Abstract
The mouse is a common and cost-effective animal model for basic research, and the number of genetically engineered mouse models with cardiac phenotype is increasing. In vivo electrophysiological study in mice is similar to that performed in humans. It is indispensable for acquiring intracardiac electrocardiogram recordings and determining baseline cardiac cycle intervals. Furthermore, the use of programmed electrical stimulation enables determination of parameters such as sinoatrial conduction time, sinus node recovery time, atrioventricular-nodal conduction properties, Wenckebach periodicity, refractory periods and arrhythmia vulnerability. This protocol describes specific procedures for determining these parameters that were adapted from analogous human protocols for use in mice. We include details of ex vivo electrophysiological study, which provides detailed insights into intrinsic cardiac electrophysiology without external influences from humoral and neural factors. In addition, we describe a heart preparation with intact innervation by the vagus nerve that can be used as an ex vivo model for vagal control of the cardiac conduction system. Data acquisition for in vivo and ex vivo electrophysiological study takes ~1 h per mouse, depending on the number of stimulation protocols applied during the procedure. The technique yields highly reliable results and can be used for phenotyping of cardiac disease models, elucidating disease mechanisms and confirming functional improvements in gene therapy approaches as well as for drug and toxicity testing.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The data supporting the findings of this study are available within the paper and its supplementary information files. Source data are provided with this paper.
References
Berul, C. I. Electrophysiological phenotyping in genetically engineered mice. Physiol. Genomics 13, 207–216 (2003).
Berul, C. I., Aronovitz, M. J., Wang, P. J. & Mendelsohn, M. E. In vivo cardiac electrophysiology studies in the mouse. Circulation 94, 2641–2648 (1996).
Li, N. & Wehrens, X.H. Programmed electrical stimulation in mice. J. Vis. Exp. https://doi.org/10.3791/1730 (2010).
Narula, O. S., Samet, P. & Javier, R. P. Significance of the sinus-node recovery time. Circulation 45, 140–158 (1972).
Rosenblueth, A. Functional refractory period of cardiac tissues. Am. J. Physiol. 194, 171–183 (1958).
Simson, M. B., Spear, J. & Moore, E. N. The relationship between atrioventricular nodal refractoriness and the functional refractory period in the dog. Circ. Res. 44, 121–126 (1979).
Strauss, H. C., Saroff, A. L., Bigger, J. T. Jr. & Giardina, E. G. Premature atrial stimulation as a key to the understanding of sinoatrial conduction in man. Presentation of data and critical review of the literature. Circulation 47, 86–93 (1973).
LaBarre, A. et al. Electrophysiologic effects of disopyramide phosphate on sinus node function in patients with sinus node dysfunction. Circulation 59, 226–235 (1979).
Saba, S., Wang, P. J. & Estes, N. A. 3rd. Invasive cardiac electrophysiology in the mouse: techniques and applications. Trends Cardiovasc. Med. 10, 122–132 (2000).
Fenske, S. et al. Sick sinus syndrome in HCN1-deficient mice. Circulation 128, 2585–2594 (2013).
Fenske, S. et al. cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells. Nat. Commun. 11, 5555 (2020).
Fenske, S. 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).
Hagendorff, A. et al. Conduction disturbances and increased atrial vulnerability in Connexin40-deficient mice analyzed by transesophageal stimulation. Circulation 99, 1508–1515 (1999).
Kaese, S. & Verheule, S. Cardiac electrophysiology in mice: a matter of size. Front. Physiol. 3, 345 (2012).
Verheule, S. et al. Cardiac conduction abnormalities in mice lacking the gap junction protein connexin40. J. Cardiovasc. Electrophysiol. 10, 1380–1389 (1999).
Coates, S. & Thwaites, B. The strength–duration curve and its importance in pacing efficiency: a study of 325 pacing leads in 229 patients. Pacing Clin. Electrophysiol. 23, 1273–1277 (2000).
Clasen, L. et al. A modified approach for programmed electrical stimulation in mice: inducibility of ventricular arrhythmias. PLoS ONE 13, e0201910 (2018).
