Chrysosplenol-C Increases Contraction by Augmentation of Sarcoplasmic Reticulum Ca2+ Loading and Release via Protein Kinase C in Rat Ventricular Myocytes.

Naturally found chrysosplenol-C (4',5,6-trihydroxy-3,3',7-trimethoxyflavone) increases the contractility of cardiac myocytes independent of ß-adrenergic signaling. We investigated the cellular mechanism for chrysosplenol-C-induced positive inotropy. Global and local Ca2+ signals, L-type Ca2+ current (ICa), and contraction were measured from adult rat ventricular myocytes using two-dimensional confocal Ca2+ imaging, the whole-cell patch-clamp technique, and video-edge detection, respectively. Application of chrysosplenol-C reversibly increased Ca2+ transient magnitude with a maximal increase of ∼55% within 2- to 3-minute exposures (EC50 ≅ 21 µM). This chemical did not alter ICa and slightly increased diastolic Ca2+ level. The frequency and size of resting Ca2+ sparks were increased by chrysosplenol-C. Chrysosplenol-C significantly increased sarcoplasmic reticulum (SR) Ca2+ content but not fractional release. Pretreatment of protein kinase C (PKC) inhibitor but not Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor abolished the stimulatory effects of chrysosplenol-C on Ca2+ transients and Ca2+ sparks. Chrysosplenol-C-induced positive inotropy was removed by the inhibition of PKC but not CaMKII or phospholipase C. Western blotting assessment revealed that PKC-δ protein level in the membrane fractions significantly increase within 2 minutes after chrysosplenol-C exposure with a delayed (5-minute) increase in PKC-α levels in insoluble membrane. These results suggest that chrysosplenol-C enhances contractility via PKC (most likely PKC-δ)-dependent enhancement of SR Ca2+ releases in ventricular myocytes. SIGNIFICANCE STATEMENT: Study shows that chrysosplenol-C, a natural flavone showing a positive inotropic effect, increases SR Ca2+ releases on depolarizations and Ca2+ sparks with an increase of SR Ca2+ loading but not L-type Ca2+ current in ventricular myocytes. Chrysosplenol-C-induced enhancement in contraction is eliminated by PKC inhibition, and it is associated with redistributions of PKC to the membrane. These indicate that chrysosplenol-C enhances contraction via PKC-dependent augmentations of SR Ca2+ release and Ca2+ loading during action potentials.