Download Comprehensive multilevel in vivo and in vitro analysis of heart rate

protocol
Comprehensive multilevel in vivo and in vitro
analysis of heart rate fluctuations in mice by ECG
telemetry and electrophysiology
Stefanie Fenske1, Rasmus Pröbstle1, Franziska Auer1, Sami Hassan1, Vanessa Marks1, Danius H Pauza2,
Martin Biel1 & Christian Wahl-Schott1
1Center for Integrated Protein Science (CIPS-M) and Center for Drug Research, Department of
Pharmacy, Ludwig-Maximilians-Universität München, and DZHK
(German Center for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany. 2Institute of Anatomy, Faculty of Medicine, Lithuanian University
of Health Sciences, Institute of Anatomy, Kaunas, Lithuania. Correspondence should be addressed to C.W.-S. ([email protected]).
© 2015 Nature America, Inc. All rights reserved.
Published online 10 December 2015; doi:10.1038/nprot.2015.139
The normal heartbeat slightly fluctuates around a mean value; this phenomenon is called physiological heart rate variability
(HRV). It is well known that altered HRV is a risk factor for sudden cardiac death. The availability of genetic mouse models makes
it possible to experimentally dissect the mechanism of pathological changes in HRV and its relation to sudden cardiac death. Here
we provide a protocol that allows for a comprehensive multilevel analysis of heart rate (HR) fluctuations. The protocol comprises a
set of techniques that include in vivo telemetry and in vitro electrophysiology of intact sinoatrial network preparations or isolated
single sinoatrial node (SAN) cells. In vitro preparations can be completed within a few hours, with data acquisition within 1 d.
In vivo telemetric ECG requires 1 h for surgery and several weeks for data acquisition and analysis. This protocol is of interest to
researchers investigating cardiovascular physiology and the pathophysiology of sudden cardiac death.
INTRODUCTION
The continuous and stable heartbeat is essential for life. It is
generated in the central region of the SAN, which is the pacemaker
region of the heart. The SAN is formed by a group of spontaneously active cells, so-called pacemaker cells, which are connected
to each other and make up a coupled network1. The rhythm of
the heart fluctuates from beat to beat; this phenomenon is called
physiological HRV. These distinct fluctuations can be quantified
in the ECG as fluctuations in the beat-to-beat duration of cardiac
cycles or RR intervals, which are the distances between consecutive
peaks of R waves of QRS complexes. In healthy individuals, HRV
is associated with several physiological processes including the
respiratory cycle (respiratory sinus arrhythmia)2,3. Physiological
HRV also includes periodic HR fluctuations corresponding to
changes in venous return, arterial blood pressure (which leads
to adjustments of the HR via the baroreceptor reflex), peripheral
vasomotor activity or thermoregulatory blood flow4–7.
Physiological HRV has traditionally been attributed to changes
in the extrinsic regulation of the HR by the autonomic nervous system (ANS). However, recently this concept has been extended, and
a more sophisticated view of HR regulation has been developed8–14.
This extended concept also considers that the HR and HRV are
affected by the concerted action of two other major components:
the intrinsic properties of the pacemaker cells of the SAN 11 and
the responses of these cells to external (autonomic and other)
inputs in the presence of unchanged activity of the ANS15–18.
Therefore, HRV can be considered in this broader context, in
which case all three components underlying physiological HRV
and HR fluctuations need to be investigated for a full understanding of HRV. In this protocol, we provide the tools that are required
to analyze each of the three components of HRV in mice.
Extended concept of HRV regulation
HRV is determined by the intrinsic properties of the pacemaker
cells within the SAN. Within the central region of the SAN (leading
pacemaker region; Fig. 1), individual pacemaker cells synchronize
in the sinoatrial network to a common rhythm by mutual electrical
interactions via gap junctions. This mechanism is called mutual
entrainment. The sequential process of impulse formation and
synchronization gives rise to a stable intrinsic HR in the absence of
autonomic regulation (the SAN rhythm) and defines the set point
of the actual HR and the baseline level of HR fluctuations. It arises
from intrinsic properties of the sinoatrial network itself—e.g., the
structure of cells (different SAN cell types) and network, connection between cells and basal ion channel activity.
HR fluctuations are also determined by the responsiveness of
the sinoatrial network to perturbating external stimuli such as
autonomic inputs to the heart8–14. In this context, the SAN acts
as a transfer element, which responds to these external perturbations19. Under physiologic conditions, the response of the SAN
(the output function of the SAN) faithfully represents the external
changes in autonomic drive (the input function19). However, this
assumption may not be valid in the presence of SAN pathology
(and pathology of the downstream conduction system). The characteristics of the response function (the input output function of
the SAN) are determined by the following: the number of receptors mediating autonomic SAN regulation; the signaling network
downstream of these receptors, including G proteins and their
effectors (including adenylyl cyclase, kinases and phosphatases);
and the targets of these signaling networks, including the ion
channels, transporters and gap junctions. Changes in each of these
parameters can lead to changes in the gain and the dynamic range
of the response function of the SAN to external stimuli (even if
the external stimulus via the ANS is completely unchanged) and,
as a consequence, they can lead to increased or decreased HR
fluctuations. There is evidence from genetic mouse models that
this mechanism strongly contributes to SAN dysfunction20,21.
HRV is also determined by a specific entrainment process
between the ANS and the SAN (neuronal entrainment; Fig. 1).
nature protocols | VOL.11 NO.1 | 2016 | 61
© 2015 Nature America, Inc. All rights reserved.
protocol
Figure 1 | Multilevel analysis of HR
a
In vivo
Network
Single cell
fluctuations. (a) The leading pacemaker
region of the SAN is formed by pacemaker
Neuronal
Intrinsic
entrainment
entrainment
cells, which are spontaneously active and
coupled to other pacemaker cells via gap
junctions (middle). Within the sinoatrial
Sympathetic
network, pacemaker cells synchronize to
a common rhythm by mutual entrainment
Parasympathetic
(intrinsic entrainment). The SAN network
is also entrained by external stimuli via the
Electrocardiography
SAN explant
Isolated SAN cells
ANS (neuronal entrainment). HR fluctuations
blood pressure
atrial preparation
can be caused by changes in neuronal
whole heart
b
entrainment, intrinsic entrainment or by
ECG
AP data
AP data
Input data
responsiveness of the SAN to neuronal
72 h recordings
up to 1 h recordings
1–5 min recordings
entrainment and other extrinsic entrainment
Time
domain
stimuli. To differentiate between these
12 h
60–120 s
60–120 s
Histogram
possibilities, three levels of analyses are
of HA and LA,
performed. In vivo telemetric ECG recordings
respectively
are used to quantify HR fluctuations at the
whole-animal level. The data can alternatively
Mean RR
2h
60–120 s
60–120 s
be extracted from telemetric blood pressure
SDNN
of HA and LA,
RMSSD
respectively
recordings. The function of the SAN network
pNN6
is analyzed either by whole-heart preparations,
by atrial preparations or by isolated whole20,000
60–120 s
60–120 s
Poincaré
mount SAN preparations. Single-cell analysis
consecutive
consecutive
consecutive
is performed by isolation of pacemaker cells.
RR intervals
NN intervals
NN intervals
(b) Input data (top row) required for the
Frequency domain
Periodogram
analysis of parameters calculated in the time
103 s
spectral analysis
103 s
60–103 s
domain (second row) and frequency domain
of HA and LA,
TP, VLF, LF and HF
respectively
(third row). (c) Differentiation of possible
causes for HR fluctuations by evaluation of the
c Changes in beat-to-beat variability
presence (+) or absence (−) of difference in
ANS or
HRV between WT and genetic mouse line.
+
–
–
response to ANS
Example: The presence of differences in
SAN network
+
+
–
fluctuation at the in vivo level and the absence
at the two other levels suggest a problem at
Single SAN cell
+
+
+
the level of the ANS or with the responsiveness
of the SAN to autonomic regulation. The presence of changes on all levels suggests an intrinsic problem in single SAN cells. Finally, alterations of more than
one level could be present in a given mouse line (e.g., alterations could be present in isolated sinoatrial pacemaker cells and also in the ANS).
The balance and the dynamic interplay between the sympathetic
and parasympathetic nervous system regulates the operating
point and the variability of the actual HR. It is well known that
regulation of the SAN by the parasympathetic nervous system is
phasic and characterized by a short delay and fast decline, and
that it thereby allows regulation of the HR effectively at high
frequency (HF). In line with this notion, the highest-frequency
oscillations of HR (HF peak; ~0.15–0.40 Hz in human) are widely
believed to reflect cardiac parasympathetic nerve activity and
can be correlated with respiration (phasic sinus arrhythmia)4.
Regulation by the sympathetic nervous system is tonic on a much
slower time scale22. Therefore, low-frequency (LF) oscillations
of HR (LF peak; ~0.04–0.15 Hz in human) are often assumed to
have a dominant but not exclusive sympathetic component22–25.
LF power is strongly affected by the oscillatory rhythm of the
baroreceptor system, and it is abolished by total autonomic blockade4. A 0.5-Hz oscillation in the LF band reflecting thermoregulation has been described26. The very-low-frequency (VLF) band,
at 0.0033–0.04 Hz, may represent the influence of the peripheral
vasomotor and renin-angiotensin systems25. Most of the power
in a 24-h recording is contained in the ultra-low-frequency band.
This frequency band reflects all HR fluctuations with a period of
>5 min (<0.0033 Hz), and it contains not only circadian but also
neuroendocrine and other poorly understood rhythms 4. Total
62 | VOL.11 NO.1 | 2016 | nature protocols
power (TP) represents all of the variance integrated over all frequency bands present in the respective recording time interval.
For mice, which are the main focus of this protocol, the frequency bands for HRV analysis differ from those mentioned
above for humans. Three major frequency components have been
reported for mice: VLF (0.0–0.4 Hz), LF (0.4–1.5 Hz) and HF
(1.5–4.0 Hz)27–30. HR fluctuations in these frequency bands are
determined by spectral analysis. The parameters calculated by this
approach are called frequency-domain parameters. In addition,
HR fluctuations can be characterized by directly applying statistical tools to RR time series to obtain time-domain parameters27.
In this protocol, we describe how to use the following timedomain parameters: first, the s.d. of all normal RR intervals in sinus
rhythm (SDNN), which reflects total variability; second, the r.m.s.
of squared differences between successive normal RR intervals
(RMSSD), which reflects short-term variation between two successive beats; and third, the percentage of normal consecutive RR
intervals differing by >6 ms of the representative episodes (pNN6),
reflecting relatively abrupt and fast changes in HR, which have been
traditionally attributed to cardiac parasympathetic activity.
Here we describe how to analyze each of the three components
of HRV. Each component of HRV needs to be assessed by a corresponding separate technique. Together, these tools allow for a comprehensive multilevel analysis of HR fluctuations. The combination
protocol
© 2015 Nature America, Inc. All rights reserved.
of in vivo telemetry, in vitro electrophysiology of intact sinoatrial
network preparations (whole-mount SAN, atrial and whole-heart
preparations) and in vitro electrophysiology of isolated single
SAN cells allows for a characterization of the mechanism underlying altered HRV and sudden cardiac deaths.
Applications and target audience
This protocol provides the information necessary to identify the
functional mechanism underlying pathological distortion of
the heartbeat. This is clinically highly relevant, as, for example,
increased HR fluctuations compared with normal HR are associated with sinoatrial dysfunction (or sick sinus syndrome), which
is connected to susceptibility to sudden cardiac arrest and sudden
cardiac death8,31–33. Low HR fluctuations are correlated to higher
morbidity and mortality in patients after cardiac diseases such as
myocardial infarction34, hypertension, left ventricular hypertrophy
and heart failure, as well as in noncardiac conditions, including
diabetic autonomic neuropathy, stroke, sepsis and cancer34,35. Low
HRV has been identified as an independent predictor of high risk
for sudden and non-sudden cardiac deaths36–39. In many of the
specified conditions, the mechanisms underlying pathologically
increased or decreased HRV are unclear. Recently, many mouse
models for cardiac arrhythmias have become available, including
models for SAN dysfunction, coronary artery disease and chronic
heart failure, which are potentially or reportedly associated with
changed HR fluctuation. Genetic mouse lines are appropriate
models to study disease pathology, because in many cases the
molecular mechanism underlying changed HR fluctuations can be
identified at the in vivo level and then tracked down to the wholeorgan tissue, and the cellular and subcellular levels. Furthermore,
molecular and cellular mechanisms identified in analyses of
genetic mouse models can be used to understand human diseases.
Recently, a number of genetic tools using the Cre/loxP system have
also become available, which allow conditional deletion of proteins in the heart, and which also have the potential to allow deletion at a particular time point40,41. For example, genetic mouse
lines carrying a floxed exon in the gene locus encoding a protein
of interest can be deleted using the SAN and cardiac conduction
system–specific Cre-deleter mouse line, which expresses the Cre
recombinase under the control of a SAN promoter (HCN4-KiT
Cre mouse)40. SAN-specific deletion of proteins allows differentiating between extrinsic and intrinsic changes as causes for pathologic HR fluctuations. This protocol presents detailed information
for a multilevel analysis of HR fluctuations in any mice, including
wild-type (WT) mice and genetic mouse models.
Comparison with alternative approaches and limitations
The present protocol includes a set of individual methods, which in
the combination outlined is optimal for the analysis of HR fluctuations. One possible limitation of the method is the relatively high
costs for telemetric ECG or blood pressure recordings. However,
HR data obtained by telemetry are more reliable than ECG recordings obtained by alternative approaches such as ECG recordings in
strained or anesthetized animals. Another limitation of the protocol is that it is optimized for studies in the mouse. However, the
protocol could be adapted to similar experiments in rats or other
rodents. In this case, optimization and adaptation is required.
Each assay in the presented protocol has alternative approaches.
For example, at the single-cell level, instead of using single isolated
SAN cells, any spontaneously beating cardiac cell such as isolated
embryonic or neonatal cardiomyocytes or spontaneously beating
induced cardiomyocyte-like cells can be used. The best cell type
to use depends on the scientific question being asked. We do not
provide a protocol for the generation of isolated embryonic or
neonatal cardiomyocytes, because full details for the generation
of induced cardiomyocyte-like cells is available in an alternative
protocol42. However, patch-clamp data obtained from these cells
can be easily analyzed and processed according to our protocol
and MATLAB scripts.
