Abstract
Diphenyleneiodonium (DPI) has been widely used as an inhibitor of NADPH oxidase (Nox) to discover its function in cardiac myocytes under various stimuli. However, the effects of DPI itself on Ca(2+) signaling and contraction in cardiac myocytes under control conditions have not been understood. We investigated the effects of DPI on contraction and Ca(2+) signaling and their underlying mechanisms using video edge detection, confocal imaging, and whole-cell patch clamp technique in isolated rat cardiac myocytes. Application of DPI suppressed cell shortenings in a concentration-dependent manner (IC(50) of ≅0.17 µM) with a maximal inhibition of ~70% at ~100 µM. DPI decreased the magnitude of Ca(2+) transient and sarcoplasmic reticulum Ca(2+) content by 20%-30% at 3 µM that is usually used to remove the Nox activity, with no effect on fractional release. There was no significant change in the half-decay time of Ca(2+) transients by DPI. The L-type Ca(2+) current (ICa) was decreased concentration-dependently by DPI (IC(50) of ≅40.3 µM) with ≅13.1%-inhibition at 3 µM. The frequency of Ca(2+) sparks was reduced by 3 µM DPI (by ~25%), which was resistant to a brief removal of external Ca(2+) and Na(+). Mitochondrial superoxide level was reduced by DPI at 3-100 µM. Our data suggest that DPI may suppress L-type Ca(2+) channel and RyR, thereby attenuating Ca(2+)-induced Ca(2+) release and contractility in cardiac myocytes, and that such DPI effects may be related to mitochondrial metabolic suppression.