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Comprehensive multilevel in vivo and in vitro analysis of heart rate fluctuations in mice by ECG telemetry and electrophysiology

Abstract

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.

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Figure 1: Multilevel analysis of HR fluctuations.
Figure 2: Subcutaneous implantation of ECG transmitter.
Figure 3: Screenshots of Dataquest A.R.T. settings for checking the circadian rhythm of mice.
Figure 4: Determination of HR histograms using ecgAUTO.
Figure 5: Time-domain analysis using ecgAUTO.
Figure 6: Frequency-domain analysis of HR fluctuations.
Figure 7: Settings for HRV analysis in ecgAUTO software.
Figure 8: Field potential recordings from Langendorff-perfused hearts.
Figure 9: Dissection of the heart.
Figure 10: Anatomic localization of the SAN.
Figure 11: Anatomic localization of the SAN and whole-mount SAN preparations.
Figure 12: Dissection of the SAN of a gelatin-inflated heart.
Figure 13: Isolated SAN cells.
Figure 14: Current-clamp recordings from whole-mount SAN preparations.
Figure 15: Time and frequency-domain parameters from telemetric ECG recordings.
Figure 16: Time- and frequency-domain parameters from isolated SAN cells.
Figure 17: Time- and frequency-domain parameters from whole-mount SAN recordings.

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Acknowledgements

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 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..

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Authors

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 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.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Step-by-step exposure and dissection of the SAN.

(a) Posterior view of the isolated heart with tissue that must be removed for SAN dissection. (b) Lungs, esophagus, large parts of the thymus and fat are removed to expose the central SAN region. At this stage it is already possible to dissect the SAN. Black arrow heads: vagus nerve, white arrow heads: sulcus terminalis. (c) Same heart after complete removal of excess tissue. The white dotted line indicates the cutting line for SAN dissection. The white dot indicates the area where the SAN should be gently hold with fine forceps for isolation. Other parts should not be touched to avoid damage of the central SAN region. Please note that removal of all surrounding tissue was done for demonstration purposes only and is not necessary for SAN dissection. (d) The SAN was dissected and is placed on the ventricles for demonstration. The white dot indicates the area where the SAN was grabbed with fine forceps. Th: thymus, Lu: parts of the lung, Es: esophagus, RAA: right atrial appendage, LV: left ventricle, RV: right ventricle, SAN: sinoatrial node, ST: sulcus terminalis, SVC: right superior vena cava, IVC: inferior vena cava, VN: vagus nerve, Ao: Aorta. Scale bar: 1 mm.

Supplementary Figure 2 Dissection of a whole mount SAN preparation of a gelatin-inflated heart.

(a) Dorsal view of the heart. All excess tissue and organs were removed. The white dotted line indicates the first cutting line along the sulcus coronarius. The second cut (yellow dotted line) is made after folding up the RAA and cutting along the sulcus coronarius up to the superior caval vein. The third cut (black dotted line) is made slightly left of the IVC along the intraatrial septum up to the SVC. The whole mount preparation is isolated completely by making a connecting cut (red dotted line) between the root of the aorta and the root of the right superior caval vein. SVC: right superior vena cava, IVC: inferior vena cava, PV: pulmonary veins, RAA: right atrial appendage, LV: left ventricle, RV: right ventricle, Ao: Aorta (b) The first incision is indicated by white arrow heads. The second and third incision cannot be seen because they lie behind the RAA and IVC, respectively. (c) The last connecting cut leads to complete isolation of the whole mount SAN preparation. (d) Heart together with whole mount SAN preparation. Scale bar: 1 mm.

Supplementary Figure 3 Ventral view of the gelatin- inflated heart.

Black dotted line indicates optimal length of Aorta for cannulation. Ao: Aorta; LCA: left carotid artery; RCA: right carotid artery; PT: pulmonary truncus; RSA: right subclavian artery; LAA: left atrial appendage; RAA: right atrial appendage, LV: left ventricle: RV: right ventricle. Scale bar: 1 mm

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 1 and 2, Supplementary Methods 1–3 (PDF 1584 kb)

Supplementary Data 1

Zip file containing 4 MATLAB scripts. (ZIP 6 kb)

Supplementary Data 2

Raw data files for the tachograms shown in Figure 15g, Figure 16f and Figure 17f. These data files were used for the data analysis shown in Figure 15h-l, Figure 16g-k and Figure 17g-k. (XLSX 21 kb)

Anatomy and localization of the SAN 1.

Panoramic view across the supraventricular part of a gelatine inflated heart from the inferior vena cava over the SAN area to the right atrium. (MP4 1994 kb)

Anatomy and localization of the SAN 2.

View of an untreated heart before removal of the surrounding tissue. Fine forceps show the location of the SAN area. (MP4 2543 kb)

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Fenske, S., Pröbstle, R., Auer, F. et al. Comprehensive multilevel in vivo and in vitro analysis of heart rate fluctuations in mice by ECG telemetry and electrophysiology. Nat Protoc 11, 61–86 (2016). https://doi.org/10.1038/nprot.2015.139

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