To obtain a time series of spontaneous activity in explanted
SAN preparations and atrial and whole-heart preparations ,there
are also alternative approaches available, such as multielectrode
array experiments and optical imaging of electrical activity using
voltage-sensitive fluorescent dyes or fluorescent Ca2+ indicators.
At the in vivo level, there are alternative methods, which do not
require surgical implantation of telemetric transmitter probes,
to making telemetric ECG or blood pressure recordings from
which HR data can be extracted. These methods include echocardiography, external ECG or external blood pressure recording.
However, these methods require anesthesia, which could change
HR and HRV, thus making results hard to interpret or meaningless. Furthermore, cardiac echocardiography requires extremely
expensive equipment, which may not be available.
The methods presented in this protocol can also be used separately from each other for purposes other than the analysis of HR
fluctuations. This opens up a broad spectrum of additional potential applications for the techniques described in this protocol.
Development of the protocol
We initially developed this protocol after a detailed review of
the literature in which we were looking for the ideal combination of methods required for a comprehensive characterization
of the cellular mechanisms leading to pathological changes of
HRV. We screened the literature for publications using telemetric
HRV analysis in mouse models and optional additional methods
such as whole-mount SAN preparations, atrial or whole-heart
preparations, as well as isolated single SAN cell experiments. We
found the 27 publications summarized in Supplementary Table 1.
In 20 of these papers, only telemetric ECG or BP recordings
were performed, whereas seven papers also presented data from
tissue and/or single-cell experiments. In only five papers, telemetric ECG recordings and isolation of single SAN cells, explant
of whole hearts, intact SAN or atria were performed. We found
that telemetric ECG analysis, despite being very valuable for the
analysis of HRV on its own, is not sufficient for the identification
of the level (ANS, SAN network and single SAN pacemaker cells)
and the cellular mechanisms causing changes in HRV. This conclusion may be particularly relevant in mouse models presenting
with increased HRV (Supplementary Table 1), because in these
cases intrinsic SAN pathology is likely and requires additional
SAN network and single-cell analysis. To identify the level and
the mechanism of HRV changes, both in vivo and ex vivo experimental approaches are mandatory (Fig. 1). At the in vivo level,
telemetric ECG or blood pressure recordings are performed. In
addition, in vivo electrophysiological study using octapolar catheters can provide initial mechanistic insights into SAN pathology
(determination of SAN recovery time and sinoatrial conduction
time)15. These studies are then complemented by analysis of
nature protocols | VOL.11 NO.1 | 2016 | 63
protocol
© 2015 Nature America, Inc. All rights reserved.
whole hearts, atrial preparations or whole-mount SAN preparations (organ, tissue, and network level) and isolated SAN cells
(cell level). The experiments on whole-network and single-cell
levels can be performed with electrophysiological, Ca2+ imaging or imaging studies using voltage-sensitive dyes. As part of
this protocol, we present the electrophysiological approach. The
protocol was developed and validated by our group as per Fenske
et al.15 and Direnberger et al.43. In Figure 1c, it is shown how
our analysis can be used to dissect whether differences in HR
fluctuations are specifically caused by ANS, response to autonomic control, the SAN network or single cells.
Experimental design
The first stage of our multilevel analysis is long-term telemetric ECG
monitoring (Steps 1–24). Alternatively, other telemetric data, which
allow for extraction of HR data such as telemetric blood pressure
recordings, can be used. For ECG data acquisition, we use Dataquest
A.R.T. acquisition software. The software coordinates the collection
of ECG signals from up to eight animals. For ECG data analysis,
we use either Dataquest A.R.T. or ecgAUTO software. By using
both software packages, standard ECG parameters can be determined. Both software packages allow for a baseline HRV analysis,
which includes the calculation time and frequency-domain
parameter. In addition, HR histograms and Poincaré plots can be
determined using the ecgAUTO software. For more complex applications (more advanced HRV, autoregressive modeling), we also
wrote MATLAB scripts (Supplementary Data 1). Here we present
HRV analysis based on RR interval statistics. For a detailed overview on standard ECG parameters, we refer to more specialist literature44,45. To obtain an initial overview of the data, two graphical
representations of the data are used: HR histograms (Steps 25–32)
and Poincaré plots (Step 40). For Poincaré plots, a graphical map is
calculated, in which each heartbeat is plotted versus the next heartbeat (n versus n+1). Both graphical representations nicely reflect
overall changes in HR regulation and HRV. Furthermore, tachograms (RR time series) can be extracted from the ECG recordings.
For time-domain analysis (Steps 25–39), mean RR interval, s.d.
of all RR intervals (SDNN), r.m.s. of the difference of successive
RR intervals (RMSSD) and percentage of normal consecutive RR
intervals differing by >6 ms (pNN6) is calculated from tachograms.
For frequency-domain analysis of HR fluctuation (Steps 41–50),
power spectral density plots are calculated and high-, low- and
very-low-frequency bands, as well as TP, are determined.
In the next stage of our protocol, hearts are dissected out of
mice (Steps 52–57) and used for extracellular field potential measurements (Step 58D), microelectrode recordings (Step 58C) or the
isolation of SAN cells for single-cell electrophysiology (Step 58B).
The signaling processing tools applied to in vivo data obtained from
the live mouse (Steps 23–50) can also be applied to data obtained
from ex vivo preparations for the calculation of histograms,
Poincaré plots, and time- and frequency-domain parameters.
The individual ex vivo preparations comprise the intact SAN network present in whole-mount SAN preparations, atrial preparations (Step 58C(i–x)), explanted whole hearts (Step 58D(i–viii))
and isolated SAN cells (Step 58B(i–xli); Fig. 1).
Experiments in isolated SAN cells (Step 58B(i–xli)) and also
explanted SAN preparations (Step 58C(i-x)) require extensive
knowledge of the mouse SAN anatomy and of anatomical landmarks of this region. To familiarize the reader with the complex
SAN anatomy, we provide details for a method to inflate the heart
with gelatin, which freezes the cardiac anatomy in a quasi in vivo
state46 (Step 58A(i–x)).
When developing the procedures described in the present
protocol, we considered how best to ensure adherence to the
‘3Rs’ (reduction, replacement and refinement) of animal testing; thus, any deviation from the procedures may have a major
impact on adherence to the 3Rs. Gelatin-inflation of the heart
familiarizes the experimenter with SAN and heart anatomy,
and it reduces the amount of preparations needed to train
SAN whole-mount preparation and single SAN cell isolation.
In addition, to minimize the number of required preparations, experimenters should be trained in electrophysiological
methods with cells grown in cell culture, such as HEK293 cells,
in advance of experiments with isolated SAN cells or explanted
SAN preparations. Full details on how to perform electrophysiology are not included here; we instead advise users to consult
the detailed information on patch clamping that can be found
in the Axon Guide (Axon Instruments). In addition, training
in the patch-clamp technique using atrial or ventricular cells
can be particularly helpful for beginners, because these cells can
be prepared in large amounts. In the TROUBLESHOOTING
section, we provide information to specifically refine the
procedure of telemetric ECG implantation. Experimenters
should consider ordering retired breeders, which are commonly
available from commercial vendors, for use in training for all
techniques described in this protocol.
MATERIALS
REAGENTS
• Mice. The protocol can be used on any mouse strain with mice of any
age. The protocol for single SAN cell preparation is optimized for young
animals (5–7 weeks). It is also possible to use older animals, but enzyme
concentrations and digestion times have to be increased. The experiments
with whole-mount SAN measurements are best performed on mice aged
12–16 weeks. Older animals have more fat at the posterior atrial wall
and around the vessels, and this makes it more difficult to dissect the
whole-mount preparation. ECG transmitter implantation is performed
at a minimum age of 12 weeks and at a minimal weight of 17 g. House
animals in single cages in a 12-h dark/night cycle environment with free
access to food and water, and allow them to socialize with littermates
before experiments ! CAUTION All experiments involving animals
must conform to relevant institutional and governmental regulations.
This protocol was approved by the German (Regierung von Oberbayern)
and Lithuanian (Lithuanian State service on food and veterinary
64 | VOL.11 NO.1 | 2016 | nature protocols
(permission number 0206)) authorities in accordance with German
and Lithuanian laws on animal experimentation, and it was performed
according to the Guide for the Care and Use of Laboratory Animals
published by the US National Institutes of Health. Effort was made to
keep the number of animals at a minimum.
• Barium chloride dihydrate (BaCl2·2H2O; Sigma-Aldrich, cat. no. B0750)
! CAUTION This is harmful if inhaled and swallowed, it is irritating to
the eyes, respiratory system and skin. It may cause sensitization of skin
upon contact. Therefore, wear protective goggles, clothing and gloves as
appropriate. Use the chemicals in a fume hood. Carefully read the MSDS
for all chemicals used in this protocol.
• Cadmium chloride (CdCl2; Sigma-Aldrich, cat. no. 439800) ! CAUTION This
is a very toxic reagent. Avoid contact with skin and eyes. Wear proper eye
and skin protection.
• Calcium chloride dihydrate (CaCl2·2H2O; Carl Roth, cat. no. 5239)
• DL-Asp potassium salt (K-Asp; Sigma-Aldrich, cat. no. A2025)
© 2015 Nature America, Inc. All rights reserved.
protocol
• DMSO (Sigma-Aldrich, cat. no. D2438) ! CAUTION DMSO is an irritant to
eyes, skin and the respiratory system. Wear proper eye and skin protection.
• EGTA (Sigma-Aldrich, cat. no. T4378)
• HEPES (Carl Roth, cat. no. 6763)
• l-glutamic acid (Sigma-Aldrich, cat. no. G1251)
• Magnesium chloride (MgCl2·6H2O; Carl Roth, cat. no. 2189)
• Magnesium sulfate (MgSO4, Sigma-Aldrich, cat. no. M2643)
• Paraformaldehyde (PFA; Sigma-Aldrich, cat. no. P6148) ! CAUTION PFA is
a toxic reagent. Avoid inhalation or contact with skin and eyes. Wear
protective gear while handling.
• Phosphocreatine di (tris) salt (Sigma-Aldrich, cat. no. P1937)
• Poly-l-lysine hydrobromide (PLL; Sigma-Aldrich, cat. no. P1399)
• Potassium chloride (KCl; Sigma-Aldrich, cat. no. P5405)
• Potassium hydroxide (KOH; Sigma-Aldrich, cat. no. P5958)
• Potassium phosphate monobasic (KH2PO4; Sigma-Aldrich, cat. no. P5655)
• Sodium bicarbonate (NaHCO3; Sigma-Aldrich, cat. no. S5761)
• Sodium chloride (NaCl; Sigma-Aldrich, cat. no. S7653)
• Sodium hydroxide (NaOH; Sigma-Aldrich, cat. no. S8045)
! CAUTION Sodium hydroxide is a corrosive alkali. Avoid contact with
eyes and skin. Wear protective gear while handling it.
• Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O, VWR Chemicals,
cat. no. 28029.260)
• Taurine (Sigma-Aldrich, cat. no. T0625)
• α-d-(+)-Glucose monohydrate (Carl Roth, cat. no. 6887)
• Adenosine 5′-triphosphate magnesium salt (Mg-ATP; Sigma-Aldrich,
cat. no. A9187)
• Guanosine 5′-triphosphate sodium salt hydrate (Na-GTP; Sigma-Aldrich,
cat. no. G8877)
• Amphotericin B from Streptomyces sp. (Sigma-Aldrich, cat. no. A4888)
! CAUTION Amphotericin B is hazardous. Avoid inhalation or contact with
skin and eyes. Wear protective gear while handling it.
• BSA (Sigma-Aldrich, cat. no. 05470)
• Collagenase B from Clostridium histolyticum (199 U/mg; Roche Diagnostics
Deutschland; cat. no. 11088807001)  CRITICAL There is great variability
between enzyme qualities of different suppliers, and thus we recommend
the Roche product.
• Elastase from porcine pancreas (≥15 U/mg; Sigma-Aldrich, cat. no. 45124)
 CRITICAL Note that there is great variability between enzyme qualities of
different suppliers, and also great batch-to-batch variability. We recommend
ordering a large amount of one batch after an initial test phase.
• Protease from Streptomyces griseus (type XIV, ≥3.5 U/mg; Sigma-Aldrich,
cat. no. P5147)  CRITICAL There is great variability between
enzyme qualities of different suppliers, and thus we recommend
the Sigma-Aldrich product.
• Carbogen (5% vol/vol CO2 in O2; Air Liquide Deutschland,
cat. no. P3750L50R5A001)
• Gelatin powder (Dr. August Oetker Nahrungsmittel)
• PU 4II resin and hardener kit (color blue; vasQtec)
• Sylgard 184 silicone elastomer kit (Dow Corning)
• Carprofen (50 mg/ml; Rimadyl, Pfizer)
• Eye ointment (5% wt/vol Bepanthen, Bayer Vital)
• Isoflurane (100%; IsoFlo; Abbott Animal Health, cat. no. 05260-05)
! CAUTION Isoflurane is harmful if it is inhaled and swallowed. It may
cause nausea, vomiting, nose/throat/respiratory irritation, headache,
drowsiness and skin irritation. Wear gloves and long sleeves to avoid skin
contact. Carbon filters should be used to scavenge waste anesthetic gas.
