Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Summary

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Abstract

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes’ terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Introduction

Cardiomyocytes are not proliferative. There are some atrial cardiomyocyte cell lines, like HL-1 and AT-1 cells derived from mouse atrial tumors; however, there are no adult ventricular cardiomyocyte cell lines available for research. Primary cell cultures of adult mouse cardiomyocytes provide a powerful model for heart research at the cellular and molecular levels. To date, they have been used extensively for biochemical, physiological, and pharmacological research¹. Additionally, the frequent use of genetically modified mice has necessitated effective methods of cardiomyocyte isolation. Pure culture allows for conditions free from interaction with other or....

Protocol

All research on mice was done according to procedures and guidelines of the National Institutes of Health, and the protocols were approved by the Institutional Animal Care and Use Committee of the University of Toledo, College of Medicine and Life Sciences.

1. Perfusion System Preparation

1. The day before isolation, fill the entire perfusion system (including reservoirs) with a solution of fast alkaline residue-free detergent. Allow the solution to soak into the system for a few hours or overnight.
2. The next morning, adjust the thermocycler temperature to 37 °C and allow the solution to warm up. Remove the deter....

Results

1. Successful Isolation Quantification

Two criteria are used to quantify the success of the isolation: first, the total number of cardiomyocytes isolated, and second, the ratio of rod-shaped calcium-tolerant to round non-tolerant myocytes. Generally this protocol takes around 75-90 min from heart removal to myocyte plating and yields around 1 million rod-shaped cardiomyocytes (70-90% of total myocytes harvested) from one adult mouse heart. This may vary with mouse body weight and strain. Typicall.......

Discussion

For the best preparation, the most critical steps are: 1) promptly hooking up the mouse heart to the cannula after excision; 2) appropriate heart perfusion. Also, note that water quality, perfusion temperature, pH of buffer, sterilization, chemical purity, and clean, non-contaminated tubing and chambers are also important factors. 18.2 mΩ molecular biology-grade H₂O is highly recommended for buffer preparation. The pH of 200 mM ATP stock solution should be adjusted to 7.2, a step which is easily missed.

It is e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isolated heart system for small rodent	Harvard Apparatus	IHSR-mouse 73-4019
Aortic Cannula, OD 1.0 mm	Harvard Apparatus	73-2816
Dumont #4 Forceps	Fine science tools	11294-00
Straight sharp serrated scissors	Fine science tools	14070-12
Serrated Graefe forceps	Fine science tools	11050-10
Curved, serrated Graefe forceps	Fine science tools	11052-10
Straight Mini Serrefines clamps	Fine science tools	18054-28
MEM	Gibco	11575-032
Collagenase II	Worthington	4176
Mucosal universal detergent	Sigma	Z637181	Mucasol is a fast alkaline residue-free detergent.