Reiffel, J. A., Bigger, J. T. Jr. & Konstam, M. A. The relationship between sinoatrial conduction time and sinus cycle length during spontaneous sinus arrhythmia in adults. Circulation 50, 924–934 (1974).
Kugler, J. D., Gillette, P. C., Mullins, C. E. & McNamara, D. G. Sinoatrial conduction in children: an index of sinoatrial node function. Circulation 59, 1266–1276 (1979).
Jalife, J. The sucrose gap preparation as a model of AV nodal transmission: are dual pathways necessary for reciprocation and AV nodal “echoes”? Pacing Clin. Electrophysiol. 6, 1106–1122 (1983).
Rosenblueth, A. The operator of the atrioventricular node. Arch. Inst. Cardiol. Mex. 25, 171–193 (1955).
Paes de Carvalho, A. & de Almeida, D. F. Spread of activity through the atrioventricular node. Circ. Res. 8, 801–809 (1960).
Efimov, I. R. et al. Structure–function relationship in the AV junction. Anat. Rec. A Discov. Mol. Cell Evol. Biol. 280, 952–965 (2004).
Hoffman, B. F., De Carvalho, A. P. & De Mello, W. C. Transmembrane potentials of single fibres of the atrio-ventricular node. Nature 181, 66–67 (1958).
Billette, J. Atrioventricular nodal activation during periodic premature stimulation of the atrium. Am. J. Physiol. 252, H163–H177 (1987).
Katritsis, D. G. & Camm, A. J. Atrioventricular nodal reentrant tachycardia. Circulation 122, 831–840 (2010).
Hoffman, B. F. & Singer, D. H. Effects of digitalis on electrical activity of cardiac fibers. Prog. Cardiovasc. Dis. 7, 226–260 (1964).
Acknowledgements
This work was supported by the German Research Foundation (FE 1929/1-1, FE 1929/2-2, WA 2597/3-1, WA 2597/3-2, BI 484/5-1, BI 484/5-2, project P06 of TRR152).
Author information
Authors and Affiliations
Contributions
K.H. carried out in vivo experiments, data analysis and figure preparation, and wrote the manuscript. R.R. carried out ex vivo experiments, data analysis and figure preparation. J.R. carried out ex vivo experiments and provided veterinary advice. Y.W. carried out ex vivo experiments. S.T. provided images for figure preparation. M.B. wrote parts of the manuscript. S.F carried out in vivo experiments, performed data analysis and composed the figures. C.W.S. and S.F. wrote the manuscript, designed the protocol and provided funding.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Protocols thanks Alicia D’Souza, Roddy Hiram and Na Li for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Related links
Key references using this protocol
Fenske, S. et al. Nat. Commun. 11, 5555 (2020): https://doi.org/10.1038/s41467-020-19304-9
Fenske, S. et al. Circulation 128, 2585–2594 (2013): https://doi.org/10.1161/CIRCULATIONAHA.113.003712
Fenske, S. et al. Nat. Protoc. 11, 61–86 (2016): https://doi.org/10.1038/nprot.2015.139
Extended data
Extended Data Fig. 1 SACT NM.
Stimulation protocol parameters, surface ECG lead I and intracardiac lead RVp are displayed. Schematic ladder diagram (middle) depicts the activation sequence during assessment of SACT with the Narula method. The red areas indicate retrograde and anterograde SACT, respectively. ATR, atrium. Representative recordings are obtained from a 4-month-old male mouse.
Extended Data Fig. 2 Discontinuous AV conduction.
Stimulation protocol parameters, surface ECG lead II and intracardiac leads HRAp, RVd and RVp are displayed during premature atrial stimulation. (Upper) The representative ECG traces show discontinuous AV conduction following a premature beat with a coupling interval of 48 ms. The impulse generated by S2 travels down the slow alpha pathway (long A2V2) and retrogradely excites the beta pathway, producing an atrial echo (Ae). (Lower) Discontinuous AV nodal refractory and recovery curves. At a critical S1S2 interval of 58 ms, an abrupt increase in AV conduction time occurs. Unfortunately, a His deflection was not visible in these recordings. Representative recordings are obtained from a 4-month-old male mouse.
Extended Data Fig. 3 AVNERP.
