Methods in cardiomyocyte isolation, culture, and gene transfer

Abstract

Since techniques for cardiomyocyte isolation were first developed 35 years ago, experiments on single myocytes have yielded great insight into their cellular and sub-cellular physiology. These studies have employed a broad range of techniques including electrophysiology, calcium imaging, cell mechanics, immunohistochemistry and protein biochemistry. More recently, techniques for cardiomyocyte culture have gained additional importance with the advent of gene transfer technology. While such studies require a high quality cardiomyocyte population, successful cell isolation and maintenance during culture remain challenging. In this review, we describe methods for the isolation of adult and neonatal ventricular myocytes from rat and mouse heart. This discussion outlines general principles for the beginner, but also provides detailed specific protocols and advice for common caveats. We additionally review methods for short-term myocyte culture, with particular attention given to the importance of substrate and media selection, and describe time-dependent alterations in myocyte physiology that should be anticipated. Gene transfer techniques for neonatal and adult cardiomyocytes are also reviewed, including methods for transfection (liposome, electroporation) and viral-based gene delivery.

Figure 1. Apparatus for ventricular cardiomyocyte isolation

Myocytes can be reliably isolated using a Langendorff setup, with cannulation of the aorta and retrograde perfusion of the heart with enzyme-containing solutions. One isolation method is to use constant pressure perfusion, where solutions are suspended above the apparatus and gravity fed to the heart. Alternatively, constant flow perfusion can be employed by pumping the perfusate (shown at right, connection by dotted lines). By both methods, the perfusate may be heated, often to 37°C, by inclusion of a heating coil connected to a water bath (shown at left). Inclusion of a drip chamber can be included to prevent air bubbles from reaching the heart. The heart is typically fixed to the cannula with a silk suture.

Figure 2. Method for cannulation of the aorta and perfusion of the coronary arteries

A) The heart and tip of the cannula are immersed in low-Ca²⁺ solution. Using fine-tipped forceps to grip the sides of the aorta, the heart is then cannulated and secured by silk thread tied around the aorta. B) Following proper cannulation, the aortic valve closes when perfusion is initiated and the perfusate is forced through the coronary arteries. Placing the cannula too deep in the aorta and through the aortic valve will prevent adequate perfusion of the coronaries and cardiomyocyte isolation will not be successful.

Figure 3. Typical alterations in cardiomyocyte morphology during cell culture

(A–C) Light images (left panels) show that myocytes become rounded at the ends with time in culture. T-tubule density is also rapidly and progressively reduced as indicated by confocal cross-sectional images of di-8-ANEPPS stained cells (A–C, right panels) and mean density measurements (D). T-tubule loss results in a reduction in cell surface area, reflected as a measured decrease in cell capacitance (E). * denotes P<0.05 vs. control, # denotes P<0.05 vs 24 hr culture. Scale bar = 10 μm. Reprinted from [65] by permission.