Figure 5

Quantitation of [Ca²⁺]_m transients under each condition and a proposed _mCa²⁺ signaling pathway in sheared MCU OX ECs. (A) The percentage of MCU OX cells that exhibited [Ca²⁺]_m transients under SS significantly decreased in the presence of either GsMTx4, MT (in a dose-dependent manner), or L-NAME (mean ± SE from n = 4 independent experiments/condition). The percentage was significantly higher in MCU OX cells (greater than 80%) compared to that in untransduced or AdβGal-transduced ECs (~ 20%). (B) The percentage of ECs that exhibited transients in each condition was binned over the total number of transients (excluding the initial peak that was present in every cell exposed to SS). (C) The total number of transients per cell was binned over 60 s intervals up to 10 min (1 min static followed by 9 min SS). Data are shown as mean ± SE for 60 cells (from n = 4 independent experiments). Only the MCU OX cells, and to a lesser extent the MCU OX cells in the presence of the lowest MT dose, exhibited persistent transients/oscillations during SS. (D) The average oscillation frequency (mHz) was calculated from n = 4 independent experiments per condition. ns, P > 0.5; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Upregulated MCU expression leads to increased _mCa²⁺ influx (step 1) that increases O₂^-. production by the ETC and mROS levels in the matrix (step 2). High [Ca²⁺]_m (and mROS) activate/open the low conductance mPTP channel (step 3). Due to redistribution of ions, mitochondria undergo a brief depolarization (ΔΨ_m flicker) that contributes to additional mROS generation, but also causes ROS emissions into the MAMs (step 4), where ROS sensitize the IP₃R to release ER Ca²⁺ leading to _mCa²⁺ uptake via the MCU (through the voltage-dependent anion channel, VDAC) and generating the next [Ca²⁺]_m peak (step 1). When the low conductance mPTP opens, opening is brief, because the resultant reduction in [Ca²⁺]_m (and lowering of matrix pH due to entry of H⁺ ions) favors mPTP closure and, hence, steps 1 through 4 (and back to 1) take place in an oscillatory fashion. SS activates Piezo1 (step 5) and Ca²⁺ entering the cytosol maintains Ca²⁺ in the MAMs, but it may also help maintain the ER Ca²⁺ content (step 6). SS-induced Piezo1-mediated Ca²⁺ influx also activates eNOS (step 7) and the produced NO, either on its own or via generation of RNS, enhances mROS production by the ETC (step 8).