Cell Death in Cultured Adult Rat Cardiomyocytes: Use of the Comet Assay to Distinguish Apoptosis from Necrosis

Apoptosis is a form of programmed cell death that has only recently been implicated in the pathogenesis of cardiac diseases, including ischemia and reperfusion damage and in response to cytokines and other apoptotic triggers. Methods to study apoptosis range from measures of early changes in morphology to late events such as DNA laddering. In studying apoptosis in the heart, the application of some of these techniques is limited due to low sensitivity of detection and difficulties in quantification, especially when working with adherent cell cultures of cardiomyocytes. We present here results on the modification of the single cell gel electrophoresis or comet assay in order to assess apoptosis of adult cardiomyocytes in primary cell cultures. The comet assay quantifies DNA fragmentation in individual cell nuclei embedded in agarose and then electrophoresed. We present data on TNFα and sphingosine-induced apoptosis in adult rat cardiomyocytes as assessed by the comet assay. By using a combination of the alkaline, neutral and subcellular versions of the comet assay, we were able to accurately differentiate apoptosis from necrosis in cardiomyocytes. We conclude that the comet assay is a sensitive and useful method for the detection of apoptosis in cardiomyocytes.