Isolation and study of cardiac nuclei from canine myocardium and adult ventricular myocytes.

The nuclear envelope encloses the genome as well as the molecular machinery responsible for both the replication and transcription of DNA as well as the maturation of nascent RNA. Recent studies ascribe a growing number of functions to the nuclear membrane, in addition to sequestering the DNA, through receptors and their effectors, ion channels, as well as ion pumps and transporters located within the nuclear membrane itself. Despite the obvious structural and functional importance of the nucleus, certain aspects remain poorly understood due to the challenges associated with its accessibility in vivo, as well as isolating nuclei intact and with sufficient purity from cardiac cells to permit studies in vitro. Here, we present a detailed protocol for isolation of intact nuclei from both myocardial tissue and freshly isolated adult ventricular cardiomyocytes. These methods are based on partial permeabilization of plasma membrane with digitonin and cell disruption, followed by differential and discontinuous sucrose density centrifugation. These preparations provide for rapid separation of nonnuclear membranes and cytosol from nuclei.