much lower enzyme concentration and perfusion rate had to
be employed to ensure the timely adjustment of perfusion time in accordance with the digestion
status. The third is to minimize the mechanical stress on the cells. These cardiomyocytes from the
immature heart are extremely vulnerable and less tolerant to enzymatic and mechanical treatment.
Normally we did not repeatedly pipette the finely chopped tissue through small pore pipette tips
unless the digestion was not complete.
Supplementary Materials ‐ AV nodal cells in neonate heart Electrophysiological Recordings
Isolated single cells were allowed to settle on poly-L-lysine coated glass-bottomed dishes and the
perforated whole-cell patch-clamp technique was used to record action potentials and ionic currents
(MultiClamp 700A, Axon Instruments, Union City, CA). Individual cells were visualized on an
inverted microscope equipped with a 63x oil immersion objective (Eclipse TE300 Nikon). Patch
pipettes (3 to 4 M) were pulled from thin-walled glass capillaries (World Precision Instruments,
Sarasota, FL) using a vertical puller (PP-830, Narashige, Japan) and liquid junction potentials were
corrected by -10 mV. 1-2 G seals were formed under the voltage clamp configuration and only
cells in which the access resistance dropped to less than 10 M within 10-20 minutes after seal
formation were used. Recordings acquired using pClamp (version 8.2, Axon Instruments, Digidata,
1322A) at 20 kHz and filtered at 1 kHz. All experiments were conducted at room temperature
(22C).
The external Tyrode’s solution contained (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10
HEPES, and 10 glucose, pH 7.4 adjusted with NaOH. Extracellular solution was superfused at 0.2
ml min-1. The internal pipette solution contained (in mM): 10 NaCl, 50 KCl, 80 KOH, 80 aspartic
acid, 1 MgCl2, 10 HEPES, pH 7.2 adjusted with KOH. Amphotericin B (Sigma) which was
solubilized in dimethyl sulfoxide (DMSO) was used in a final concentration of 250 g/ml in the
patch pipette and the final DMSO concentration was 0.2%.
Immunocytochemistry
The excised enzyme-digested rectangular tissue was chopped in a tissue culture dish containing 2%
paraformaldehyde for 10 minutes. Cells were quenched by 100 mM glycine for 10 minutes,
permeabilized with 0.1% Triton X-100 for 10 minutes, washed with PBS 2x for 10 minutes each,
then allowed to settle onto poly-L-lysine (#P5899, Sigma) coated coverslips for one hour at room
temperature. Cells were labeled with primary and secondary antibodies (see Table S2) following
the same protocols as for immunohistochemistry. Single cell images were acquired on a Zeiss LSM
5 Pascal laser scanning confocal microscope equipped with Zeiss 63x/1.4 Plan-Apochromat oil
immersion objective. Images were deconvolved to maximize available resolution5 using a
maximum likelihood estimation (MLE) algorithm (Huygens Pro 2.4.1, Scientific Volume imaging,
Supplementary Materials ‐ AV nodal cells in neonate heart Hilversum, Netherlands) in conjunction with an empirically determined point spread function6.
Colocalization analyses were then conducted to determine the frequency of colocalization events.