• Ketamine hydrochloride (100 mg/ml; Ketavet Pfizer)
• kodan tincture forte (Schülke & Mayr, cat. no. 104 005)
• Povidone-iodine (100 mg/g; PVP-Jod ratiopharm Salbe Ratiopharm)
• Saline solution (0.9% wt/vol NaCl; B. Braun Melsungen, cat. no. 23580720)
• Xylazine (50 mg/ml; Rompun-TS Bayer Healthcare)
EQUIPMENT
Dissecting set and ECG transmitter implantation
• Mayo scissors (Fine Science Tools (FST), cat. no. 91401-14)
• Fine forceps (Dumont no. 5 forceps; FST, cat. no. 11251-20)
• Blunt forceps (Standard Pattern Forceps; FST, cat. no. 11000-13)
• Spring scissors; 8 mm blades (FST, cat. no. 15024-10)
• Tungsten-Carbide Iris scissors (FST, cat. no. 14568-12)
• Fine Iris scissors, curved (FST, cat. no. 14095-11)
• Blunt dissecting scissors (Lexer-Baby scissor; FST, cat. no. 14078-10)
• Iris scissors, delicate (FST, cat. no. 14061-09)
• Vannas spring scissors, 2.5 mm blades (FST, cat. no.15000-08)
• Needle holder, Halsey (FST; cat. no. 12501-13)
• Needle holder, Olsen-Hegar with scissor action (FST, cat. no. 12002-14)
• Hot bead sterilizer (FST, cat. no. 18000-45)
• Trimmer, Wella Contura type 3HSG1 (Procter & Gamble)
• Homeothermic blanket systems with flexible probe (small; Harvard
Apparatus, cat. no. 507221F)
• Needle-suture combination, sterile (silk, gauge no. 4-0 USP, metric 1.5,
braided; Resorba Medical, cat. no. 4024)
• Needle-suture combination, sterile (absorbable polyglycolic acid (PGA)
Resorba 6-0 USP, metric 0.7 braided; Resorba Medical, cat. no. PA 10273)
• Suture (silk; sterile precut suture, non-needled; 5-0 USP metric 1; braided
Resorba Medical, cat. no. G 2105)
• Sensitive plasters (5 m × 1.25 cm; Beiersdorf)
Patch-clamp and microelectrode recording setup
• Axiovert 135 TV microscope (Carl Zeiss)
• AxioCam color MR (Carl Zeiss)
• AxioVision software (Carl Zeiss)
• Recording/perfusion chamber RC-26GLP (Warner Instruments,
cat. no. 64-0236)
• Glass coverslip CS-22/40 (22 × 40 mm; Warner Instruments, cat. no. 64-0707)
• Stage-adapter SA20-LZ (16.5 × 10 mm for Zeiss Microscope; Warner
Instruments, cat. no. 64-2413)
• PH-1 platform for series 20 chambers, heater (Warner Instruments,
cat. no. 64-0284)
• Temperature controller TC344B (Warner Instruments)
• SHM-6 six-line solution in-line heater (Harvard Apparatus; cat. no. 640104)
• Polyethylene tubing PE-160/10 (10 feet; 1.57 mm outer diameter (o.d.) ×
1.14 mm inner diameter (i.d.); Warner Instruments, cat. no. 64-0755)
• MPII mini-peristaltic pump (Harvard Apparatus, cat. no. 702027)
• HEKA patch-clamp amplifier, EPC10 USB single (HEKA instruments,
cat. no. EPC 10 USB)
• IX2-700 dual intracellular preamplifier with headstage 7001 (specify
N = 0.1; Dagan)
• Axon Instruments Digidata 1440A (Molecular Devices)
• Micromanipulator (LN Mini-25XR; Luigs & Neumann Feinmechanik &
Elektrotechnik)
• For microelectrode recordings, we use a Dagan IX2-700 dual intracellular
preamplifier with headstage 7001 and pClamp 10.4 software. Analysis is
done off-line using pClamp 10.4, Origin2015 and MATLAB software
• For current-clamp recordings in isolated SAN cells, we use a HEKA
amplifier and HEKA software. Analysis is performed off-line with
pClamp 10.4, Origin2015 and MATLAB
• For extracellular field potential measurements, we use bipolar electrodes
attached to regular surface ECG electrodes. The signals are recorded by the
portable EP tracer system (Cardiotek). Analysis is done off-line with EP
tracer software and MATLAB
Patch-clamp electrodes
• DMZ universal puller (Zeitz Instruments)
• Borosilicate glass capillaries GC150TF-8 for current-clamp recordings
(o.d. 1.5 × i.d. 1.17 × length (L) 80 mm; Harvard Apparatus). Pull the
electrodes and fire-polish the tips. The resistance of the electrodes should
be ~2–3 MΩ
• Borosilicate glass capillaries BM150F-10P for microelectrode recordings
(o.d. 1.50 × i.d. 0.86 × L 100 mm; BioMedical Instruments, cat. no. 10718).
Pull the electrodes and fire-polish the tips. The resistance of the electrodes
should be ~20–30 MΩ  CRITICAL The SAN preparation continuously
contracts. Contraction-related movements are transferred to the
microelectrode. The tip of the electrodes must be long and very flexible
to guarantee that the tip, after being impaled into the cell, can smoothly
move together with the tissue. Otherwise, it will perforate the cell or slip
out of the cell.
• Microfil (28 gauge/67 mm long; World Precision Instruments,
cat. no. MF28G67-5)
Data acquisition and analysis
• DSI Dataquest A.R.T. 4.00 (Data Sciences International)
• DSI APR-1 ambient pressure reference (Data Sciences International)
• DSI Data Exchange Matrix (Data Sciences International)
• DSI PhysioTel TA11ETA-F10 for mice (Data Sciences International)
• DSI PhysioTel receivers RPC1 (plastic cages; Data Science International)
• EMKA ecgAUTO v3.3.012 (emka TECHNOLOGIES)
• MATLAB R2014b (The MathWorks)
• pClamp 10.4 (Molecular Devices)
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• HEKA PatchMaster v32×73.2 (HEKA Instruments)
• Excel 2013 (Microsoft Corporation)
• Origin2015 (OriginLab)
• EP TRACER Portable (Schwarzer Cardiotek)
• EP-Tracer_V1.05 software (Schwarzer Cardiotek)
Other
• Stereomicroscope Stemi 2000 (Carl Zeiss)
• Petri dish (100 × 15 mm; VWR International, cat. no. 710-4105)
• Petri dish (140 × 20.6 mm; VWR International, cat. no. 391-1503)
• Sterican hypodermic needle (10G × ½ inch B. Braun Melsungen,
cat. no. 4657519)
• Sterican hypodermic needle (26G; B. Braun Melsungen, cat. no. 4657683)
• Eppendorf Research Plus pipette (1,000 µl; Eppendorf, cat. no. 3120000062)
• Eppendorf Research Plus pipette (20 µl; Eppendorf, cat. no. 3120000038)
• Eppendorf Research Plus pipette (200 µl; Eppendorf, cat. no. 3120000054)
• Pipette tips (1,000 µl, Sarstedt, cat. no. 70.762)
• Pipette tips (200 µl, Sarstedt, cat. no. 70.760.001)
• Syringe (5 ml; Terumo cat. no. 6SS05S)
• Syringe (1 ml; Terumo cat. no. 6SS01T)
• Syringe (50 ml, Luer-lock tip; Terumo, cat. no. 1086524)
• Syringe filter, sterile (0.2 µm; VWR International, cat. no. 10708S1G43BD)
• Coverslips (12 mm; VWR International, cat. no. 631-1577)
• Lighter
• Reaction tube, 1.5/2.0 ml (Eppendorf Safe-Lock tubes; Eppendorf,
cat. no. 0030120086/0030120094)
• Conical tube 15/50 ml (Sarstedt, cat. no. 62.554.001/62.548.004)
• Thermomixer compact (Eppendorf)
• Water bath Lauda A 100 (Lauda-Brinkmann)
• Centrifuge 5415D (Eppendorf)
• Minutien pin (diameter 0.1 mm; Fiebig Lehrmittel, cat. no. 1184)
• Safety-Multifly needle (21G × 3/4 inch needle length; Sarstedt,
cat. no. 851638235)
• Peristaltic pump MINIPULS 3 (Gilson)
• Heating coils (10–45 mm; Radnoti, cat. no. 158821)
REAGENT SETUP
PBS Dissolve 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4·2H2O and
1.8 mM KH2PO4. Adjust the pH to 7.4 with NaOH and autoclave the
solution. PBS can be stored at room temperature (19–21 °C) for 1 month.
Tyrode III Dissolve 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM
CaCl2, 5 mM HEPES and 5.5 mM glucose. Adjust the pH to 7.4 with NaOH.
This solution can be stored at 4 °C for up to 1 week.
Tyrode low pH 6.0 Dissolve 140 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2,
0.2 mM CaCl2, 5 mM HEPES, 5.5 mM glucose, 1.2 mM KH2PO4 and 50 mM
taurine. Adjust the pH to 6.0 with NaOH. This solution can be stored at 4 °C
for up to 1 week.
Tyrode low pH 6.9 Prepare the solution as for Tyrode low pH 6.0, and adjust
the pH to 6.9 with NaOH. This solution can be stored at 4 °C for up to 1 week.
Kraftbrühe (KB) Dissolve 80 mM l-glutamic acid, 25 mM KCl, 3 mM
MgCl2, 10 mM KH2PO4, 20 mM taurine, 10 mM HEPES, 10 mM glucose
and 0.5 mM EGTA. Adjust the pH to 7.4 with KOH. This solution can be
stored at 4 °C for 5 d.
Collagenase B stocks Dissolve collagenase B in Tyrode low pH 6.9. The
aliquots (150 µl) for one preparation containing 0.54 U can be stored
at −20 °C for up to 1 week.
Protease stocks Dissolve protease (activity 5.6 U) in Tyrode low pH 6.9.
The aliquots (125 µl) for one preparation containing 1.79 U can be stored
at −20 °C for up to 6 months.
Elastase stocks Dissolve elastase in Tyrode low pH 6.0. The aliquots (40 µl) for
one preparation containing 18.87 U can be stored at −20 °C for up to 1 week.
Poly-l-lysine (PLL)–coated coverslips Dissolve 100 mg of PLL hydrobromide in 2 ml of ddH2O for a PLL stock solution. Store this solution at 4 °C
for up to 1 month. Wash the coverslips (diameter 12 mm) with 70% (vol/vol)
ethanol for 30 min and let them dry completely. Coat every coverslip with
100 µl of a 1:500 PLL solution for 30 min and incubate at 37 °C. Remove the
PLL solution and wash the coverslips two times with PBS, pH 7.4, and allow
them to dry. The coated coverslips can be stored for up to 1 week.
Paraformaldehyde (PFA) tissue fixation Dissolve PFA in PBS to a final concentration of 4% (wt/vol) and adjust the pH to 7.4 with HCl. The aliquots can
be stored at −20 °C for up to 6 months. ! CAUTION PFA is a toxic reagent. Avoid
inhalation or contact with skin and eyes. Wear protective gear while handling.
Action potential (AP) intracellular solution Dissolve 130 mM K-Asp,
10 mM NaCl, 0.04 mM CaCl2, 10 mM HEPES, 0.1 mM Na-GTP, 2.0 mM
Mg-ATP and 6.6 mM phosphocreatine, and adjust the pH to 7.2 with KOH.
Aliquots of 1,980 µl can be stored at −20 °C for up to 6 months.
AP intracellular solution with amphotericin B Dissolve 2 mg of amphotericin B in 100 µl of DMSO on the day of use, and sonicate the solution for
~10 min until it is completely dissolved. Thaw one aliquot of AP intracellular
solution and add 20 µl of amphotericin B stock solution (final concentration
200 µg/ml).  CRITICAL Amphotericin B must be completely dissolved.
Store the solution on ice and protect it from light. Renew the solution after
1 h of use because amphotericin B comes out of solution.
Microelectrode intracellular solution This solution is 3 M KCl in ddH2O.
It can be stored at 4 °C for 1 month.
Gelatin solution Dissolve 5 g of gelatin in 50 ml of boiling ddH2O, and
then place it into a water bath at 37 °C. Prepare this on the day of use.
Sylgard coating Mix Sylgard 184 components according to the manufacturers’
instructions, and fill 20 ml in a 100-mm Petri dish and 0.1 ml in a RC-26GLP
recording chamber. Both can be used after 1 d of polymerization at 37 °C.
Anesthesia mix Prepare a mixture of 100 mg/kg ketamine and 10 mg/kg
xylazine in 0.9% (wt/vol) NaCl. Prepare this mixture on the day of use.
Carprofen for analgesia Prepare carprofen (5 mg/g) on the day of use.
Inject this subcutaneously (s.c.); should postoperative analgesia be required,
administer a repeat dosage every 12–24 h.
BSA stock solution BSA stock solution is 100 mg/ml BSA in ddH2O for a
final concentration within the digestion of 1 mg/ml. The aliquots (10 µl) for
one preparation containing 1.79 U can be stored at −20 °C for up to 6 months.
Langendorff perfusion buffer (Krebs Henseleit buffer) Langendorff
perfusion buffer is 118.5 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 1.2 mM
MgSO4, 1.8 mM CaCl2, 1.2 mM KH2PO4 and 11 mM glucose. This solution
must be aerated with carbogen to adjust the pH to 7.4. This solution can be
stored at 4 °C for 1 week.
Perfusion cannula (custom made) Cut the sharp ending of a cannula
(10G × 1/2 in). Grind down the endings and engrave it two times at
distances 1 and 2 mm from the tip.
Incubation chamber Dampen two paper towels and place them within a
Petri dish (diameter 140 mm). The lid of a second Petri dish (diameter 100 mm)
is placed upside-down into the first dish with the opening facing upwards.
Attach, crosswise, two strips of tape across the top edge of the open lid. Place
the PLL coverslips on tape and close the incubation chamber
Dissociation tip Cut off the first quarter from a 200- and 1,000-µl pipette
tip, and fire-polish the resulting sharp edges using a lighter. These tips are
used to triturate the SAN tissue.  CRITICAL Sharp edges can cause damage
of the cells during the cell separation step.
PROCEDURE
Surgical implantation of telemetric transmitters for ECG telemetry ● TIMING 45–60 min
1| Before surgery, autoclave all instruments and materials. Disinfect the workbench to assure aseptic conditions. Sterilize
the surgical instruments regularly during surgery in a hot-bead sterilizer. The use of a surgical microscope and a workbench
equipped with a laminar flow is not necessary.
2| Remove ECG transmitters from their sterile package.
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3| Shorten the leads of the transmitter to a length appropriate for the size of the mouse. For a 12- to 16-week-old
male B6 (C57BL/6) mouse weighing 25–30 g, we usually shorten the positive (red) lead with an old pair of scissors
to ~40 mm and the negative (white) lead to a length of ~35 mm. These values are given as reference for orientation,
and they need to be adapted to the size of the mouse.
