Optical Imaging of Isolated Murine Ventricular Myocytes

J Vis Exp. 2020 Jan 17;(155). doi: 10.3791/60196.

Abstract

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

Publication types

  • Video-Audio Media

MeSH terms

  • 4-Aminopyridine / pharmacology
  • Action Potentials / physiology
  • Animals
  • Calcium / metabolism
  • Cell Separation / methods*
  • Electricity
  • Fluorescent Dyes / chemistry
  • Heart Ventricles / cytology*
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Myocytes, Cardiac / cytology*
  • Myocytes, Cardiac / drug effects
  • Optical Imaging*
  • Pressure
  • Rats, Sprague-Dawley
  • Sarcomeres / metabolism

Substances

  • Fluorescent Dyes
  • 4-Aminopyridine
  • Calcium