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

The NADPH oxidase inhibitor diphenyleneiodonium suppresses Ca2+ signaling and contraction in rat cardiac myocytes

“…Cardiac myocytes were isolated from male Sprague-Dawley rats (200–300 g) as previously described [ 30 ]. The experiments were performed in accordance with the principles for the care and use of experimental animals published by the Korean Food and Drug Administration.…”

“…Ventricular myocytes attached at the bottom of experimental chamber were continuously superfused with normal Tyrode’s solutions (pH = 7.4) containing 2 mM Ca 2+ and field-stimulated at 1 Hz with two paralleled Pt wires connected to an electrical stimulator (D-7806; Hugo Sachs) at room temperature. Cell shortenings were detected with a video edge detector (Model VED-105; Crescent Electronics) connected to a CCD camera (LCL902C; Till Photonics) and video monitor (ViewFinder III, Polychrome V system; Till Photonics) as previously reported [ 30 ]. Changes of cell length were monitored using A/D converter (Digidata 1322A; Molecular Devices) connected to a PC software pClamp (9.0; Molecular Devices) and analyzed with Clampfit 9.0 (Molecular Devices) and Origin software (8.0; OriginLab Corporation).…”

“…Dyes were excited at 488 nm using Ar ion laser, and fluorescence emission at > 510 nm was detected. Cytosolic Ca 2+ fluorescence signals were recorded in 2-D images using a laser scanning confocal imaging system (A1; Nikon) attached to an inverted microscope (Eclipse Ti; Nikon) fitted with a ×60 oil immersion objective lens (Plan Apo, Numerical Aperture 1.4; Nikon) [ 30 ]. Acquisition and analysis of images were performed using a workstation software (NIS Elements AR, v3.2; Nikon).…”

“…The average diastolic fluorescence intensity (F 0 ) was measured from several frames captured before the upstroke of Ca 2+ transient. The time-courses of Ca 2+ transients were evaluated as the average fluorescence of each area normalized relative to the F 0 (F/F 0 ) [ 30 ]. To measure Ca 2+ spark frequency, the whole-cell images were recorded at 30 Hz.…”