4| Measure ~6–7 mm from the distal part of each electrode, and score the insulation with a sharp scalpel blade. Prepare tip
covers with the excessive insulation tubing by pulling it ~2 mm away. Tie a piece of nonabsorbable 5-0 silk suture material
around the tip, and cut the excess tubing that extends beyond the distal end of the lead. Tie another piece of suture material
around the silicone tubing just proximal to the exposed portion of the wire to avoid intrusion of liquid along the lead.
5| Place the transmitter in warm sterile saline. The transmitter is now ready for surgical implantation.
6| Anesthetize a mouse by intraperitoneal (i.p.) injection of the anesthesia mix.
© 2015 Nature America, Inc. All rights reserved.
7| To protect the animal’s eyes during anesthesia, apply eye ointment.
8| Confirm loss of the toe-pinch reflex, and then place the animal in dorsal recumbence on a temperature-controlled
surgery stage, which is adjusted to 37 °C. Introduce the temperature probe into the animal’s rectum to enable a
temperature-control feedback loop.
9| Shave the abdomen and chest of the animal using a trimmer and disinfect the skin. Place a sterile drape over the
animal (to visualize the surgical procedure, this was omitted in Fig. 2).
10| Incise the abdominal skin using small, blunt dissecting scissors. Make the incision 1.5–2.5 cm long; it should extend
from the epigastric area 1 cm below the caudal tip of the sternum along the midline toward the chest (Fig. 2a).
11| Form a s.c. tunnel directed toward the left flank of the animal using small, blunt dissecting scissors, and form a small
pouch (Fig. 2b).
 CRITICAL STEP The tunnel and s.c. pouch must be as small as possible to keep the transmitter in place. Otherwise, you will
need to lock it into position with suture material or tissue adhesive.
12| Irrigate the tunnel with a 1-ml syringe and introduce ~300 µl of prewarmed sterile saline into the pouch (Fig. 2c).
13| Introduce the transmitter into the pouch (Fig. 2d).
14| Form a thin tunnel to the right chest muscle, and place the negative (white or colorless) lead into the tunnel using blunt
forceps (Fig. 2e,f). If the position of the lead is not as desired by simply introducing the lead into the tunnel, it can be
additionally fixed with a stitch. To do this, make a small skin incision near the right pectoral muscle, and then fix the lead with a
stitch to the abdominal wall muscle using 5-0 absorbable suture material. Close the skin with 6-0 nonabsorbable suture material.
 CRITICAL STEP If the lead is too long to fit into the tunnel, it is necessary to shorten the lead to the proper length and to
form a new tip. The lead must lie flat against the body for the whole length of the lead. Mouse skin is very delicate, and the
long lead will disturb the animals and they will try to remove the transmitter. You will risk irritation of the tissue and wound
dehiscence (wound rupture along the surgical suture).
15| Form a loop with the positive (red) lead, and secure its position by placing a suture tie (Fig. 2g,h).
16| Close abdominal skin using 5-0 absorbable suture material (Fig. 2i).
17| For postoperative pain relief, inject carprofen s.c.
18| Place one-half of the cage on a warm (39 ± 1 °C) platform for 12 h, and then transfer the animal onto the warm area.
As soon as the animal is awake, it can decide whether to stay in the warm area or to move into the colder part of the cage.
19| As soon as the animal is fully awake, transfer one of its littermates into the cage to allow social interaction.
20| During the recovery period of 4–10 d, carefully and daily monitor progress in wound healing, general health conditions,
vital parameters and body weight, and food and water consumption. The monitoring score sheet (Supplementary Table 2)
defines criteria for humane endpoints.
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protocol
a
b
c
d
e
f
g
h
i
Figure 2 | Subcutaneous implantation of ECG transmitter. (a–i) Step-by-step image sequence depicting ECG transmitter implantation. (a) Abdominal skin
incision. (b) Subcutaneous tunnel directed toward the left flank of the animal. (c) Irrigation of the tunnel with a 1-ml syringe and introduction of ~300 µl of
prewarmed sterile saline into the pouch. (d) Transmitter placement in the subcutaneous pouch; inset: tip of the positive ECG lead. The insulation is tied to
the electrode with nonabsorbable suture material. (e) Placement of the negative ECG lead. (f) The tip of the negative lead is placed on the pectoral muscle.
If the white lead is too long and does not lie flat against the body, it must be shortened and a new tip has to be prepared during the surgery procedure.
(g,h) Placement of the positive lead including suture. (i) Red circles indicate approximate positions of the lead tips. Experiments were performed according
to institutional and governmental regulations and approved by the Regierung von Oberbayern.
21| Allow the mice to recover for at least 2 weeks before proceeding to the first ECG measurement.
? TROUBLESHOOTING
22| To start ECG data acquisition, switch on telemetric transmitters by touching the animal with a magnet, and place
the receiver plate below the animals’ cage.
Data pre-processing for HRV analysis ● TIMING 4 d
23| Acquire continuous ECG recordings over 72 h.
24| Check the presence of a regular circadian rhythm by inspecting HR, activity and temperature of the animals using
Dataquest A.R.T. software (for settings, see Fig. 3).
 CRITICAL STEP If the mice do not display a normal circadian rhythm (and this is not the phenotype of your mouse), you
should give them more recovery time after the transmitter implantation. Alternatively, the acoustic isolation of the animal
facility may not be not optimal.
Time-domain analysis: mean RR, HR and HR histogram ● TIMING 45–60 min
 CRITICAL Steps 25–32 describe determination of the mean RR, HR and HR histogram using ecgAUTO software.
25| Use a 12-h segment of light phase (low-activity phase) and a 12-h segment of dark phase (high-activity phase) from the
total recording period, and analyze each of the two segments separately. For R-wave detection, it is reasonable to perform a
protocol analysis and to divide the 12-h analysis into six steps, each taking 2 h (for settings, see Figs. 4 and 5a).
68 | VOL.11 NO.1 | 2016 | nature protocols
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Figure 3 | Screenshots of Dataquest A.R.T.
settings for checking the circadian rhythm of
mice. (a,b) Settings to visualize and review a
time period of 72 h of ECG, temperature and
activity recordings. (c) The animal displays a
regular circadian rhythm. HR (top), temperature
(middle) and activity (bottom) are higher during
the dark/high-activity phase indicated by the
blue boxes. The mouse was very active during
the second low-activity/light phase, because it
was agitated by a person who entered the room
several times. Such episodes must be discarded
and cannot be used for analysis. Experiments
were performed according to institutional and
governmental regulations and approved by the
Regierung von Oberbayern.
26| Check the quality of the detection
using the success frozen view window.
If the detection percentage is low
(<90% of detection), check whether
a different 12-h episode has higher
quality (Fig. 4a).
 CRITICAL STEP Phases of high HRs
are often accompanied by high activity
of the animals. This could lead to
very-low-quality signals with superimposed movement artifacts and missed
detection of R waves. Exclusion of
epochs with insufficient quality or
low detection percentage, as it is often
done or recommended by others,
most likely leads to calculation of
erroneously low mean HR values.
a
b
c
27| If necessary, remove ectopic beats,
sinus pauses or artifacts; however, this
is not usually necessary because of the
large number of data points.
 CRITICAL STEP When the quality of
your ECG data is low, it might, however,
be necessary to remove artifacts. We recommend that results obtained after manual exclusion of these artifacts be compared
with results obtained without removal of artifacts for one animal to get an idea of the differences in the two HR histograms.
28| Open the HRV analysis window.
29| Make appropriate settings for the HR histogram (Fig. 4b).
30| Set the parameter to be analyzed from default RR to HR (Fig. 4c).
31| Set the epoch definition mode to one per analysis (i.e., 1/analysis).
32| Export the HR histogram data and open it in calculation software such as Origin, Excel or MATLAB to plot the graph.
Time-domain analysis: SDNN, RMSSD and pNN6 ● TIMING 45–60 min
 CRITICAL In Steps 33–39, we describe time-domain analysis using ecgAUTO software or a combination of RR detection
in ecgAUTO and further analysis of the data using the MATLAB script (Supplementary Data 1).
33| Perform R-wave detection of a 2-h interval during a low- or high-activity phase (Fig. 5a).
nature protocols | VOL.11 NO.1 | 2016 | 69
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Figure 4 | Determination of HR histograms using
ecgAUTO. (a) Success frozen view window, for
checking the detection percentage, as indicated
by the height of the red bars. (b) Histogram
settings in the HRV module. (c) HR histogram of
a WT mouse from 12 h of the light/low-activity
phase. Experiments were performed according to
institutional and governmental regulations and
approved by the Regierung von Oberbayern.
a
b
c
34| Manually inspect the tachogram
and select at least three representative
episodes of 10 min in duration.
We usually calculate time-domain
parameters separately for episodes
of relatively low HR—for example,
at rest (average HR <480 bpm)
corresponding to an RR interval of
125 ms and episodes of relatively
high HR (average HR >600 bpm)
corresponding to an average
RR interval of 100 ms. Parameters
obtained from phases of high HR versus those of low HR can be compared between WT and genetically modified mice. This
approach is very convenient because even subtle changes in HRV in either a high- or low-activity phase can be specifically
detected and might be overlooked if these phases are not separately analyzed. The individual episodes should display a
stable sinus rhythm characterized by the absence of sinus pauses and of obvious atrial and ventricular arrhythmia, ectopic
beats or artifacts. Furthermore, HR fluctuations should decrease in phases of high HR and increase in phases of low HR18.
35| Confirm stationarity of the trace. For tachograms, stationarity refers to a series of RR intervals without a trend and
with constant mean and variance over time, and with a constant autocorrelation structure over time and no periodic
fluctuations18. Trends are defined as slow linear or more-complex shifts in the baseline of a tachogram. So that meaningful
HRV data can be obtained18, we usually choose time epochs on the basis of subjective stationarity, thereby making a
compromise between overprocessing the signal (and therefore risking the loss of data characteristics) and applying rules
too weakly for data pre-processing18.
36| Manually inspect these episodes for beats that are not properly detected, and remove them. Incorrect R-wave detection
occurs particularly during phases of high activity of the animals, in areas with artifacts or low signal-to-noise ratios, or in
areas with a low detection percentage (Fig. 5b).
 CRITICAL STEP To determine SDNN, RMSSD and pNN6, it is indispensable to manually remove artifacts because they would
have a marked effect on the results.
37| Open the multiple trend graphs window to see the corresponding raw tachogram (Fig. 5c,d, inset).
38| Manually inspect the raw tachogram, search for sinus pauses and ectopic beats, and invalidate them. It is also
possible to use Poincaré plot presentation in the HRV analysis window to manually search for outliers (Fig. 5e, inset).
Alternatively, it is possible to exclude ectopic beats, sinus pauses and outliers using our MATLAB script (Supplementary
Data 1). Import all detected RR intervals into the script and exclude RR intervals, which are above or below a defined
threshold value relative to a moving average (see ANTICIPATED RESULTS) or by excluding beats that differ more than x-fold
from the s.d. This is not as exact as the manual inspection, but it also gives reliable results and is less time consuming.
39| Calculate the mean value (±s.e.m.) of SDNN, RMSSD and pNN6, and then plot the graphs.
Time-domain analysis: Poincaré plot ● TIMING 20–30 min
40| Use 20 000 consecutive data points (RR intervals) for Poincaré plots. Plot RR intervals (n; x axis) against the next
RR interval (n + 1; y axis). This can be performed by exporting the RR time series to ordinary spreadsheet calculation
software such as Origin or Excel. Simply shift the time series by one time interval (n + 1; y axis) and plot it versus n (x axis).
Alternatively, use the MATLAB script (Supplementary Data 1) or the Poincaré module of the ecgAUTO software.
70 | VOL.11 NO.1 | 2016 | nature protocols
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protocol
Figure 5 | Time-domain analysis using ecgAUTO.
(a) Settings for reliable RR detection in mice.
(b–e) Removal of artifacts, ectopic beats and
sinus pauses. Blue lines indicate detected
RR intervals. Turquoise boxes indicate marked
beats. Red boxes indicate areas in which no
RR intervals could be detected or manually
invalidated beats. Manually invalidated beats
are additionally represented in green boxes.
(b) Incorrect RR detection in noisy areas with
low detection percentage. (c) Sinus pauses
can easily be detected by inspection of the
raw tachogram (inset, red circle) and then be
manually invalidated. (d) Ectopic beats can be
found in the raw tachogram by searching for a
very short RR interval that is followed by a very
long RR interval (inset, red circles). In the ECG
trace, the ectopic beat can be identified as a QRS
complex without preceding P wave followed by
a compensatory pause. (e) Incorrectly detected
RR intervals can also be easily found in Poincaré
plot presentation (inset, red circle). Experiments
were performed according to institutional and
governmental regulations and approved by the
Regierung von Oberbayern.
a
Frequency-domain HRV parameters:
fast Fourier transform (FFT) spectra,
TP, low-frequency and high-frequency
power ● TIMING 1–2 h
 CRITICAL In Steps 41–51, we
describe frequency-domain analysis
using the ecgAUTO software (for
settings see Fig. 6).
d
41| As already mentioned in Step 38,
we usually compare parameters of WT
mice and genetically modified mouse
lines during a phase of relatively
low HR—for example, at rest (average
HR <480 bpm) and during a phase
of relatively high HR (average
HR >600 bpm). Manually inspect raw
ECG traces and search for episodes
with stable sinus rhythm, which are
characterized by the absence of sinus
pauses and obvious atrial and ventricular arrhythmia, ectopic beats or artifacts.
b
c
e
42| Perform a 103-s local analysis to detect RR intervals (Fig. 7a).
43| Open the HRV window and inspect the raw tachogram in the data panel (Fig. 7b). After RR detection in ecgAUTO
(Steps 41 and 42), an alternative option to proceeding with this step and Steps 44–50 is to further analyze the data
using the MATLAB script (Supplementary Data 1). An advantage of the script is that better detrending algorithms are
available (linear, smoothness priors and polynomial47), and that the user can choose among diverse methods for ectopic
beat exclusion. In addition to the actual MATLAB code (Supplementary Data 1), we also provide a PDF file containing the
fully commented and structured code (Supplementary Method 1). Analysis can also be performed in Dataquest A.R.T. (for
settings, see Fig. 6); this is not further described here, but it is explained in detail in the Dataquest A.R.T. user manual.
44| Confirm stationarity of the trace (see Step 40) and manually identify and remove ectopic beats and sinus pauses.
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A.R.T.
