We analyzed contraction (sarcomere shortening), Ca2+ transients (Indo-1) and ROS formation (DFF fluorescence) of ventricular myocytes from young (2 mo.) and aged (24 mo.) mice.

160 ms voltage clamp pulses (-80 to 0 mV; 0.5–4 Hz; 37UC) induced Ca2+ stress in both young and aged cells as indicated by aftercontractions. Fractional shortening was similar in both groups. Time to peak shortening and time to 70% relengthening were prolonged in aged cells but were normalized by tiron (O2- scavenger) and apocynin (NADPH oxidase inhibitor). Similarely, Ca2+ transients showed a decelerated decline in aged cells that was normalized by apocynin. Compared to non-paced cells, pacing (1 Hz) increased ROS formation; an effect that was augmented in aged cells. Apocynin partially reduced the effect of pacing on ROS formation. Chelation of intracellular free Ca2+ (BAPTA) completely attenuated the DFF fluorescence increase. Analysis of NADPH oxidase subunit mRNA showed increased expression of NOX4, NOX2 and p47phox in aged hearts, whereas expression of p22phox and rac1 remained unchanged.

We conclude that slowing of Ca2+ transients and contractions in aged ventricular myocytes results from an increased ROS formation due to an increased expression of NADPH oxidases that are activated in response to Ca2+ stress.