Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Summary

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Abstract

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.

The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Introduction

Cardiac diseases are one of the leading causes of death worldwide. Almost all types of cardiac injury result in a significant loss of adult cardiomyocytes (CMs). Adult mammalian hearts are unable to repair their cardiac injury due to the senescent nature of the adult CM¹. Thus, any insult to the adult mammalian heart results in a permanent loss of CMs, leading to reduced cardiac function and heart failure. Unlike adult mammals, small animals like zebrafish and newt hearts can regenerate their cardiac injury through existing CM proliferation^2,3,4. A wor....

Protocol

All experiments should be performed in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health (NIH). All protocols displayed in the video were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati, College of Medicine.

1. Preparation before heart extraction from adult mice (and rats)

1. Prepare the corresponding perfusion, enzyme, and stop solutions, according to the recipes given in Table 1 and Table 2, for rat and mouse isolation, respectively. Filter the solutions thro....

Results

The current modified protocol allows efficient isolation and culture of rat and mice CMs in vitro. For rat CM isolation, a total of 3 adult (12-week-old) male Fischer 344 rats were used in the procedure. Figure 1 shows the surgical apparatus and isolation setups that are required in the procedure; each part has been marked and described in the figure legend. Collagenase type 2 was used for digestion, which yields a high quantity of high quality CMs from successful isolation (

Discussion

There is a critical need to establish a protocol for adult cardiomyocyte isolation and long-term culture to perform cell-specific mechanistic studies. There are only a few reports discussing adult CM isolation protocols, and even fewer of them are used for long-term culture of adult mice CM^15,16,17. It is been shown that the adult rat CM has a higher tolerance to in vitro culture than the adult mice CM¹⁰<.......

Materials

Name	Company	Catalog Number	Comments
2,3-Butane Dione monoxime	Sigma-Aldrich	B-0753
Blebbistatin	APExBIO	B1387
Bovine serum albumin	Sigma-Aldrich	A3059
CaCl2	Sigma-Aldrich	449709
Cell culture plate	Corning Costar	3526
Cell strainer	BD Biosciences	352360
Cel-miR-67	Dharmacon	CN-001000-01-50
Collagenase type2	Worthington	LS004177
Disposable Graduated Transfer Pipettes	Fisherbrand	13-711-20
Disposable polystyrene weighing dishes	Sigma-Aldrich	Z154881-500EA
Dulbecco's Modified Eagle's medium	Thermo Scientific	SH30022.01
EdU	Life Technologies	C10337
Fetal bovine serum	Corning	35-015-CV
Fine Point High Precision Forceps	Fisherbrand	22-327379
Glucose	Sigma-Aldrich	G-5400
Hemocytometer	Hausser Scientific	1483
Heparin	Sagent Pharmaceuticals	PSLAB-018285-02
HEPES	Sigma-Aldrich	H3375
High Precision Straight Broad Strong Point Tweezers/Forceps	Fisherbrand	12-000-128
Hyaluronidase	Sigma	H3506
Insulin	Sigma-Aldrich	I0516-5ML
K2HPO4	Sigma-Aldrich	P-8281
KCl	Sigma-Aldrich	746436
Light Microscope	Nikon
Lipofectamine RNAiMAX	Life Technologies	13778-150
MgSO4	Sigma-Aldrich	M-2643
NaCl	Sigma-Aldrich	S9888
NaOH	Fisher Scientific	S318-500
Natural Mouse Laminin	Invitrogen	23017-015
Penicillin/Streptomycin	Corning	30-002-CI
Pentobarbital	Henry Schein	24352
Phosphate buffered saline	Life Technologies	20012-027
Protease XIV	Sigma-Aldrich	P5147-1G
Selenium	Sigma-Aldrich	229865+5G
siMeis2	Dharmacon	s161030
siRb1	Dharmacon	s128325
Straight Blunt/SharpDissecting Scissors	Fisher Scientific	28252
Straight Very Fine Precision Tip Forceps	Fisherbrand	16-100-120
Taurine	Sigma-Aldrich	T0625
Transferrin	Sigma-Aldrich	T8158-100MG
Ultra-smooth, beveled-edge finish scissor	Fisherbrand	22-079-747
Water Bath	Fisher Scientific	3006S