MATLAB
Raw tachogram
Data length: 103 s
stationary signal
w (n) = α – β cos [(2�n) ⁄ (N–1)]
Data processing
Remove ectopic beats
Resampling step: 50 ms
Resampling max
Point #: 2,048
Interpolation mode:
third-degree spline
Data processing
Strip NANs
Detrend
Suppress mean
Interpolate: 50 ms
Order: 3 (cubic)
Data processing
Remove ectopic beats
Detrend
Resampling: 50 ms
Interpolation mode:
cubic spline
FFT
Start offset shift : 50%
Point number: 1,024
Windowing type: Hamming
Remove: average of each
FFT data or linear trend
of FFT input
exported spectrum
undersample: 1
Periodogram
Count: 1,024
windowing method:
Hamming
# subseries: 3
Welch periodogram
# Data points FFT: 2,048
# Data points/window: 1,024
# Windows: 3
Overlap: 512
Window function: Hamming
Power spectrum
# Data points: 512
Frequency range: 0–10 Hz
VLF: 0–0.4 Hz
LF: 0.4–1.5 Hz
HF: 1.5–4 Hz
HF
LF
3
VLF
45| If linear trends are present,
remove them using the detrending
algorithm of the ecgAUTO software
(remove linear trend of tachogram,
which will serve as input for further
FFT processing). This option also
subtracts the mean RR interval length
from the tachogram in order to extract
HR variation in the data (for HRV
analysis settings, see Figs. 6 and 7c).
 CRITICAL STEP We prefer discarding
epochs that do not fulfill the
subjective stationarity criteria,
and we usually search for epochs
without ectopic beats.
ecgAUTO
Power (a.u.)
© 2015 Nature America, Inc. All rights reserved.
Figure 6 | Frequency-domain analysis of HR
fluctuations. Left, workflow illustrating sequential
steps of signaling processing involved in
calculating the HRV spectra from ECG telemetry
and action potential input data. Right, for three
different software options (ecgAUTO, A.R.T.
and MATLAB scripts (Supplementary Data 1)),
specific parameter settings are given.
a.u., arbitrary units.
2
1
0
0
1
2
Frequency (Hz)
3
4
Power:
mV/Hz
Power: ms2/Hz
Power: s2/Hz
46| Interpolate tachograms by third-degree spline interpolation at 50-ms intervals to create equidistant points that
are suitable for FFT.
47| Split the tachogram sequence in three half overlapping subsequences (50% start offset shift), and apply Hamming
windowing function to each subsequence in order to reduce spectral leakage. The number of subsequences and the amount
of overlap can be adapted to meet individual analysis needs. See Supplementary Method 2 for the mathematical relation
between number N of sample points contained in a tachogram, lengths of subsequence windows, number of spectral points and
percentage of overlap. Window type can be chosen among Hamming, Hanning, triangle or rectangle windows. Power spectral
densities for each windowed sequence are averaged by the software to obtain the periodogram with 512 spectral points.
a
c
48| Calculate 3–6 individual
periodograms within 12 h in the
high- or low-activity phase.
49| From each periodogram (each
time segment), calculate the TP as
the integral of total variability over
the entire frequency range recorded
(0–10.0 Hz).
b
72 | VOL.11 NO.1 | 2016 | nature protocols
Figure 7 | Settings for HRV analysis in ecgAUTO
software. (a) ECG trace displaying regular sinus
rhythm without artifacts. R peaks are indicated
by red dots. (b) Stationary raw tachogram
without ectopic beats. Brown tiles indicate
the three subsequences with 50% overlap that
will be used for FFT. Time-domain parameters
calculated by the software for this epoch are
shown on the left. A magnification of the
calculated parameters is shown in the inset.
(c) Settings for FFT in the HRV module.
Experiments were performed according to
institutional and governmental regulations and
approved by the Regierung von Oberbayern.
protocol
© 2015 Nature America, Inc. All rights reserved.
50| For each time segment, further subdivide the periodogram into three major frequency components: VLF (0.0–0.4 Hz),
LF (0.4–1.5 Hz) and HF (1.5–4.0 Hz). Average the data obtained for each time segment.
Preparations required before heart dissection ● TIMING depends on the option followed
51| If proceeding to gelatin-inflation of the heart for anatomical studies, prepare as described in option A. If preparing
for isolation and enzymatic digestion of the SAN for electrophysiological measurements, prepare as described in option B.
If preparing for whole-mount dissection of the SAN for microelectrode recordings, prepare as described in option C.
If preparing for whole-heart preparation for extracellular field measurements, prepare as described in option D.
(A) Preparations for gelatin-inflation of the heart ● TIMING 5 min
(i) Prepare 5 ml of ice-cold PBS, 50 ml of gelatin solution (37 °C) and 50 ml of PBS (at RT).
(ii) Set the flow rate of the perfusion pump to 4 ml/min.
(B) Preparations for isolation and enzymatic digestion of the SAN for single-cell electrophysiology ● TIMING 20–30 min
(i) Prepare 3 ml of ice-cold KB solution in a conical 15-ml tube.
(ii) Warm 50 ml of Tyrode III solution to 37 °C in a water bath.
(iii) Warm 2 ml of Tyrode low pH 6.9 in a 2-ml reaction tube to room temperature.
(iv) Warm 675 µl of Tyrode low pH 6.9 in a 2-ml reaction tube to 37 °C in a water bath.
(v) Heat a dry block heater to 36 °C.
(vi) Thaw the enzyme and BSA aliquots on ice (150 µl of collagenase B containing 0.54 U, 40 µl of elastase containing
18.87 U, 125 µl of protease containing 1.79 U and 10 µl of BSA (100 mg/ml)).
(vii) Fill the 100 mm Sylgard-coated Petri dish with 40–50 ml of Tyrode III at 37 °C.
(C) Preparations for whole-mount SAN dissection for microelectrode recordings ● TIMING 5 min
(i) Fill the 100 mm Sylgard-coated Petri dish with 40–50 ml of Tyrode III (RT).
(D) Preparations for whole-heart preparation for extracellular field measurements ● TIMING 30 min
(i) Warm the perfusion buffer to 37 °C in a water bath before use.
(ii) Prepare the perfusion apparatus. Set the circulating water bath so that the outflow from the tip of the cannula
is 37 °C. Set the flow rate of the pump to 3 ml/min (Fig. 8).
 CRITICAL STEP Check the flow rate and the temperature of the perfusion buffer on a regular basis. Replace the
peristaltic pump tubing every month to maintain constant flow.
(iii) Run 100 ml of purified water through the perfusion system.
(iv) Prime the perfusion system with perfusion buffer, and run the perfusion buffer through the system for at least 5 min.
 CRITICAL STEP Be sure that no air bubbles are in the perfusion system, because these can cause an air embolism.
(v) Add 40–50 ml of room temperature perfusion buffer to a 100 mm Petri dish for heart cannulation, and place it below
a stereo microscope (Fig. 8a).
(vi) Mount the perfusion cannula on the system so that the tip of the cannula is 1 mm below the liquid surface. Run a
small amount of perfusion buffer through the cannula to remove air bubbles that might enter the cannula when it
touches the surface.
(vii) Cut two small pieces of 6-0 surgical silk, and knot them loosely around the upper part of the cannula.
Step-by-step dissection of the heart ● TIMING 10–20 min
52| Anesthetize a mouse deeply using isoflurane inhalation and kill it by cervical dislocation. Decapitate the mouse
rapidly with large Mayo scissors to release blood from the circulation and to reduce blood accumulation in the thorax
during preparation.
53| Place the mouse in supine position on the preparation dish. Tape down the front and hind paws using surgical tape.
Disinfect the chest with 70% (vol/vol) ethanol (Fig. 9).
54| Make a 1-cm transverse incision caudally to the sternum using blunt forceps and tungsten-carbide Iris scissors,
and pull the skin caudally and cranially apart (Fig. 9a).
55| Open the peritoneum by a 4- to 5-cm transverse incision 2 mm caudally of the sternum. To obtain free access to the
diaphragm, carefully separate the liver from the diaphragm and displace abdominal organs caudally (Fig. 9b,c).
56| Incise the diaphragm along the thorax to expose the pleural cavity. On both sides, cut the lateral wall of the rib
cage from the costal arches up to the clavicles. Make sure that you carefully displace the lungs (Fig. 9c–e).
 CRITICAL STEP Cutting the costal arches should be performed with caution to avoid damage of the heart and the
surrounding blood vessels.
nature protocols | VOL.11 NO.1 | 2016 | 73
protocol
Figure 8 | Field potential recordings from
Langendorff-perfused hearts. (a) Experimental
setup for the heart isolation and cannulation.
(b) Custom-made cannula with two circular
notches at distances 1 and 2 mm from the
tip. (c) Cannulated heart tied with silk suture
material to the cannula. (d) Experimental setup
for the recording of field potentials from the
Langendorff-perfused heart. (e) Langendorffperfused heart with bipolar recording electrode.
The electrode is attached to regular ECG
electrodes (d). Gassing of the buffer reservoir was
omitted for better visibility.
© 2015 Nature America, Inc. All rights reserved.
57| Lift the sternum and displace the
sternum down cranially with a Halsey
needle holder to obtain free access to
the heart (Fig. 9f).
a
d
b
c
e
Instructions for level-specific
in vitro preparations ● TIMING
variable
58| Proceed with one of the
following options, depending on the
specific experiment being performed:
option A, gelatin-inflation of the
heart for anatomical studies; option B,
isolation and enzymatic digestion
of the SAN for single-cell electrophysio­
logy; option C, whole-mount SAN
dissection for microelectrode
recordings; or option D, whole-heart
preparation and extracellular field
potential recordings.
(A) Gelatin-inflation of the heart for anatomical studies ● TIMING 3 h
(i) Insert a Safety-Multifly needle into the left ventricular cavity (Fig. 9g).
(ii) Make small incisions into the lobes of the liver (Fig. 9h).
(iii) Perfuse the heart with PBS at room temperature via the Safety-Multifly needle until the blood is washed out.
An important sign for optimal washout is that the liver gets pale (Fig. 9h). This takes ~3–4 min.
(iv) To inflate the flabby atrial walls and vessels, pressure-inflate in situ by injection of warm gelatin solution into the
atria and ventricles via the same needle until all organs look inflated. This takes ~3 min.
? TROUBLESHOOTING
(v) Pour 5 ml of ice-cold PBS directly onto the heart while the perfusion cannula is still in the left ventricle.
(vi) Remove the cannula. Place the mouse into a fridge at 4 °C for at least 1.5 h to solidify the gelatin.
 PAUSE POINT It is also possible to solidify it at 4 °C for a longer time—e.g., overnight.
(vii) Once gelatin is solidified, remove the heart from the chest with Fine Iris scissors. Make sure that cardiac outflow
tract and long cardiac arteries and veins are still connected to the heart.
(viii) Immerse the heart in a Sylgard-coated Petri dish filled with PBS at room temperature.
(ix) Gently separate the pericardium, pulmonary arteries and mediastinal fat from the base of the heart (Figs. 10–12).
(x) Identify typical landmarks of the SAN—e.g., the superior and inferior caval vein, the sulcus terminalis and the sinus
node artery (Figs. 10 and 11; Supplementary Figs. 1 and 2; Supplementary Videos 1 and 2).
(B) Isolation and enzymatic digestion of the SAN for single-cell electrophysiology ● TIMING 4 h for isolation and
up to 5 h for current clamping
(i) Hold the heart at the apex, and gently lift it with blunt standard pattern forceps (Fig. 9f). Using curved Iris scissors,
gently remove the heart by transecting cardiac blood vessels and mediastinal tissue en bloc (Supplementary Fig. 1a).
 CRITICAL STEP Avoid stretching of the heart and vessels. Pay extreme attention to avoid damaging the delicate
posterior wall of the atria and the connected right atrial veins. Be sure to cut the right superior caval vein as long as
possible. To obtain the best cell quality, the time for removal of the heart and isolation of the SAN should be <10 min.
74 | VOL.11 NO.1 | 2016 | nature protocols
protocol
© 2015 Nature America, Inc. All rights reserved.
Figure 9 | Dissection of the heart. (a–f) Individual steps during dissection of
the heart, which then can further be used for SAN isolation, whole-mount
SAN preparation, whole-heart preparation and gelatin-perfusion of the heart.
(a) Transverse skin incision from the left to the right costal arch. (b) Transverse
incision of the peritoneum. (c,d) Incision of the diaphragm along the thorax
to expose the pleural cavity. (e) Cutting the ribcage from the costal arches up
to the clavicles. (f) Removal of the heart. (g) Perfusion of the mouse with PBS
followed by perfusion with gelatin solution to pressure-inflate the heart. The
tip of the needle is located in the left ventricular cavity. Before the perfusion
is started, the liver appears dark red. (h) After gelatin perfusion, the color of
all organs and especially of the liver is pale, indicating optimal perfusion. Small
incisions in the lobes of the liver can be seen. Experiments were performed
according to institutional and governmental regulations and approved by the
Regierung von Oberbayern.
a
b
c
d
(ii) Transfer the heart into the Petri dish, and turn it
e
f
by 180°, so that the right atrium and the SAN are
directly accessible. Fix the heart by placing a 26G
needle through the apex.
(iii) As quickly as possible while working with a steromicroscope, use fine Vannas spring scissors and fine forceps
to remove fat tissue and the pericardium, as well as the
lungs, thymus and esophagus (Supplementary Fig. 1a,b).
g
h
(iv) Identify the typical landmarks of the SAN area
(Figs. 11 and 12; Supplementary Figs. 1 and 2;
Supplementary Videos 1 and 2). The right lateral
border of the SAN is marked by the sulcus terminalis,
and the cranial border is marked by the right superior
caval vein. Furthermore, the SAN artery centrally
extends through the longitudinal axis of the SAN
in the direction of the inferior caval vein. However, this artery is not consistently visualized using a stereomicroscope.
(v) Dissect the SAN by first cutting along the sulcus coronarius beginning from the basis of the sulcus terminalis into the
direction of the inferior vena cava (Fig. 12 and Supplementary Fig. 1c,d). A second incision is made in the direction
of the superior vena cava. A third cut is made slightly lateral to the sulcus terminalis. To isolate the SAN completely,
fold up the SAN and make a connecting cut along the root of the right superior vena cava.
