Mitochondria are subcellular organelles that synthesize ATP, generate reactive oxygen species (ROS), and control cellular fates such as apoptosis and aging. Mitochondria generate different amounts of ROS in association with their morphologies. Cyclic stretch is a mechanical stimulation exerted on cells due to arterial pulsation, and induces cells to generate mitochondrial ROS. Therefore, one can speculate that morphological changes of mitochondria may play a role in mitochondrial ROS generation in cells under cyclic stretch. However, whether the morphologies of mitochondria are actually altered under cyclic stretch remains unclear. This study attempted to answer this question by time-lapse imaging the morphological dynamics of mitochondria in bovine aortic endothelial cells (BAECs) subjected to two levels of uniaxial cyclic stretch: (1) a physiologic level (5% at 1 Hz) for 1 hour, and (2) a supra-physiologic level (20% at 1 Hz) for 1 hour. Mitochondria were stained with Mito-tracker Orange, and MicroP software and FibrilTool were used for mitochondrial alignment and length analyses. No clear changes in the average length of mitochondria were observed at the physiological level of stretch (5%) compared to no stretch (0%), while the average length was decreased by the supra-physiological level of stretch (20%). In addition, cellular alignment was not different between 0% and 5% stretches, but the cells became perpendicularly aligned in the direction of stretch when 20% stretch was applied. Cellular circularity was not signi cantly different among the three levels of cyclic stretch. Thus, BAECs exhibited changes in both mitochondrial dynamics and cellular remodeling dynamics under 20% stretch, but showed no changes in both under 5% stretch. The results indicate that changes in morphological dynamics of mitochondria correlate with changes in cellular dynamics, particularly change in cellular alignment.

Cells are continuously exposed to the mechanical stimulus suGh as the cyclic stretch due to heart beats， The cells are aligned nearly perpendicular to the direction ofthe cyclic stretch． Recent reports ｛ ndicate that the pathological levels of cyclic stretch cause apoptosis ． The balance of the mitochondrial fUsion and fission influences the state of the ce1L

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Mitochondria in endothelial cells remodel morphologically when supraphysiological cyclic stretch is exerted on the cells. During remodeling, mitochondria become shorter, but how they do so remains elusive. Drp1 is a regulator of mitochondrial morphologies. It shortens mitochondria by shifting the balance from mitochondrial fusion to fission. In this study, we hypothesized that Drp1 activation is involved in mitochondrial remodeling under supraphysiological cyclic stretch. To verify the involvement of Drp1, its activation was first quantified with Western blotting, but Drp1 was not significantly activated in endothelial cells under supraphysiological cyclic stretch. Next, Drp1 activation was inhibited with Mdivi-1, but this did not inhibit mitochondrial remodeling. Intracellular Ca 2+ increase activates Drp1 through calcineurin. First, we inhibited the intracellular Ca 2+ increase with Gd 3+ and thapsigargin, but this did not inhibit mitochondrial remodeling. Next, we inhibited calcineurin with cyclosporin A, but this also did not inhibit mitochondrial remodeling. These results indicate that mitochondrial remodeling under supraphysiological cyclic stretch is independent of Drp1 activation. In endothelial cells under supraphysiological cyclic stretch, reactive oxygen species (ROS) are generated. Mitochondrial morphologies are remodeled by ROS generation. When ROS was eliminated with N-acetyl-L-cysteine, mitochondrial remodeling was inhibited. Furthermore, when the polymerization of the actin cytoskeleton was inhibited with cytochalasin D, mitochondrial remodeling was also inhibited. These results suggest that ROS and actin cytoskeleton are rather involved in mitochondrial remodeling. In conclusion, the present results suggest that mitochondrial remodeling in endothelial cells under supraphysiological cyclic stretch is induced by ROS in association with actin cytoskeleton rather than through Drp1 activation.