Abstract 1232: Regulation of Mitochondria membrane Potential by Shear Stress: Implication of NO Signaling

Abstract

Introduction : Mitochondria are the most important source of intracellular reactive oxygen species (ROS) in vascular endothelial cells (EC). Mitochondrial membrane potential (ΔΨm) is an indicator of mitochondrial energy state and mtROS production. Shear stress is known to induce eNOS and produce nitric oxide (NO). Hypothesis: We assessed whether NO is implicated in shear stress-induced ΔΨm and mtROS generation. Methods : Confluent HAEC were subjected to three conditions: static control (τ ave = 0 dyn.cm −2 at ∂τ/∂t = 0); pulsatile shear stress (PSS) at a mean shear stress (τ ave ) of 23 dyn.cm −2 with a temporal variation (∂τ/∂t) at 71 dyn.cm −2 .s −1 ; and oscillatory shear stress (OSS) at τ ave = 0.02 with ∂τ/∂t at ±3 dyn.cm − 2.s −1 . Tetramethyl rhodamine methyl ester (TMRM) and Mitosox red fluorescent dyes were used to assess mitochondrial ΔΨm and O 2 ·− production respectively. Intracellular H 2 O 2 production was assessed by carboxy-H 2 DCFDA. HAEC were treated with DETA-NO (NO donor) and L-NAME (eNOS inhibitor) to assess how shear stress-induced NO production regulates ΔΨm. Results: Delta;Ψm was increased by 12±5.3 mV in response to OSS, and by 33.5±8.9 mV in response to PSS (n=3, P <0.05) relative to static control. DETA-NO at 0.21mM and 0.94mM was used to simulate PSS-induced NO production during 1 hour and 4 hours of flow and it increased Delta;Ψm by 9.6±4.9 mV and 18.9 ±3.2 mV, respectively (n=3, P<0.05). L-NAME reduced Delta;Ψm (n=3, P<0.05) suggesting that NO is involved in Delta;Ψm regulation. PSS-induced Delta;Ψm was accompanied by a 1.4 fold increase in mtROS production (n=3, P<0.05) and a concomitant 10 fold upregulation in mitochondrial manganese superoxide dismutase (MnSOD) mRNA expression (n=6, P<0.05). Conclusion: Nitric oxide modulates Delta;Ψm and mtROS production. PSS, which upregulates eNOS, is more potent than OSS at increasing Delta;Ψm and mtROS, implicating NO as a signaling molecule in mitochondrial and endothelial function.