Stimulation protocol parameters, surface ECG lead II and intracardiac leads HRAp, RVd and RVp are displayed during premature atrial stimulation. The representative ECG traces depict that at a critical S1S2 interval of 42 ms a block of AV conduction occurs. The stimulus S2 induces an atrial signal A2 that is not followed by a ventricular signal. AVNERP is defined as the longest S1S2 interval with loss of AV nodal conduction. Representative recordings are obtained from a 2-month-old male mouse.
Extended Data Fig. 4 WBP and 2:1 conduction.
a, Stimulation protocol parameters to identify the anterograde WBP and 2:1 cycle length. b, Determination of WBP. Surface ECG lead II and intracardiac leads HRAp, RVd and RVp are displayed during atrial stimulation. At a critical S1S1 interval of 54 ms, the first AV block occurs. The sixth stimulus is not conducted from the atria to the ventricles. c, Determination of 2:1 conduction. Surface ECG lead II and intracardiac leads HRAp, RVd and RVp are displayed during atrial stimulation. At a critical S1S1 interval of 48 ms, 2:1 conduction occurs. Every second atrial activation is conducted to the ventricles. For clarity, the timepoints of stimulus application in b and c are connected to the corresponding R peaks in the surface ECG. Representative recordings are obtained from a 2-month-old male mouse.
Extended Data Fig. 5 AERP.
(Upper) Stimulation protocol parameters, surface ECG lead II and intracardiac leads HRAp, RVd and RVp are displayed during atrial stimulation. At a critical S2S3 interval, no atrial response is elicited because the atrial tissue is still refractory. AERP is determined as the first (longest) S2S3 interval with loss of atrial depolarization. (Lower) Representative refractory curve and recovery curve of the atrial myocardium. The curves are determined by premature stimulation using the protocol described in the upper panel. Representative recordings and graphical analysis are obtained from a 4-month-old male mouse.
Extended Data Fig. 6 VERP.
(Upper) Stimulation protocol parameters, surface ECG lead II and intracardiac leads HRAp, RVd and RVp are displayed during ventricular stimulation. At a critical S1S2 interval, no ventricular response is elicited because the ventricular tissue is still refractory. VERP is determined as the first (longest) S1S2 interval with loss of ventricular depolarization. (Lower) Representative refractory curve and recovery curve of the ventricular myocardium. The curves are determined by premature stimulation using the protocol described in the upper panel. Representative recordings are obtained from a 4-month-old male mouse. Representative graphical analysis is derived from a 4-month-old male mouse.
Extended Data Fig. 7 Burst stimulation protocols to test vulnerability to arrhythmia.
a, Stimulation protocol parameters to induce atrial tachycardia. b, Atrial flutter following atrial burst stimulation. Surface ECG lead II and intracardiac leads HRAp and RVp are displayed. Example of sustained atrial tachycardia with a typical saw-tooth pattern of P waves on the surface ECG induced by atrial burst pacing with 100 stimuli with an interval of 10 ms. Atrial flutter can be distinguished from atrial fibrillation, since in atrial fibrillation the atrial rate is so fast that the P waves are no longer identifiable. Regular AV conduction with a 3:1 conduction pattern can best be identified in lead RVp. Atrial and ventricular cycles were regular with a cycle length of 26 ms and 104.3 ± 0.5 ms, respectively. Representative recordings are obtained from a 4-month-old male WT mouse. c, Stimulation protocol parameters to induce ventricular tachycardia. d, Nonsustained ventricular tachycardia (NSVT) following ventricular burst stimulation. Surface ECG lead I and intracardiac leads HRAd and RVp are displayed during ventricular burst stimulation. NSVT was induced by eight burst stimuli with an interval of 32 ms in a WT mouse. This example shows a run of NSVT, which lasted for 1.8 s and was spontaneously terminated and converted to normal sinus rhythm. Representative recordings are obtained from a 2-month-old male mouse.
