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 β-adrenergic signaling. We investigated the cellular mechanism for chrysosplenol-C-induced positive inotropy. Global and local Ca²⁺ signals, L-type Ca²⁺ current (I_Ca), 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 I_Ca 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.