Isolation of Adult Mouse Cardiac Myocytes

For the isolation of adult mouse cardiac myocytes, we developed a protocol based on established procedures in rat and rabbit, and we recently expanded the protocol to include mouse(14,15,16). Briefly, this protocol entails removing the heart, cannulating the aorta on a perfusion system, arresting the heart with a retrograde perfusion of calcium-free buffer, dissociating myocytes using a collagenase-based enzymatic solution, and reintroducing calcium to produce isolated, quiescent, rod-shaped myocytes (Table 1). Using this protocol, we routinely obtained 1.5 to 1.7 million myocytes per heart, of which 65% to 74% were rod-shaped (Table 2). The total myocyte yields were approximately two- to three-fold higher than previously published reports for the isolation of mouse myocytes(14,15,16). Similar results were obtained in both the Laboratory for the Development of Signaling Assays (LDSA) and the Cell Preparation and Analysis Laboratory (CPAL) (Table 2), demonstrating that the procedure was transferable between laboratories. Further, myocytes were isolated from multiple hearts (up to four), permitting the isolation of up to six million myocytes for a single experiment.

We identified several critical factors in the protocol that improved myocyte isolation (summarized in Table 1). First, using isoflurane, an inhalation anesthetic, significantly improved myocyte yields compared to using a combination of ketamine and xylazine. Anesthetics like ketamine reduce respiratory rate and have a long onset period. This increases the risk of ischemia due to respiratory depression, which can reduce myocyte yields. Second, including 2, 3-butanedione monoxime, a contractile inhibitor, in the perfusion buffer improved yields by preventing myocyte contracture during the isolation procedure. Third, perfusion buffer pH was maintained with HEPES, and, contrary to expectations, we found no beneficial effect on myocyte yield of oxygenating the perfusion buffer. Fourth, liberase blendzyme, a recombinant enzyme mix containing collagenase and other proteases (Roche Molecular Biochemicals, Indianapolis, IN), supplemented with trypsin was used to digest hearts. Crude collagenases are the conventional choice of enzyme, and we had very good results with different collagenase preparations (collagenase type II, Worthington Biochemicals, Lakewood, NJ, and a combination of collagenases B and D, Roche). However, the defined blendzyme mix reduced variation between preparations of collagenase, improving standardization between laboratories. Fifth, constant flow perfusion (3 ml/min) was used during the myocyte isolation rather than constant pressure. The constant pressure system (70 mm Hg) was satisfactory, but flow rates varied because coronary resistance typically increased at the onset of perfusion with digestion buffer. This increase in vascular resistance varied from heart to heart, and the consequent inconsistency in flow rates caused variations in the enzymatic digestion of the heart. Finally, calcium was reintroduced at room temperature, rather than 37 °C, which reduced spontaneous myocyte contracture. Calcium reintroduction was performed in a 60-mm sterile bacterial dish, which allowed visualization of the myocytes and helped reduce clumping that might occur if the myocytes were left in a tube.