The Effect of Aging on Calcium Transients in Rat Cardiomyocytes: Impact of NOX Inhibition

Abstract

Cardiac aging is characterized by alterations in contractility and calcium handling. It has been suggested that oxidative stress may be involved in this process. The superoxide generating system NADPH oxidase (NOX) is expressed in the heart (NOX2 and 4). We and others have reported that in cardiac failure, the NOX-derived superoxide is increased, with a negative impact on calcium and contractility. We tested the hypothesis that calcium transients and contractility in aged rat cardiomyocytes are disturbed by NOX.Cardiomyocytes were obtained form adult (5 months-old) and aged (20 months-old) Sprague-Dawley rats. Aged myocytes were treated with apocynin (50 μmol/L). Cells were field-stimulated from 0.5 to 4 Hz. Contractility was evaluated as sarcomere shortening and [Ca2+]i was monitored with fura-2.Sarcomere shortening was similar in adult and aged myocytes. [Ca2+]i transients amplitude was increased in aged cardiomyocytes (p<0.005) and was further increased by apocynin treatment. Time-50 to peak [Ca2+]i was increased in aged myocytes (p<0.05), suggesting impairment in the ryanodine receptor RyR2, and was improved by apocynin treatment. Time-50 to maximal relaxation was increased in aged myocytes (p<0.05) and reduced towards normal by NOX inhibition.Using thapsigargin to block the sarcoplasmic reticulum calcium pump SERCA2, we submitted myocytes to tetanic stimulation to evaluate the myofilaments Ca2+ sensitivity. By comparing the amplitude of the tetanic contraction achieved, with the level of [Ca2+]i evoked, we found that myofilaments Ca2+ sensitivity was reduced in aged myoctes (p<0.05).In conclusion, contractility was preserved in aged myocytes, but at a higher [Ca2+]i level, as a result of diminished myofilaments Ca2+ sensitivity. NOX inhibition increased [Ca2+]i transients amplitude and improved Ca2+ kinetics, without changes in contractility. These results suggest a NOX-dependent effect in aged myocytes at the level of RyR2 SERCA2 and myofilaments.