Endothelial mitochondria regulate the intracellular Ca²⁺ response to fluid shear stress

Shear stress is known to stimulate an intracellular free calcium concentration ([Ca²⁺]i) response in vascular endothelial cells (ECs). [Ca²⁺]i is a key second messenger for signaling that leads to vasodilation and EC survival. Although it is accepted that the shear-induced [Ca²⁺]i response is, in part, due to Ca²⁺ release from the endoplasmic reticulum (ER), the role of mitochondria (second largest Ca²⁺ store) is unknown. We hypothesized that the mitochondria play a role in regulating [Ca²⁺]i in sheared ECs. Cultured ECs, loaded with a Ca²⁺-sensitive fluorophore, were exposed to physiological levels of shear stress. Shear stress elicited [Ca²⁺]i transients in a percentage of cells with a fraction of them displaying oscillations. Peak magnitudes, percentage of oscillating ECs, and oscillation frequencies depended on the shear level. [Ca²⁺]i transients/oscillations were present when experiments were conducted in Ca²⁺-free solution (plus lanthanum) but absent when ECs were treated with a phospholipase C inhibitor, suggesting that the ER inositol 1,4,5-trisphosphate receptor is responsible for the [Ca²⁺]i response. Either a mitochondrial uncoupler or an electron transport chain inhibitor, but not a mitochondrial ATP synthase inhibitor, prevented the occurrence of transients and especially inhibited the oscillations. Knockdown of the mitochondrial Ca²⁺ uniporter also inhibited the shear-induced [Ca²⁺]i transients/oscillations compared with controls. Hence, EC mitochondria, through Ca²⁺ uptake/release, regulate the temporal profile of shear-induced ER Ca²⁺ release. [Ca²⁺]i oscillation frequencies detected were within the range for activation of mechanoresponsive kinases and transcription factors, suggesting that dysfunctional EC mitochondria may contribute to cardiovascular disease by deregulating the shear-induced [Ca²⁺]i response.