Extended Data Fig. 8 Ex vivo surface and intracardiac ECG recording under baseline conditions and during VNS.
a, HR tachogram before, during and after VNS. Duration of nerve stimulation is indicated by the yellow line. Basal mean HR and mean HR during VNS are indicated by gray lines. The timepoints, for the representative ECG traces shown in b and c, are marked by blue circles. b,c, Surface ECG lead II and intracardiac leads HRAd, HRAp, RVd, and RVp are displayed during basal conditions (b) and during VNS (c). VNS stimulation artifacts are indicated (yellow). Representative recordings are obtained from a 4-month-old male mouse.
Extended Data Fig. 9 Ex vivo SNRT without and during VNS.
a, Representative HR tachograms derived from surface ECG recordings of a Langendorff-perfused heart before, during and after applying the SNRT stimulation protocol. SNRT recordings were performed without (left) and during VNS (right). Overdrive pacing duration is indicated by a gray line, and duration of VNS is depicted by a yellow line. Blue dashed rectangles represent the areas magnified in b. b, Surface ECG lead II and intracardiac leads HRAp RVd and RVp are displayed from measurements without (1) and during VNS (2). VNS stimulation artifacts are indicated (yellow). SNRT is measured as the interval between the last stimulation spike S1 and the first spontaneous, sinus-node-triggered atrial activation A2 (SNRT = S1A2 interval). c, To calculate cSNRT, subtract the average SCL from SNRT. VNS significantly increases cSNRT values at a pacing cycle of 120 ms. Representative recordings are obtained from a 4-month-old male mouse. Statistical data are obtained from 3–4-month-old male mice.
Extended Data Fig. 10 Ex vivo WBP and 2:1 conduction during VNS.
a, Representative HR tachogram derived from the surface ECG recording during VNS. Blue dashed squares indicate pacing cycles at which the WBP or the first 2:1 conduction occur. The duration of the VNS is depicted by a yellow line. The basal mean HR and mean HR under VNS are presented by gray lines. b, Magnification of the timepoints indicated in a. Data points are shown with circles. WBP is reflected in a shortened/unstable paced HR in the HR tachogram, and 2:1 conduction shows a paced HR reduced by half. c, Duration of S1S1 coupling intervals where the WBP and first 2:1 conduction occurred under basal and VNS condition. d, Detailed surface and intracardiac ECG of the WBP (left) and 2:1 conduction (right) from the recording shown in a. Representative recordings are obtained from a 3-month-old male mouse. Statistical data are obtained from 3–4-month-old male mice.
Supplementary information
Supplementary Information
Supplementary Figs. 1–4.
Supplementary Video1
Video recording of the surgical procedure required for performing in vivo EPS.
Supplementary Data 1
Statistical source data for Table 1.
Supplementary Data 2
Statistical source data for Table 2.
Supplementary Data 3
Statistical source data for Table 3.
Supplementary Data 4
Statistical source data for Table 4.
Source data
Source Data Fig. 3
Representative traces.
Source Data Fig. 7
Representative traces and graphical analysis data.
Source Data Fig. 8
Representative traces.
Source Data Fig. 10
Representative traces and graphical analysis data.
Source Data Extended Data Fig. 1
Representative traces.
Source Data Extended Data Fig. 2
Representative traces and graphical analysis data.
Source Data Extended Data Fig. 3
Representative traces.
Source Data Extended Data Fig. 4
Representative traces.
Source Data Extended Data Fig. 5
Representative traces and graphical analysis data.
Source Data Extended Data Fig. 6
Representative traces and graphical analysis data.
Source Data Extended Data Fig. 7
Representative traces.
Source Data Extended Data Fig. 8
Representative traces.
Source Data Extended Data Fig. 9
Representative traces and statistical source data.
Source Data Extended Data Fig. 10
Representative traces and statistical source data.
Rights and permissions
About this article
Cite this article
Hennis, K., Rötzer, R.D., Rilling, J. et al. In vivo and ex vivo electrophysiological study of the mouse heart to characterize the cardiac conduction system, including atrial and ventricular vulnerability. Nat Protoc 17, 1189–1222 (2022). https://doi.org/10.1038/s41596-021-00678-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41596-021-00678-z
This article is cited by
-
Paradigm shift: new concepts for HCN4 function in cardiac pacemaking
Pflügers Archiv - European Journal of Physiology (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
