Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Summary

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (V_m) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Abstract

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

Introduction

A unique calcium (Ca) and voltage (V_m) dual channel optical mapping technique^1-5 is emerging as an efficient tool to simultaneously record V_m and Ca signals in both intact hearts and cultured cell monolayers. This technique makes it possible to obtain powerful information regarding the relationship between calcium transients and action potentials to better understand the underlying electrophysiological mechanisms of cardiac arrhythmias.

Cultured cell monolayers have proven to be a useful cellular model to study cardiac electrophysiology and the underlying mechanism of arrhythmias.^4,6-8 HL-1 cells ....

Protocol

1. Solution Preparation

1. Norepinephrine (NP) solution
   1. Dissolve 40 mg of NP into 25 ml of 30 mM ascorbic acid (0.1475 g ascorbic acid in 25 ml of purified Milli-Q (MQ) water. Avoid direct light exposure during this solution preparation because norepinephrine (NP) is light sensitive.
   2. Filter the NP solution with a 0.22 µm syringe filter. Aliquot the solution into 1 ml sterile microtubes and store at -20 °C. Once frozen, NP is stable for one month.
2. Supplemented Wash and Final Claycomb media
   1. Start with 43.5 ml of Claycomb medium and supplement with 5 ml of fetal bovine serum (FBS), 0.5 ml of penicillin/s....

Results

A cultured confluent monolayer exhibits a regular intrinsic rhythm as demonstrated in Movie 1. We then performed V_m/Ca dual channel optical mapping in a fully confluent HL-1 monolayer. Figure 1A shows example traces of V_m and Ca signals from a recorded single beat. Representative isochronal maps of uniformly propagated V_m and Ca signals using the dual channel optical mapping system are shown in Figures 1B and 1C. Represen.......

Discussion

This article describes the key aspects of optical mapping in a cultured HL-1 atrial myocyte monolayer stained with calcium and voltage sensitive fluorescent dyes. It includes culturing an optimal HL-1 cell monolayer, setup of the mapping equipment, mapping a cultured monolayer, and data analysis.

To successfully map cultured cells, the key is to prepare a uniformly distributed cell monolayer. When seeding the cells on to the coverslips, be sure to evenly disperse the cells. Always map fully-gr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rhod2 dry powder	AAT Bioquest	21062
pluronic	AAT Bioquest	20050
DMSO	Sigma	276855
Rh237	Invitrogen	S-1109
NaCl	Sigma	S7653
KCl	Sigma	P3911
KH2PO4	Sigma	P0662
NaH2PO4	Sigma	S9638
MgSO4	Sigma	M7506
D-Glucose	Sigma	G8270
NaHCO3	Sigma	S6014
CaCl2	Sigma	C3881
HEPEs	Sigma	H3375