Published: September 24th, 2013
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.
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.
Murine models of cardiovascular disease have served as effective tools for elucidating fundamental disease mechanisms1,2 as well as for identifying potential therapeutic targets1,3. 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 disease6-8. The use of cell culture techniques to study signaling cascades3,9,10 and alterations in individual proteins that underlie cellular excitability and excitation-contraction coupling in the heart11-13 at the level of the single cell have comp....
1. Cardiomyocyte Isolation
Materials (Figure 1)
Delicate 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
Wax coated, braided silk 4-0, 1.......
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 .......
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 .......
|Name of Reagent/Material
|Sodium Phosphate Monobasic
|Roche Applied Science
|Roche Applied Science
|Protease XIV from Streptomyces griseus
|Albumin from Bovine Serum
|Minimum Essential Media
|Albumin solution from bovine serum
|Insulin-transferrin-sodium selenite media supplement
|Adenosine 5'-triphosphate magnesium salt
|Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid
|Cesium Hydroxide Solution
|Tetraethylammonium hydroxide solution
|OptiVisor optical glass binocular visor
|Dohegan Optical Company Inc.
|Tissue forceps, 5.5", 1x2 teeth
|Moloney forceps - 4.5" (11.5 cm) long slight curve, serrated
|Dumont #3 Forceps, Dumostar, tip size 0.17 x 0.10mm
|Packer Mosquito Forceps 5" Straight Flat
|Micro Dissecting Scissors 4.5" Curved Sharp/Sharp
|Micro Dissecting Scissors 3.5" Straight Sharp/Sharp20mm
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