 CRITICAL STEP It is important to avoid stretching of the delicate SAN tissue, as it can be mechanically damaged
very easily.
(vi) Gently hold the isolated tissue and slightly incise the tissue.
 CRITICAL STEP Incision of the isolated SAN can be helpful to increase the surface area and to improve the contact
between the tissue and the digesting enzymes (collagenase, elastase and protease), in particular in the central part
of the SAN. The tissue can also be separated in several parts, but caution must be taken during the dissociation steps
because the small pieces tend to stick to the tip of the dissociation pipettes and might be lost for further dissociation. If you need to obtain cells from the periphery of the node, we do not recommend incisions.
(vii) Transfer the SAN preparation rapidly into the 2-ml reaction tube containing Tyrode low pH 6.9 at 37 °C, and place it
into the dry block heater (36 °C, without shaking).
(viii) Incubate the tube for 5 min.
(ix) Add 10 µl of BSA to the tube, followed by the enzyme aliquots of elastase (40 µl, 18.87 U), protease (125 µl,
1.79 U) and collagenase B (150 µl, 0.54 U) to the 675 µl of Tyrode low pH 6.9 that was preheated in a 2-ml reaction
tube. These components enzymatically digest the SAN.
(x) Incubate the SAN for 30 min in the dry block heater (36 °C, 600 r.p.m.). Remove the reaction tube from the heater
every 7 min, and gently shake the tube manually until the tissue floats up.
 CRITICAL STEP To prevent the SAN tissue from sticking to the cap of the tube, never invert the reaction tube during
digestion or washing steps.
(xi) To terminate the enzymatic digestion, centrifuge the SAN tissue at 200g for 2 min at 4 °C. Discard the supernatant
(a volume of ~990 µl) and carefully add 990 µl of fresh Tyrode low pH 6.9 (RT) to dilute the enzyme concentration and
wash the tissue. The tube should again be slightly shaken until the SAN tissue floats up.
 CRITICAL STEP Take caution not to suck the SAN tissue into the pipette tip during washing steps, as this causes
cell damage through shear stress.
nature protocols | VOL.11 NO.1 | 2016 | 75
protocol
a
c
LAA
Anterior wall
of RA
RAA
PT
Ao
C
D
RPA
E
S ul cu s te r m i n a l i s
LPA
d
Fat pad adjacent to
root of Ao
SVC
LPV
MPV
RPV
RA
F
Root IVC
Root SVC
Root SVC
b
f
cu
s
te
rm
ina
lis
li s
mina
Sulcus ter
© 2015 Nature America, Inc. All rights reserved.
e
l
Su
Figure 10 | Anatomic localization of the SAN. (a) Schematic caudal view of the heart. The course of the sinus node artery is drawn in red. Red boxes indicate
the location of the magnifications shown in (c–f) SVC, right superior vena cava; IVC, inferior vena cava; Ao, aorta; PT, pulmonary truncus; LPA, left pulmonary
artery; RPA, right pulmonary artery. RPV, MPV and LPV, pulmonary veins; RAA, right atrial appendage; RA, right atrium; LAA, left atrial appendage. (b) Caudal
view of a gelatin-inflated heart. Scale bar, 1 mm. White arrow heads (c–f) indicate sinus node artery. (c–f) Magnified sections of the heart. Scale bars, 0.5 mm.
(xii) Centrifuge the SAN tissue at 200g for 2 min at 4 °C. Discard the supernatant and add 990 µl of Tyrode low pH 6.9.
Repeat this step two more times with ice-cold KB.
(xiii) Centrifuge again for 2 min at 200g at 4 °C and discard the supernatant. Add 350 µl of ice-cold KB and shake gently
until the tissue floats up. Placement of the tube containing the SAN in a refrigerator at 4 °C for 2.5–3 h to let it recover
from enzymatic digestion may increase the cell quality compared with immediate dissociation of the cells, and it is an
optional additional step.
SVC
(xiv) Preheat a water bath to 37 °C.
SVC
a
b
c
SVC
SNA
AO
PA
PA
Figure 11 | Anatomic localization of the SAN
and whole-mount SAN preparations. (a,b) Dorsal
view of the heart (a) and magnification of the
central SAN area of a gelatin-inflated heart (b).
(c) Whole-mount SAN preparation of a gelatininflated heart. (d–f) Right dorsolateral view of
a gelatin-inflated heart (d) and magnifications
of the sinus node artery (e,f). (g,h) Dorsal
view of the heart (g) and magnification of the
central SAN area in a regular preparation as it
is used for experiments (h). (i) Whole-mount
SAN preparation as it is used for microelectrode
experiments. SNA, sinus node artery; RSVC, right
superior vena cava; LSVC, left superior vena
cava; IVC, inferior vena cava; RA, right atrial
appendage; AO, aorta; PA, pulmonary artery; PV,
pulmonary vein; ST, sulcus terminalis; CT, crista
terminalis; RV, right ventricle; LV, left ventricle.
White and black arrow heads indicate sinus node
artery. Scale bars, 0.5 mm.
76 | VOL.11 NO.1 | 2016 | nature protocols
ST
SNA
PV
SNA
ST
CT
RAA
RAA
RAA
LSVC PV
IVC
LV
IVC
RV
d
e
SVC
f
SVC
SVC
SNA
PV
SNA
RAA
ST
RAA
SNA
RAA
g
h
AO
i
SVC
SVC
LSVC
RAA
ST
PV
SVC
RAA
CT
ST
RAA
IVC
IVC
protocol
© 2015 Nature America, Inc. All rights reserved.
a
b
SVC
PV
SAN
rminalis
us te
Sulc
Figure 12 | Dissection of the SAN of a gelatininflated heart. (a) Dorsal view of the heart.
All excess tissues and organs were removed.
The white dashed line indicates the cutting line
for SAN dissection. The white dot indicates the
area in which the SAN should be gently held with
fine forceps for isolation. Other parts should
not be touched in order to avoid damage to the
central SAN region. SVC, right superior vena cava;
IVC, inferior vena cava; PV, pulmonary vein;
RAA, right atrial appendage; LV, left ventricle;
RV, right ventricle. (b) The first incision, which is
indicated by white arrowheads, is made from the
sulcus terminalis into the direction of the
IVC along the sulcus coronarius. (c) A second
incision is made into the direction of the SVC.
(d) A third cut is made slightly lateral of the
sulcus terminalis. (e) Fold up the SAN and
make a connecting cut along the root of the
right superior vena cava. (f) The SAN is cut out
completely. Scale bar, 1 mm.
RAA
IVC
LV
c
RV
d
(xv) Place seven PLL coverslips into
the incubation chamber.
e
f
SAN
(xvi) Incubate the tube for 10 min
in the water bath (37 °C).
(xvii) Mechanically separate SAN
cells by pipetting the SAN
tissue 4–8 times with the
1,000-µl dissociation tip.
To increase the efficiency of
the separation process, proceed
using the 200-µl dissociation
tip 3–5 times.
 CRITICAL STEP The tissue should almost fall apart on its own, which indicates optimal digestion. Avoid intense
agitation, as this will cause too much shear stress. Take 50 µl of the cell suspension after every pipetting step, and
inspect dissociation progress and cell quality using a microscope.
(xviii) Let large cell clusters sediment by gravity for 15 s. Place 25 µl of the supernatant containing already-dissociated
cells on each coverslip.
(xix) Continue to dissociate the remaining cell clusters using the 200-µl dissociation tip 3–5 times.
(xx) Let the large cell clusters settle for 15 s. Place an additional 25 µl of the supernatant on each coverslip.
(xxi) Allow the cells to sediment by gravity and to attach to the coverslip for 10–15 min in the incubation chamber.
(xxii) Transfer the coverslip with KB solution carefully into the recording chamber.
(xxiii) Add 5 µl of Tyrode III (final Ca2+ concentration is increased to 0.34 mM) and wait for 2 min.
 CRITICAL STEP In this and the next steps, Ca2+ concentration is gradually increased to gently re-adapt the cells
to physiological Ca2+ levels. This is done by a stepwise increase of the Ca2+ concentration within the KB.
(xxiv) Add 11 µl of Tyrode III (final Ca2+ concentration is increased to 0.59 mM) and wait for 2 min.
(xxv) Add 25 µl of Tyrode III (final Ca2+ concentration is increased to 0.92 mM) and wait for 2 min.
(xxvi) Add 86 µl of Tyrode III (final Ca2+ concentration is increased to 1.35 mM) and wait for 2 min.
(xxvii) Start to superfuse the cells continuously with Tyrode III solution (final Ca2+ concentration is increased to 1.8 mM)
and slowly warm them to 32 °C. The single SAN cells are now under physiological conditions, and they can be used
for current-clamp recordings.
 CRITICAL STEP The exchange of the extracellular solution has to be done carefully because the cells are only
slightly attached to the coverslip.
(xxviii) Check the quality of the cell isolation. Our criteria for optimal cell quality are as follows: Cells have a striated
pattern and are not granulated (Fig. 13). In current-clamp measurements, cells have a maximal diastolic potential
around −60 mV and a slow diastolic depolarization, and they fire spontaneous and regular action potentials
(~300 bpm at 32 °C)15. In voltage-clamp experiments, cells display large If currents (>500 pA). We recommend
extensive training in patch-clamp experiments using WT SAN cells that beat regularly to help experimenters
nature protocols | VOL.11 NO.1 | 2016 | 77
protocol
b
a
get a feeling for optimal cell quality, as well as
physiological and biophysical properties. After this
training period, measurements with cells from
d
e
c
genetically modified mice, which might display
arrhythmic beating, can be performed.
? TROUBLESHOOTING
(xxix) Identify single isolated SAN cells. SAN cells of
optimal quality beat and contract regularly in
warm, physiological solutions. Wave-like contraction of the cells indicates poor cell quality. There
are several SAN cell subtypes, including spindle
cells, elongated cells and spider cells (Fig. 13). The SAN preparation will also contain various amounts of atrial and
atrial-like cells. These cell types differ in size and cell capacitance, action potential shape and frequency, current
amplitudes, half-maximal activation voltage (V0.5), and activation kinetics of If and other ion channels.
 CRITICAL STEP We recommend analyzing electrophysiological parameters for each cell type separately in order
to reduce variation in these parameters. When the remaining variability in electrophysiological properties is still
an issue, the number of cells for data analysis should be increased.
(xxx) C urrent clamping. Place SAN cells into the recording chamber and heat up slowly to 32 °C while continuously
superfusing with Tyrode III.
 CRITICAL STEP This and subsequent steps in this section describe how to analyze isolated SAN cells using the
perforated-patch technique in the current-clamp mode.
(xxxi) Dip the tip of the recording electrode briefly in intracellular solution without amphotericin B.
(xxxii) Back-fill the recording electrode with intracellular solution containing amphotericin B.
(xxxiii) Select an individual SAN cell to patch and position it at the center of the microscope’s field of view.
 CRITICAL STEP SAN cells have small cell bodies (<4 µm) that resemble somata of neurons and have at least
two processes. The patch electrode needs to be attached to the soma-like part of SAN cells. Do not patch cells
that are located in cell clusters.
(xxxiv) Set the amplifier to voltage-clamp mode.
a
(xxxv) Approach the SAN cell body with the electrode
from above, applying just a little positive pressure
to push away any debris.
(xxxvi) Stop applying pressure when you are close to the
SAN cell.
(xxxvii) Go down with the pipette until you are in contact
with the cell surface (the striated grid-like pattern
1s
of the cell widens as you press the pipette to the
b
membrane surface), and establish cell-attached
configuration by applying negative pressure.
20 mV
© 2015 Nature America, Inc. All rights reserved.
Figure 13 | Isolated SAN cells. (a) Cells isolated from the SAN, including
single cells, cell aggregates, dead cells and cell debris. Scale bar, 200 µm.
(b) Atrial-like cell. (c) Elongated cell. (d) Spider cell. (e) Spindle cell.
Scale bar, 50 µm (b–e).
Figure 14 | Current-clamp recordings from whole-mount SAN preparations.
(a) Small regular voltage deflections indicate that the microelectrode
is close to the SAN tissue (left). Impalement of the cell causes a rapid
voltage drop (right). Gray line, 0 mV. (b) Once the resting membrane
potential and the peak potential are stable, you can start measurements.
Note that action potentials are recorded in an atrial cell that lacks SDD.
It displays a relatively negative resting membrane potential (RMP) and a
high upstroke velocity. The atrial cell is paced regularly by neighboring SAN
cells. (c) Action potentials recorded in the periphery of the central SAN are
characterized by the presence of SDD and a relatively high upstroke velocity.
The minimum diastolic potential (MDP) is more positive compared with that
of atrial cells. (d) Action potentials recorded in the center of the SAN are
characterized by SDD, lower upstroke velocity and a more-positive MDP.
78 | VOL.11 NO.1 | 2016 | nature protocols
c
d
10
10 s
1.5
1
0.5
PSD Welch
PSD Burg
AR method
pNN6
i
400
j
12
10
8
6
4
2
500
600
HR (bpm)
700
60
50
40
30
20
10
LF
h
Raw data
Interpolated
data
RR
AIC value ×10
4
Figure 15 | Time and frequency-domain parameters from telemetric
0
1
2
3
4
5
ECG recordings. (a) Representative ECG recording from a freely moving
Frequency (Hz)
WT mouse. To resolve individual ECG complexes, a 10-s interval is shown
k
l –5
1
from an ECG trace of 103 s. (b) Poincaré plot from 20,000 consecutive
RR intervals during low-activity phase. (c) Mean RR interval ±s.e.m.
–5.5
0.5
determined from 12 h during the low-activity phase. (d,e) Mean values
of SDRR, RMSSD and pNN6 ±s.e.m. were calculated from three
–6
0
representative 10-min intervals during the low-activity phase. Beats that
differed more than 20% of the moving average were excluded from the
–6.5
–0.5
calculation. (f) 12 h HR histogram during the low-activity phase.
(g) Tachogram for a time series of 103 s. (h) Periodogram and superimposed
–7
0
20
40
60
80
20 40 60 80 100
AR model (model order: 39) determined from detrended tachograms.