Supplementary Materials ‐ AV nodal cells in neonate heart Table S1. Primary (1o) and secondary (2o) antibodies used for immunohistochemistry and
immunocytochemistry
Antibodies
Vendor
Catalog #
Dilution
Mouse anti NF160 IgG1
DSHB
2H3
Supernatant
Chicken anti-NF160 IgY
SCT
1452
1:1000
Mouse anti-NCX IgM
TS
MA3-926
1:3000
Mouse anti-HCN4 IgG1
NM
75-150
1:10
Mouse anti-Cx43 IgG1
MP
MAB3068
1:500
Mouse anti-RyR IgG1
TS
MA3-916
1:200
Mouse anti-SERCA IgG1
TS
MA3-910
1:3000
Mouse anti-PLB IgG2a
TS
MA3-922
1:200
Goat anti-mouse IgG1
IV
A-21121
1:500
o
1 Ab
2o Ab
congugated to Alexa Fluor 488 or 555
AffiniPure Goat Anti-chicken IgY
A-21127
JIR
103-485-155
1:3000
IV
A-21426
1:500
conjugated to DyLight 488
Goat anti-mouse IgM
conjugated to Alexa Fluor 555
DSHB - Developmental Studies Hybridoma Bank; SCT - StemCell Technologies; TS - Thermo Scientific
NM - Neuromab; MP - Millipore; IV - Invitrogen; JIR - Jackson ImmunoResearch
Supplementary Materials ‐ AV nodal cells in neonate heart Table S2. Parameters for single cell isolation
10d AVNCs
10d SANCs
10d ACs
Solution A
Perfusion rate
(ml/min)
18
18
18
Enzyme solution
perfusion rate
(ml/min)
3.6
3.6
3.6
Enzyme solution
concentration
(mg/ml)
Collagenase 0.10
Collagenase 0.20
Collagenase 0.10
Protease 0.05
Protease 0.10
Protease 0.05
Elastase 0.00
Elastase 0.40
Elastase 0.00
BSA 1.00
BSA 1.00
BSA 1.00
Enzyme solution
volume
(ml)
65-95
50
65-95
Enzyme solution
recirculation?
no
yes
no
Supplementary Materials ‐ AV nodal cells in neonate heart Table S3. Action Potential Parameters – Range of Observations
10d AVNC
MDP
(mV)
Overshoot
(mV)
Amplitude
(mV)
APD50
(ms)
APD75
(ms)
min
-70
-1
69
33
80
max
-60
40
105
66
230
MDP
(mV)
Overshoot
(mV)
Amplitude
(mV)
APD50
(ms)
APD75
(ms)
min
-95
-4
85
29
58
max
-60
50
110
141
192
MDP
(mV)
Overshoot
(mV)
Amplitude
(mV)
APD50
(ms)
APD75
(ms)
min
-100
-7
87
72
141.7
max
-65
30
105
213
354
10d SANC
10d AC
Figure S1 - Measurement of diastolic depolarization rate (DDR). Figure S2 - AP50 and AP75
Figure S1. The measurement of DDR was performed by determining the maximum diastolic
potential (MDP) as indicated in the above diagram with the arrow labeled 1 and connecting it to the
threshold as indicated in the above diagram with the arrow labeled as 2.
Figure S2. Measurement of Action Potential Duration (AP50 and AP75). The AP amplitude was
determined as the difference between the MDP and the point of maximum overshoot. The times
required to repolarize to 50% (AP50) and 75% (AP75) of this value were then calculated.
Supplementary Materials ‐ AV nodal cells in neonate heart Figure S3.
S3A
S3B
S3C
Figure S3. Figures S3A, S3B, and S3C are the original images of Figures 2A, 2B, and 2C, respectively.
Images shown in the text were slightly modified with Adobe Photoshop CS5.5 to remove artifactual staining
of debris on slides (Figures S3A-C) and of the aorta (Ao) (top right of Figure S3C). Using a 40x objective,
the staining of the debris was found to be extracellular in origin thereby constituting non-specific staining.
Likewise, staining of the aorta was found to be extracellular and appears to constitute the well-established
“edge effect” 7 thereby constituting non-specific staining.
Movie S1. A rotating 3D AVNC cell dual-labeled with NF160 and NCX clearly indicating that
NF160 (red) shows a striated appearance in the intracellular compartment that corresponds to the
sarcomere pattern of the AVNC sarcomere structures and very little co-localization was observed
with NCX (black).
Supplementary Materials ‐ AV nodal cells in neonate heart References for Supplementary Material
1.
2.
3.
4.
5.
6.
7.
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