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Channelrhodopsins for Cell-Type Specific Illumination of Cardiac Electrophysiology

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 2191)

Abstract

Optogenetic approaches have evolved as potent means to investigate cardiac electrophysiology, with research ranging from the study of arrhythmia mechanisms to effects of cardiac innervation and heterocellular structural and functional interactions, both in healthy and diseased myocardium. Most commonly, these studies use channelrhodopsin-2 (ChR2)-expressing murine models that enable light-activated depolarization of the target cell population. However, each newly generated mouse line requires thorough characterization, as cell-type specific ChR2 expression cannot be taken for granted, and the electrophysiological response of its activation in the target cell should be evaluated. In this chapter, we describe detailed protocols for assessing ChR2 specificity using immunohistochemistry, isolation of specific cell populations to analyze electrophysiological effects of ChR2 activation with the patch-clamp technique, and whole-heart experiments to assess in situ effects of optical stimulation.

Key words

Cardiac optogenetics Transgenic mice Cell-type specific gene targeting Electrophysiology Electrocardiogram Optical pacing Cardiomyocytes Fibroblasts Macrophages 

Abbreviations

AP

Action potential

BSA

Bovine serum albumin

ChR2

Channelrhodopsin-2

Cx3Cr1

Fractalkine receptor

DMEM

Dulbecco’s Modified Eagle Medium

ECG

Electrocardiogram

eYFP

Enhanced yellow fluorescent protein

FACS

Fluorescence-activated cell sorting

FCS

Fetal calf serum

LED

Light-emitting diode

OCT

Optimal cutting temperature compound

PBS

Phosphate-buffered saline

PCR

Polymerase chain reaction

PFA

Paraformaldehyde

RT

Room temperature

Tcf21

Transcription factor 21

TTL

Transistor-transistor logic

αMHC

α-Myosin heavy chain

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Notes

Acknowledgments

We thank Prof Dr. Ernst Niggli and Ruben Jose Lopez Dicuru (University of Bern) for providing their protocol for cardiomyocyte isolation. We thank Stefanie Perez-Feliz, Cinthia Buchmann, Pia Iaconianni, and Max Giese for excellent technical assistance. We acknowledge the Lighthouse Core Facility of the Medical Center – University of Freiburg for access to and technical support with FACS. Confocal imaging was performed at the SCI-MED facility (Super-Resolution Confocal/Multiphoton Imaging for Multiparametric Experimental Designs) at the Institute for Experimental Cardiovascular Medicine. This work was funded by the German Research Foundation (SPP1926, FS1486/1-1 and an Emmy-Noether fellowship, FS1486/2-1). Marbely C. Fernández, Ramona A. Kopton, and Ana Simon-Chica contributed equally to this work.

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© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg—Bad Krozingen, Medical Center—University of FreiburgFreiburgGermany
  2. 2.Faculty of MedicineUniversity of FreiburgFreiburgGermany
  3. 3.Faculty of BiologyUniversity of FreiburgFreiburgGermany
  4. 4.Department of Cardiology IUniversity Heart Center Freiburg—Bad KrozingenFreiburgGermany

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