Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Summary

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

Abstract

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca²⁺ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Introduction

Murine models of cardiovascular disease have served as effective tools for elucidating fundamental disease mechanisms^1,2 as well as for identifying potential therapeutic targets^1,3. In particular, the use of both murine models of acquired heart disease (such as pressure-overload)^4,5 and transgenic mouse models have advanced our understanding of heart disease^6-8. The use of cell culture techniques to study signaling cascades^3,9,10 and alterations in individual proteins that underlie cellular excitability and excitation-contraction coupling in the heart^11-13 at the level of the single cell have comp....

Protocol

1. Cardiomyocyte Isolation

Materials (Figure 1)

Microdissecting forceps
Tissue forceps
Delicate hemostatic forceps
Hemostatic forceps
Microdissecting, serrated, curved forceps
Operating scissors, straight
Operating scissors, curved
15 ml Falcon tubes (5)
60 mm Petri dish
Phosphate Buffered Saline (PBS)
Nylon Mesh - 400 μm pore size
Small funnel
Wax coated, braided silk 4-0, 19 mm, 7-10 cm long
Non-hypodermic needle, blunt end, 24 G or commercial animal-feeding needle (24 x 1 in, W/1-1/4)
Heparin sodium
Ke....

Results

The isolation of adult cardiomyoctyes results in rod-shaped, striated, and quiescent (not spontaneously beating) cells (Figure 5A). Dead cells will look rounded and no striations will be present. Quiescent cells can be cultured and transfected with adenovirus to manipulate gene expression (Figures 5B and 5C). After 24 hr of culture, the morphology of the live cells does not change, they are still Ca²⁺-tolerant, and they can be paced by field stimulation. With .......

Discussion

In this report, we have described the techniques necessary for successful isolation and culture of adult CMs from the mouse heart. Our technique allows for subsequent study of CM function and excitability using the methods described above. The critical parameter for studying functionality of adult CMs is the health and quality of the isolated CMs. As described above, our techniques allow for a high yield of functional cells that are amenable to manipulation of gene expression using adenoviral/lentiviral infections in .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium Chloride	Sigma	S7653
Potassium Chloride	Sigma	P9333
Magnesium Chloride	Sigma	M8266
HEPES	Sigma	H3375
Sodium Phosphate Monobasic	Sigma	S8282
D-glucose minimum	Sigma	G8270
Taurine	Sigma	T0625
2,3-Butanedione monoxime	Sigma	B0752
Collagenase B	Roche Applied Science	11088807001
Collagenase D	Roche Applied Science	11088858001
Protease XIV from Streptomyces griseus	Sigma	P5147
Albumin from Bovine Serum	Sigma	A2153
Calcium Chloride	Sigma	C8106
Minimum Essential Media	Sigma	51411C
Albumin solution from bovine serum	Sigma	A8412
L-glutamine	Sigma	G3126
Penicillin-Streptomycin	Sigma	P4333
Insulin-transferrin-sodium selenite media supplement	Sigma	I1884
Cesium Chloride	Sigma	289329
Glutamate	Sigma	G3291
Adenosine 5'-triphosphate magnesium salt	Sigma	A9187
Ethylene glycol-bis(2-amin–thylether)-N,N,N',N'-tetraacetic acid	Sigma	E3889
Cesium Hydroxide Solution	Sigma	232041
Tetraethylammonium hydroxide solution	Sigma	86643
OptiVisor optical glass binocular visor	Dohegan Optical Company Inc.	N/A
Tissue forceps, 5.5", 1x2 teeth	Roboz Scientific	RS-8164
Moloney forceps - 4.5" (11.5 cm) long slight curve, serrated	Roboz Scientific	RS-8254
Dumont #3 Forceps, Dumostar, tip size 0.17 x 0.10mm	Roboz Scientific	RS-4966
Packer Mosquito Forceps 5" Straight Flat	Roboz Scientific	RS-7114
Micro Dissecting Scissors 4.5" Curved Sharp/Sharp	Roboz Scientific	RS-5917
Micro Dissecting Scissors 3.5" Straight Sharp/Sharp20mm	Roboz Scientific	RS-5907