Formation of reactive oxygen species at increased contraction frequency in rat cardiomyocytes

Objective Reactive oxygen species (ROS) play an ambivalent role in cardiomyocytes: low concentrations are involved in cellular signaling, while higher concentrations contribute to cellular injury. We studied ROS formation during increases in contraction frequency in isolated cardiomyocytes. Methods Rat ventricular cardiomyocytes were loaded with dichlorodihydrofluorescein and electrically stimulated (37 °C). ROS formation was assessed by the rate of oxidation-dependent fluorescence increase (OxR). Oxygen consumption (VO2) and NAD(P)H autofluorescence were measured in parallel experiments. Results Increases in contraction frequency were accompanied by an increase in VO2 and a decrease in NAD(P)H fluorescence. OxR increased to 124±4%, 146±8%, 204±25% and 256±29% of OxR at baseline during 1, 2, 3 and 4 Hz stimulation, and subsequently returned to baseline values with 0.2 Hz. The OxR increase was dose-dependently inhibited by the antioxidant NAC (10 and 100 mM), but unaffected by the NO synthase inhibitor l-NAME (200 μM and 10 mM). The OxR increase was attenuated when myosin ATPase activity was inhibited by butanedione monoxime (BDM; 5 mM). Conclusion Increased contraction frequency induces ROS formation in rat cardiomyocytes.