ABSTRACT

In this study, it was aimed to investigate the regulatory effect of N-acetylcysteine (NAC), an antioxidant, on the aging-related reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) in cardiomyocytes via exosomes released from cardiomyocytes at the molecular level.

The aging model was performed by incubating rat left ventricular cell line H9c2 with D-galactose (D-Gal; 50 mg/mL) for 48 hours. Exosomes released from H9c2 pretreated with NAC were incubated with aged cardiomyocytes and their effects on the enhanced ROS production and MMP depolarization with aging were compared with both normal control cells and aged cells without exosomes. ROS and MMP measurements were calculated relative to the confocal microscope using specific fluorescent dyes (DCFDA and FCCP) by visualizing the change in fluorescence intensity. MiR-21 expression, which is also accepted as a ROS marker, and cDNA synthesis qRT-PCR measurements were performed following exosomal miRNA isolation.

Seventy-two-hour incubation of aging-modeled cardiomyocytes with exosomes isolated from H9c2 incubated with NAC significantly suppresses the increased ROS production associated with aging and normalizes depolarized MMP. On the other hand, NAC treatment significantly suppressed exosomal miR-21 expression in normal healthy cardiomyocytes.

These findings indicate that ROS-targeted agents used in the treatment of heart damage may specifically suppress mitochondrion-derived ROS production, which increases uncontrollably through an exosome-based paracrine mechanism. Our study also demonstrates, for the first time in the literature, that NAC induces exosomal miRNA modulation in pretreated cardiomyocytes. These findings indicate that the administration of exosomes obtained from cardiomyocytes with enhanced antioxidant system is a new treatment approach for aging-related inadequate heart function.

Keywords: Aging, Cardiomyocyte, Exosome, Antioxidant, N-acetylcysteine, miR-21