Abstract
It has been reported that shear stress enhances Ca2+-induced Ca2+ release during depolarization in ventricular myocytes. To know molecular basis for the increase in global Ca2+ releases we assessed the effects of shear stress (∼16 dyn/cm2) on local Ca2+ release events (Ca2+ sparks) and examined cellular mechanisms for the shear-mediated Ca2+ spark regulation using confocal Ca2+ imaging in rat ventricular myocytes. The frequency of Ca2+ sparks was immediately (within 1 s) increased by shear stress to about 1.5-fold, and further increased to about 2-fold by prolonged (20 s) shear exposure. Inhibition of nitric oxide synthetase and interference of cytoskeletal integrity using L-NAME (1 mM) and colchicine (10 μM), respectively, did not affect the shear-mediated enhancement in spark frequency. Blockers for Na+-Ca2+ exchanger and L-type Ca2+ channel, KB-R7943 (5 μM) or Ni2+ (5 mM), did not alter the shear effect on spark occurrence. Inhibition of NADPH oxidase (NOX) and mitochondrial uncoupling using diphenyleneiodonium (3 μM) and carbonyl cyanide 3-chlorophenylhydrazone (plus oligomycin, 1 μg/ml), respectively, reduced shear mediated spark enhancements. Pretreatment of reducing agent dithiothreitol (2 mM) also significantly reduced the shear-mediated spark enhancement. Measurement of intracellular reactive oxygen species (ROS) using its specific dye 2′,7′-dichlorofluorescein revealed increase in ROS level by shear stress. Sarcoplasmic reticulum (SR) Ca2+ content was not altered immediately after the application of shear stress, but significantly increased after 20-s long shear exposure. Our data suggest that shear stress enhances the frequency of Ca2+ sparks partly by producing ROS via mitochondrial NOX, and that prolonged enhancement in spark frequency by shear stress may be also mediated by increased SR Ca2+ loading. These mechanisms may partly explain the shear-mediated enhancement in Ca2+ transient in ventricular myocytes.
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