Lag (ms)
Model order
(i) Total power (TP), very-low-frequency (VLF), low-frequency (LF) and
high-frequency (HF) power determined from the periodogram shown in h, and VLF, LF and HF are shown as a percentage of TP (j). (k,l) Determination of
AR model order using the partial autocorrelation (k) or the Akaike information criterion (AIC) (l). Experiments were performed according to institutional and
governmental regulations and approved by the Regierung von Oberbayern. Raw data files for the tachogram shown in g and used for data analysis in h–l are
given in Supplementary Data 2.
HF
100 120 140 160 180
RRn (ms)
1
VLF
80
5
2
Percentage of total power
20
2
10
LF
80
4
3
HF
100
40
4
Counts ×10
60
Counts
(ms)
120
(ms)
80
140
0
20
15
VLF
10
6
TP
20
160
Sample
partial autocorrelation
© 2015 Nature America, Inc. All rights reserved.
RR interval (ms)
g
f
Power (s /Hz) ×10
500 ms
e 20
100
Power (s2/Hz) ×10–5
RRn+1 (ms)
180
d8
–4
c 120
2
b
SD
R RR
M
SS
D
a
4
protocol
120
(xxxviii) Wait until a low-resistance access develops.
? TROUBLESHOOTING
(xxxix) Switch from voltage-clamp mode to current-clamp mode.
(xl) Record the firing of spontaneous action potentials.
? TROUBLESHOOTING
(xli) Record for a period of at least 60 s.
(C) Whole-mount SAN dissection for microelectrode recordings ● TIMING 10–15 min for mounting and 15–45 min for recording
(i) Expose the SAN area as described in ‘Isolation and enzymatic digestion of the SAN for single cell electrophysiology’
(Step 58B(i–iv)).
(ii) Dissect the SAN together with the right atrium, the right superior caval vein and the inferior caval vein
(Supplementary Fig. 2). First, cut along the sulcus coronarius starting at the inferior caval vein and ending at the
right atrial appendage. Second, after folding up the right atrial appendage, cut further along the sulcus coronarius
up to the root of the right superior caval vein. Finally, cut slightly left to the inferior caval vein and along the
intra-atrial septum up to the root of the right superior caval vein. Isolate the whole-mount preparation by making
a connecting cut between the root of the aorta and the root of the right superior caval vein.
(iii) Transfer the SAN with fine forceps into the Sylgard-coated recording chamber filled with Tyrode III solution (at RT).
(iv) Pin the SAN onto the Sylgard layer with 4–7 minutien pins with the endocardial side facing upward (Fig. 11i).
 CRITICAL STEP To avoid damage, the SAN should not be stretched too much. In addition, the microelectrode
recordings are easier when the tissue is pinned loosely.
(v) Check the quality of the preparation under a stereomicroscope. Our criteria for optimal preparations are as follows:
the crista terminalis and atrial trabeculae are not damaged and the length of right superior vena cava should be
at least 0.2 mm. The SAN beats and contracts regularly over a long time period (up to 1 h). To achieve optimal
preparation quality, we recommend extensive training in microelectrode experiments using WT whole-mount SAN
preparations that beat regularly. After this training period, measurements with preparations from genetically
modified mice, which might display arrhythmic beating, can be performed.
nature protocols | VOL.11 NO.1 | 2016 | 79
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c
40
200
R
0.5
–2
80
8
Of total power (%)
1
6
4
2
400
i
10
LF
1.5
h
VLF
10 s
2
PSD Welch
PSD Burg - AR method
TP
–0.1
2.5
Power (s2/Hz) ×10
0
g
–3
Raw data
Interpolated
Power (s2/Hz) ×10
0.1
60
40
20
AIC value ×104
Figure 16 | Time- and frequency-domain parameters from isolated
0
1
2
3
4
5
Frequency (Hz)
SAN cells. (a) Representative action potential trace measured by
current-clamp experiments of isolated SAN cells from a WT mouse.
1
k –3
j
To resolve individual action potentials, a 10-s interval is shown
–4
from a trace of 100 s. (b–e) Poincaré plots (b), mean NN
0.5
interval ±s.e.m. (c), time-domain parameters (d) and histogram
–5
0
(e) were determined from 100 s of consecutive action potentials.
–6
(f) Tachograms for a time series of 100 s. (g) Periodogram and
–0.5
–7
superimposed AR model (model order: 29) determined from
tachograms. (h) Total power (TP), very-low-frequency (VLF),
–1
–8
0
20
40
60
80
20 40 60 80 100 120
low-frequency (LF) and high-frequency (HF) power determined
Lag (ms)
Model order
from periodograms similar to those shown in g, and VLF, LF and
HF shown as a percentage of TP (i). (j,k) Determination of AR model order using the partial autocorrelation (j) or the Akaike information criterion (AIC) (k).
Raw data files for the tachogram shown in f and used for data analysis in g–k are given in Supplementary Data 2.
Sample
partial autocorrelations
© 2015 Nature America, Inc. All rights reserved.
NN interval (s)
f
250 300 350
NN (ms)
HF
SD
NN
LF
150 200 250 300 350 400 450
NNn (ms)
20
10
N
50
10
N
100
150
30
SS
D
200
20
VLF
150
M
250
Counts
30
200
300
1s
e 50
40
250
350
(ms)
NNn+1 (ms)
20 mV
d
300
400
HF
b 450
(ms)
a
(vi) Microelectrode recordings in the whole-mount SAN. Transfer the recording chamber containing the whole-mount SAN
preparation bathed in Tyrode III to an inverted microscope. Place the temperature sensor near the SAN and heat the
chamber slowly to 30 ± 1 °C and continuously superfuse the SAN with warm Tyrode III solution.
(vii) Back-fill the microelectrode with 3 M KCl solution using the Microfil.
(viii) Move the microelectrode slowly to the SAN region. On an inverted microscope, it is not possible at this stage to see
the position of the microelectrode. Small voltage deflections indicate that you are now close to the tissue (Fig. 14a).
(ix) Once you are near the SAN tissue, you should set the micromanipulator to fast mode and try to impale the cells by
pressing the z axis button several times very briefly until you impale a sinoatrial cell or an atrial cell (Fig. 14a).
Once the cell is impaled, wait until the signal is stable (Fig. 14b).
 CRITICAL STEP It is sometimes difficult to find the small central SAN region with cells that display a slow diastolic
depolarization (SDD) phase (Fig. 14c,d). The SAN artery and the SVC are useful anatomical landmarks46. For time- and
frequency-domain analyses, it is sufficient to measure action potentials in atrial cells in close proximity to the SAN,
because these cells are paced by the neighboring SAN cells and do display the same inter-beat intervals. Therefore,
atrial cells provide a sufficient readout for SAN pacemaker activity.
? TROUBLESHOOTING
(x) Record spontaneous action potentials for at least 103 s.
(D) Whole-heart preparation for extracellular field potential measurements ● TIMING 40 min for heart isolation
and 15–45 min for extracellular field potential recordings
(i) Place the heart into the Petri dish.
 CRITICAL STEP Perform Steps 53–58D(i) as fast as possible. Do not lift the heart at the apex (Fig. 9f), because
this will damage small vessels. Instead, lift the thymus gently using forceps, and then remove the heart together with
the thymus. Be sure to cut the aorta at about 2 mm from its entry into the heart.
(ii) Remove the thymus by pulling each lobe to the side using forceps, and remove excess tissue.
(iii) Identify the aorta (Supplementary Fig. 3) and cannulate the heart using fine-tip forceps to slide the aorta onto the
cannula so that the tip of the cannula is just above the aortic valve. Check the 1- and 2-mm notch on the cannula
(Fig. 8b,c) to ensure proper cannulation.
 CRITICAL STEP Do not push the cannula through the aortic valve, as this will inhibit optimal perfusion.
? TROUBLESHOOTING
80 | VOL.11 NO.1 | 2016 | nature protocols
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0.5
50
0
j
1
2
3
Frequency (Hz)
4
1.0
0.5
258
262
60
Of total power (%)
1
1.5
VLF
2
254
NN (ms)
i
LF
Figure 17 | Time- and frequency-domain parameters from whole-mount
SAN recordings. (a) Action potential trace measured by current-clamp
experiments using whole-mount SAN preparations. To resolve
individual action potentials, a 10-s interval is shown from a trace
of 103 s. (b–e) Poincaré plots (b), mean NN interval ±s.e.m. (c),
time-domain parameters (d) and histogram (e) were determined from
103 s of consecutive APs. (f) Tachograms for a time series of 103 s.
(g) Periodogram and superimposed AR model (model order: 29)
determined from tachograms. (h) Total power (TP), very-low-frequency
(VLF), low-frequency (LF) and high-frequency (HF) power determined
from periodograms similar to those shown in g, and VLF, LF and HF
shown as a percentage of TP (i). (j,k) Determination of AR model
order using the partial autocorrelation (j) or the Akaike information
criterion (AIC) (k). Raw data files for the tachogram shown in f and
used for data analysis in g–k are given in Supplementary Data 2.
250
2.0
50
40
30
20
10
5
k
1
0.5
AIC value ×10
10 s
3
Power (s2/Hz) ×10
–6
2
0
h
PSD Welch
PSD Burg-AR method
TP
g
6
246
SD
N
M N
SS
D
NN
260
R
250
255
NNn (ms)
Power (s /Hz) ×10
245
HF
245
VLF
1.0
100
Raw data
Interpolated
–6
(ms)
(ms)
250
150
90
80
70
60
50
40
30
20
10
4
© 2015 Nature America, Inc. All rights reserved.
RR interval
–3
(s) ×10
f
1.5
200
Sample
partial autocorrelations
NNn+1 (ms)
20 mV
1s
255
e
2.0
LF
d
250
HF
c
260
Counts
b
–4
a
0
–0.5
–1
–6
–7
–8
–9
–10
0
20
40
60
80
20
40
60
80
100 120
Model order
Lag (ms)
(iv) Pull the loosely knotted 6-0 silk thread over the aorta, and then tie the aorta to the cannula (Fig. 8c).
(v) O
nce the aorta is securely tied to the cannula, mount the cannulated heart to the Langendorff apparatus and
start perfusion (Fig. 8e).
(vi) Extracellular field potential measurements. Place the bipolar platinum-iridium recording electrode onto the
heart (Fig. 8e), and attach the recording electrode to the ECG electrode.
(vii) Record the extracellular signals for several minutes, and check the stability of the frequency.
? TROUBLESHOOTING
(viii) Use a recording episode of 103 s for analysis (analysis and results not shown).
Data analysis for spontaneously beating cells, whole-mount SAN preparations, whole atria and hearts using
MATLAB ● TIMING several days
 CRITICAL In Steps 59–61, we describe time- and frequency-domain analyses using the MATLAB scripts provided in
Supplementary Data 1. In addition to the preparations described above, any spontaneously beating cardiac cells, such as
isolated embryonic or neonatal cardiomyocytes, or spontaneously beating induced cardiomyocyte-like cells, can be analyzed
using the MATLAB scripts. For testing and practicing data analysis, example data files are provided in Supplementary Data 2
with time series of NN distances of a single SAN cell and a whole-mount SAN measurement, as well as RR distances of a
telemetric ECG recording (Figs. 15–17).
59| Import your time series of action potentials obtained from one of the options in Step 59 into MATLAB, and run the
‘peak detection’ script (Supplementary Data 1) to extract the NN interval series.
60| Determine the autoregressive model order using the Akaike information criterion in the ‘model order’ script
(Supplementary Data 1). Usually, the model order is between 16 and 40 (ref. 48). For a detailed description of the
model-based autoregressive spectral analysis, see Supplementary Method 3.
61| Import your NN interval series into MATLAB, and then run the ‘HRV’ script (Supplementary Data 1).
nature protocols | VOL.11 NO.1 | 2016 | 81
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? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
© 2015 Nature America, Inc. All rights reserved.
Table 1 | Troubleshooting table.
Step
Problem
Possible reason
Solution
21
Subcutaneous dislocation of
the transmitter and displacement of the electrodes
Subcutaneous placement of the
transmitters bears the risk of ventral
transmitter dislocation, and this might
lead to electrode displacement
Perform ECG measurements immediately
after 2–3 weeks of recovery time
For long-term measurements, we
recommend i.p. transmitter placement
rather than s.c. placement
Tissue necrosis along the
wound
Mouse skin is very delicate and prone
to necrosis when too much pressure or
traction is applied with forceps during
transmitter implantation and suture
Use blunt forceps and apply only
slight pressure
Handle the skin as carefully as possible
Scratching and chewing at
the surgical site
The suture knots are tied too tightly
The sutures should be tied just tightly
enough to bring the two edges together
58A(iv)
Insufficient inflation of the
ventricles and/or atria
Be careful not to puncture the right
ventricular wall during transmyocardial
injection, as this will lead to pressure
loss
Re-inject a small volume of gelatin
solution with a 26G needle directly into
the atria or ventricle if the inflation is
incomplete
58B(xxviii)
Low SAN cell yield, large
aggregates of nondissociated
cells
Insufficient digestion, activity loss of
the enzyme during storage, variability
between different enzyme batches, or
enzyme concentrations out proportion
to each other
Increase the digestion time, adjust the
concentration and/or composition of
the enzyme mix or use new batches of
enzymes. Despite a relatively low cell
yield, there will be enough cells to patch
for 4–6 h
Contamination of the SAN
preparation with atrial cells
Contamination with atrial cells is
normal because atrial cells invaginate
into the SAN. If the amount of atrial cells
is very high, the isolated tissue piece is
too large
Contamination is usually not a
problem because atrial cells can easily
be identified. It is possible to cut out
a smaller piece of tissue during SAN
preparation to increase the ratio of
SAN to atrial cells
Cell borders are round and
not sharp or cells die during
Ca2+ reintroduction
The SAN is a very delicate tissue that
can be easily damaged during isolation,
enzymatic digestion and trituration
The experimenter should be extremely
cautious during the isolation of the
SAN. Use very fine and sharp scissors
and do not stretch the tissue piece
during isolation
Decrease digestion time and/or
collagenase amount. Use less force for
cell trituration
No low-access resistance
develops after seal formation
with amphotericin B in the
intracellular solution, or the
time for perforation is very
long (<15 min)
Amphotericin B is not in solution
The intracellular solution containing
amphotericin B needs to be vortexed
vigorously or sonicated to get into
solution. Fresh intracellular solution
should be prepared every hour,
because amphotericin B will come out
of solution
The tip opening of the recording
electrode is too small
Widen the tip opening of the recording
electrode to increase the access area for
amphotericin B. The resistance should
be 1–2 MΩ
58B(xxxviii)
(continued)
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Table 1 | Troubleshooting table (continued).
