Cardiac apoptosis through TUNEL and caspase-3 assay and MMP-9 expression

Cardiac sections measuring 6 μm were examined for the presence of apoptotic cardiomyocytes by TdT-mediated dUTP nick-end labelling (TUNEL) assay using a Promega Dead End™ colorimetric TUNEL system (Promega, Madison, WI, USA) with a streptavidin-peroxidase system. Controls were obtained by omitting the TdT enzyme from the reaction mixture. The percentage of TUNEL-positive myocytes was determined by counting 10 random fields per section under a microscope (Nikon NIS Elements). Using this procedure, apoptotic nuclei were stained dark brown. Labelled nuclei were counted and expressed as the percentage of positively stained cells. The Caspase-3 activity in heart tissue was measured using a Caspase-3 colorimetric assay kit (Clontech, USA) according to the manufacturer’s instructions (results were reported as ng of Caspase-3/g of tissue). MMP-9 were associated with collagen maturation in heart failure, demonstrating the important role of these enzymes in fibrosis through collagen configuration, activation, and deposition. Therefore, we quantified MMP-9 in heart tissues through Mouse MMP9 ELISA Kit (ab253227, AbCam, Milan, Italy) following the manufacturer’s instructions; results are expressed as pg of MMP-9/mg of protein (determined through Bradford assay).