Isolated adult cardiomyocytes maintained in primary culture have been used as an adult cardiomyocyte model for more than 30 years. With recent progress and the current increasing interest in the use of molecular biology techniques to study cardiac physiology, culturing muscle cells is becoming an increasingly important technology.
Animal models have been used as tools to expand our knowledge of cardiac physiology and disease. Although these models were originally developed for the study of intact heart surgery and pharmacological interventions, later advances have led to the development of intact muscle preparations, allowing more direct experimental manipulation of muscles. However, these preparations do not allow easy access to the muscles for treatment or recording. In addition, the large size of these preparations hinders the electrophysiological examination of intact myocardium, which makes it impossible to adequately control the membrane potential during the voltage clamp experiment. This limitation is the main driving force for the development of single cardiomyocyte separation technology.
Using isolated cardiomyocytes has many additional advantages, such as being able to select cells from different areas of the heart (including the atrium, left and right ventricles, conduction system, or specific areas of the heart after myocardial infarction). Similarly, although imaging techniques are usually limited to thick tissues, isolated cells are well suited for experiments aimed at visualizing cell structure and precise positioning of molecules within the cell. Isolated cardiomyocytes are also often used to examine intracellular calcium ions. Although imaging techniques are usually limited to thick tissues, isolated cells are very suitable for experiments aimed at visualizing cell structure and precise positioning of intracellular molecules. Isolated cardiomyocytes are also often used to examine intracellular calcium ions. Although imaging techniques are usually limited to thick tissues, isolated cells are very suitable for experiments aimed at visualizing cell structure and precise positioning of intracellular molecules.
Isolation of high-quality cardiomyocytes may be the most important factor for successful experiments with fresh or cultured cells. However, even though cardiomyocyte isolation has been carried out for nearly 40 years, there is still no single universal method that can easily produce a large number of high-quality living cells without some adjustments. The protocols used by different laboratories are different, and may depend not only on the species of the isolated cells, but also on the type of experiment planned.
In recent years, various gene transfer techniques have been increasingly used in the study of isolated cardiomyocytes. These technologies have been successfully used to study the mechanisms that regulate the structure and function of cardiomyocytes. Targeted genes include genes related to cardiomyocyte Ca2+ homeostasis, contractile devices, cytoskeleton, and signaling pathways. Successful gene transfer requires the DNA encoding the target protein to reach the nucleus of the host cardiomyocyte. This can be done by transfection, which is a process in which DNA is administered in the form of a plasmid (so-called “naked DNA”). Alternatively, the DNA can be integrated into the genome of the virus and then inserted into the host cell through a process called transduction.
Cardiomyocytes isolated from small rodents are valuable tools for studying cell-level functions and electrophysiological regulation, intracellular calcium flux, contractile mechanics, and protein expression. The ability to cultivate muscle cells, even for short periods of time, further benefits researchers by allowing transgenes to be incorporated into cells and facilitating long-term treatment. In conjunction with the recent availability of transgenic animals, a series of techniques for manipulating cell physiology in the study of molecular mechanisms regulating function have been greatly expanded.
Although standard techniques have been used to isolate and culture muscle cells for many years, it is still an art to consistently produce high-quality cells. Understanding the described reagents and protocols, as well as common precautions and key points in the procedures, will enable researchers to more easily establish cell-based research strategies and promote the pursuit of human disease therapies.
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