© 2015 Nature America, Inc. All rights reserved.
Step
Problem
Possible reason
Solution
Tip dipping in amphotericin B–free
intracellular solution
Do not dip the tip of the recording
electrode in amphotericin B–free
solution before back-filling with
amphotericin B–containing solution.
It will be more difficult to form a seal
with the cell surface, but access to the
cell develops faster
Cells die after establishing
the whole-cell mode
Extracellular and/or intracellular solution
Check the composition, pH and
osmolarity of the solution
58B(xl)
Membrane potential is too
depolarized. Cells do not
beat regularly
Cell quality is not sufficient
Optimize the isolation of single SAN cells
as stated above (Step 58B(xxviii))
58C(ix)
It is not possible to impale the
cells with the microelectrode
Geometry of the microelectrode is not
optimal
Use smaller electrode tips. The resistance
of the electrode tips should be 30–50 MΩ
Arrhythmic beating of the SAN
The isolated section of the RSVC is
too short; the crista terminalis or atrial
trabeculae are damaged
Isolate the delicate SAN tissue very cautiously with enough surrounding tissue,
taking care not to transect larger bundles
of the crista terminalis or atrial trabeculae
58D(iii)
Insufficient perfusion of the
heart
Penetration of the aortic valve or air
embolism
Be sure not to introduce the cannula
too deeply into the aorta. Damage of the
aortic valve will inhibit good perfusion.
Tie the aorta securely to the cannula
Eliminate all air bubbles from the
perfusion buffer to avoid air embolism
58D(vii)
Irregular beating of the heart
Heart isolation
Be careful not to damage the SAN
region during heart isolation and
aorta cannulation
● TIMING
Steps 1–22, surgical implantation of telemetric transmitters for ECG telemetry: 45–60 min
Steps 23–50, analysis of HR and HRV: several days
Step 51, preparations required before heart dissection
Step 51A, preparations for gelatin-inflation of the heart: 5 min
Step 51B, preparations for isolation and enzymatic digestion of the SAN for single-cell electrophysiology: 20–30 min
Step 51C, preparations for whole-mount SAN dissection for microelectrode recordings: 5 min
Step 51D, preparations for whole-heart preparation for extracellular field measurements: 30 min
Steps 52–57, step-by-step dissection of the heart: 10–20 min
Step 58, protocols for different preparations, depending on the experiment being performed
Step 58A, gelatin-inflation of the heart for anatomical studies: 3 h
Step 58B, isolation and enzymatic digestion of the SAN for single-cell electrophysiological measurements: 4 h for isolation
and 5 h for current clamping
Step 58C, whole-mount SAN dissection for microelectrode recordings: 10–15 min for mounting and 15–45 min for recording
Step 58D, whole-heart preparation for extracellular field potential measurements: 40 min for heart isolation and 15–45 min
for extracellular field potential recordings
Steps 59–61, data analysis for spontaneously beating cells, whole-mount SAN preparations, whole atria and hearts using
MATLAB: several days
ANTICIPATED RESULTS
Here we present a comprehensive protocol that combines in vivo telemetry, in vitro electrophysiology of intact whole-mount
SAN preparations and isolated single SAN cells. There are technical challenges concerning the experimental side (surgery, lead
nature protocols | VOL.11 NO.1 | 2016 | 83
protocol
placement, whole-mount SAN preparation and isolation and patch clamping of isolated SAN cells), as well as concerning
the acquisition and the analysis of ECG and electrophysiological data using comprehensive time- and frequency-domain
signal processing tools. In this section, we provide some examples of typical results obtained using the protocol alongside
guidelines for the optimization and setting of parameters required for in vivo, whole-mount SAN preparation and isolated
SAN cell physiology.
© 2015 Nature America, Inc. All rights reserved.
Time- and frequency-domain parameters from telemetric ECG recordings
During the learning and setup phase of ECG telemetry, it is very important to manually practice the surgical procedures
for telemetric probe implantation. There are several types of telemetry probes available that allow for direct ECG recording
with and without combination with blood pressure recording, as well as blood pressure–only probes. Even though HRV data
could be extracted from blood pressure data, we usually use ECG telemetry probes because they can be reused several times,
whereas blood pressure probes cannot. In addition, blood pressure telemetry probes are twofold more expensive than regular
ECG telemetry probes. Depending on the mouse model being used, combined ECG and blood pressure recording may be
necessary. This combination provides a maximum set of parameters that comprise—in addition to the parameters derived
from ECG telemetry—metrics for absolute blood pressure, blood pressure variability, and respiratory rhythm variability,
which can be extracted from the blood pressure recording.
Parallel recording of these three parameters allows for the calculation of in vivo phase-response curves, which measure spontaneous autonomic regulation49. The most crucial part is the positioning of the ECG leads, as it correlates with the quality of
data acquisition and the signal-to-noise ratio of the ECG. For data acquisition, we use the DSI software package A.R.T. EMKA
also provides acquisition hardware and software. The decisions regarding which hardware and acquisition software to choose
depend on the individual preferences of the experimenter. Once the quality of the acquired telemetric ECG raw data is sufficiently high, further analysis is straightforward. For analysis, we provide three different options. The MATLAB script presented
in the Supplementary Data 1 is the most flexible one. The user can choose among several different options and also can
easily extend the script by adding published MATLAB subroutines. The disadvantage is that, in particular, long ECG traces are
difficult to review and analysis might get very time-consuming. Among commercially available ECG analysis software, we have
experience with ecgAUTO (EMKA) and A.R.T. (DSI). EcgAUTO is very powerful, and it provides many options for analysis.
In addition, .txt files containing ECG or action potential recordings can be imported into ecgAUTO, which makes it is possible
to analyze any ECG, patch-clamp data and microelectrode data. From these data, RR (NN) intervals are extracted by the peakdetection algorithm, and subsequently tachograms are generated and the complete downstream analysis can be performed.
This is not possible using the A.R.T. software, which is restricted to telemetric ECG analysis of DSI data files.
Independently of the software used for analysis, correct R-wave detection is the next important point in the HRV analysis
workflow. This might be quite challenging for long ECG traces, as are required for time-domain analysis and, in particular, for
recording during periods of high physical activity, which can cause movement artifacts and noisy traces. We recommend that
during a validation phase, the experimenter should visually inspect the complete recording, which is very time-consuming.
At later stages, after the method is established and the experimenter has developed a feeling for the parameter settings
and the detection efficiency of the software package in use, R-wave detection can be performed semiautomatically. Parts of
the traces without movement artifacts and with high signal-to-noise ratios can be analyzed by automated detection.
By contrast, stretches of the trace with low signal-to-noise ratios and large movement artifacts need visual inspection.
Once the R-wave detection is completed correctly, artifacts, sinus pauses and ectopic beats need to be removed. In the
validation phase, we recommend trying out the individual ectopic beat exclusion methods. The best option is to define an
exclusion criterion with respect to a moving average. Setting the threshold represents a compromise between losing too
many correctly detected heartbeats and not eliminating ectopic beats. In the literature, threshold values are given as two
times the s.d. of the mean RR interval, or alternatively as RR intervals that are above or below a certain percentage50.
This compromise and the respective parameters have to be individually set for a given series of experiments and for a given
mouse line. It is not possible to give any universally valid settings that are perfect for all mouse lines. As a guideline for
identifying the correct settings, we recommend performing Poincaré plots before and after ectopic beat exclusion to control
and optimize the robustness of the ectopic beat and artifact exclusion algorithm by repeatedly adjusting parameters.
For frequency-domain analysis, the time series has to be analyzed for stationarity18. This, again, is a particular problem
for long ECG traces. We recommend testing one of the three detrending algorithms specified in the MATLAB script and the
one provided in ecgAUTO side by side. We usually double-check the effect of detrending by inspection of the tachogram
and also the spectrum in order to confirm that, after detrending, the VLF power is reduced without affecting the LF and HF
power. We prefer detrending using the smoothness prior method47. After detrending the data, frequency-domain parameters
can be determined. Representative results are shown in Figure 15.
We present the power spectrum calculated by FFT periodograms. We usually use an ECG trace of 103 s, because resampling
of the tachogram by 50 ms results in 2,048 FFT points. We recommend using three windows, each containing 1,024 FFT
points and an overlap of 50%. This gives an optimal frequency resolution with 512 points for the periodogram. FFT parameters
84 | VOL.11 NO.1 | 2016 | nature protocols
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© 2015 Nature America, Inc. All rights reserved.
can be individually changed and optimized as needed by the experimenter. Rules for calculating the FFT point number and
the number of subepisodes, as well as the frequency resolution, are presented in Supplementary Method 2.
The FFT periodogram is subdivided into three major frequency components: VLF (0–0.4 Hz), LF (0.4–1.5 Hz) and
HF (1.5–4.0 Hz). In addition, TP is calculated. TP of spectral analysis is mathematically proportional to the variance of
the NN interval (=square of the s.d. of the NN interval (SDNN)). Therefore, SDNN reflects all the cyclic components.
For correspondence of time- and frequency-domain parameters, see ref. 24.
In addition to the periodogram, we usually calculate the AR model and superimpose it on the periodogram power
spectrum, as described in Supplementary Method 3. For the AR model calculation, the model order has to be determined
first using the partial autocorrelation function or the Akaike information criterion. It is important to mention that the
model order needs to be individually determined for every single mouse (or cell), and it usually differs for each individual
mouse (or cell) in the series.
Once the complete HRV analysis is established, WT mice and genetic mouse lines can be analyzed in parallel and
parameters can be compared. In addition, these experiments can be performed in the absence and the presence of drugs
such as propranolol or atropine, which are used to block autonomic control of the HR. As outlined in the INTRODUCTION,
HF power mainly reflects parasympathetic HR control, whereas LF power reflects sympathetic control of the HR. The responsiveness of HR regulation can be determined by application of additional drugs such as isoproterenol, carbachol and
the A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA).
Time- and frequency-domain parameters from isolated SAN cells
For successful and efficient preparation of the SAN and subsequent isolation of pacemaker cells, it is essential that the
researcher be familiar with SAN anatomy. We suggest using the inflated heart preparations in order to acquire the necessary
anatomical knowledge (Figs. 10 and 11). By using this method, an experimenter who is skilled at basic dissection methods
should be able to successfully carry out the SAN preparation within a few days. After becoming familiar with SAN anatomy,
the next step is practicing the SAN dissection without inflating the heart, as well as optimizing the SAN cell isolation protocol
(see TROUBLESHOOTING). A crucial step is the reintroduction of extracellular Ca2+ (see Step 58B(xxiii–xxvii)), which commonly
leads to substantial loss of SAN cells (up to 70%). Once Ca2+-tolerant SAN cells are obtained, an experimenter who is skilled
at basic electrophysiological methods should be able to carry out these experiments. By using the protocol, it is possible to
obtain 1–1.5 GΩ seals in 70% of attempts. After achieving whole-cell configuration, spontaneous pacemaker potentials can
be recorded in isolated SAN cells as long as 5 min. Experienced experimenters are able to obtain 5–10 whole-cell recordings
per 4-h recording session. For single-cell experiments, we usually record pacemaker potentials for 60–103 s depending on the
quality of the cell. We recommend adjusting the resampling rate to meet the 2,048 FFT points. The data are stored in Axon or
HEKA format and imported into the MATLAB script, or alternatively into ecgAUTO. Importing data into ecgAUTO requires
the .txt data format. We would like to stress that it is not necessary to purchase the monophasic action potential analyzer
(MAP analyzer). During import into ecgAUTO, the action potential shape is distorted by a filter, which is set by default, but
this does not affect proper action potential peak detection by simply using the RR detection mode and further processing.
Rules for setting and optimizing the individual parameters for detrending are exactly the same, as outlined in the ECG
telemetry section above (ectopic beats are not an issue in a single-cell preparation). Either the MATLAB script or ecgAUTO
can be used for analysis. From preprocessed data, time- and frequency-domain parameters can be determined from isolated
SAN cells (Fig. 16). The analysis is robust and straightforward.
Time- and frequency-domain parameters from whole-mount SAN recordings
For whole-mount SAN recordings, the most important prerequisite is the anatomically correct dissection of the SAN area.
The microelectrode experiments themselves are very straightforward and robust. With some practice, a time series of
spontaneous pacemaker or atrial action potentials can be recorded in this preparation, usually for as long as 10 min, and
sometimes for up to 1 h. The data are recorded and stored in Axon or HEKA format and imported into the MATLAB script
for peak detection and data analysis. Similar data can be acquired from biatrial preparations or isolated whole-heart
preparations (Fig. 17). For experiments in whole-mount SAN preparations or whole-heart preparations, we usually analyze
action potentials for 103 s, similarly to the ECG analysis. Specific optimization may be necessary for the removal of artifacts
such as ectopic atrial beats or premature ventricular beats in whole-heart contractions. Rules for optimizing the required
parameters are specified in the telemetry section (Steps 35–39).
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
from the German Research Foundation (DFG grant nos. BI 484/5-1, WA 2597/3-1
and SFB TRR 152 TP12) to M.B. and C.W.-S..
Acknowledgments We thank K. Hennis for technical assistance with the
Langendorff perfusion. The work of D.H.P. was supported by the Research Council
of Lithuania (grant no. MIP-13037). This work was supported, in part, by funding
AUTHOR CONTRIBUTIONS S.F. carried out the experiments and data analysis
that formed the basis of the protocol, wrote the manuscript and composed all
figures. R.P. provided images of isolated SAN cells, as well as images and videos
nature protocols | VOL.11 NO.1 | 2016 | 85
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of the anatomic localization of the SAN and isolation of the SAN, and wrote
the manuscript. F.A. wrote the MATLAB script and performed data analysis.
S.H. performed data analysis. V.M. and M.B. wrote the manuscript. D.H.P.
developed the technique for inflation of the heart with gelatin and provided
images of the anatomic localization of the SAN. C.W.-S. wrote the manuscript
and designed the protocol.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial
interests.
© 2015 Nature America, Inc. All rights reserved.
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com/reprints/index.html.
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