Detection of acetylcholine in transmitter vesicles from mouse ventricular cardiomyocytes. (A) Left, western blot confirmed that the vesicles within mouse ventricular cardiomyocytes expressed the vesicular acetylcholine transporters. Right, the bar graph summarizes the expression levels of vesicular transmitter transporters. *P < .05, calculated by one-way ANOVA; n = 3 per group. (B) Effect of the proton ionophore FCCP on the transmitter content in the extracted transmitter vesicles. *P < .05, calculated by Student’s t-test; n = 6–10 per group. (C, D) Immunogold electron microscopy demonstrated vesicle-like structures (arrows) with vesicular acetylcholine transporter immunoreactivity in ventricular cardiomyocytes from both humans and mice. The triangles indicate gap junctions. The diameter of each gold particle was 12 nm. (E, F) Immunofluorescence staining of vesicle-associated membrane protein 2 and vesicular acetylcholine transporter in human and mouse ventricular myocardial sections. Scale bar, 50 μm. Veh, vehicle