Previous studies showed an abundance of functional Connexin43 (Cx43) hemichannels consequent to loss of plakophilin-2 (PKP2) expression in adult murine hearts. This study combined pre-embedding immunolabeling with serial block-face scanning electron microscopy (SBF-SEM) to visualize and examine the gap junction plaque morphology. They used a murine model of cardiomyocyte-specific Tamoxifen (TAM) activated knockout of PKP2. For this study, adult murine hearts were harvested 14 days post-TAM, and thick vibratome slices were immunolabelled for Cx43 and treated with nanogold. Left or right ventricular free walls were dissected and three-dimensional localization of Cx43 in cardiac muscle was performed using SBF-SEM. This dataset contains electron microscopy data.