Arterial Smooth Muscle Mitochondria Amplify Hydrogen Peroxide Microdomains Functionally Coupled to L-Type Calcium Channels

Abstract

Rationale: Mitochondria are key integrators of convergent intracellular signaling pathways. Two important second messengers modulated by mitochondria are calcium and reactive oxygen species. To date, coherent mechanisms describing mitochondrial integration of calcium and oxidative signaling in arterial smooth muscle are incomplete.

Objective: To address and add clarity to this issue, we tested the hypothesis that mitochondria regulate subplasmalemmal calcium and hydrogen peroxide microdomain signaling in cerebral arterial smooth muscle.

Methods and results: Using an image-based approach, we investigated the impact of mitochondrial regulation of L-type calcium channels on subcellular calcium and reactive oxygen species signaling microdomains in isolated arterial smooth muscle cells. Our single-cell observations were then related experimentally to intact arterial segments and to living animals. We found that subplasmalemmal mitochondrial amplification of hydrogen peroxide microdomain signaling stimulates L-type calcium channels, and that this mechanism strongly impacts the functional capacity of the vasoconstrictor angiotensin II. Importantly, we also found that disrupting this mitochondrial amplification mechanism in vivo normalized arterial function and attenuated the hypertensive response to systemic endothelial dysfunction.

Conclusions: From these observations, we conclude that mitochondrial amplification of subplasmalemmal calcium and hydrogen peroxide microdomain signaling is a fundamental mechanism regulating arterial smooth muscle function. As the principle components involved are fairly ubiquitous and positioning of mitochondria near the plasma membrane is not restricted to arterial smooth muscle, this mechanism could occur in many cell types and contribute to pathological elevations of intracellular calcium and increased oxidative stress associated with many diseases.

Keywords: calcium channels; hypertension myocytes, smooth muscle; oxidative stress; reactive oxygen species.

Figure 1. Subplasmalemmal mitochondria are present but sparse in rat cerebral arterial smooth muscle cells

A, Representative confocal images showing the plasma membrane (Alexa 555-WGA fluorescence, red) and mitochondria (MitoTracker fluorescence, green) in an isolated rat cerebral arterial smooth muscle cell. In panel 3, subplasmalemmal mitochondria (≤0.5μm from the plasma membrane) are highlighted yellow. The inset in panel 3 shows z projections at the x and y positions indicated by the dashed yellow lines. B, Plot of the mean±SEM mitochondrial and non-mitochondrial volumes (% of total cell volume; n=5 cells). C, Plot of the mean±SEM non-peripheral (>0.5 μm from the plasma membrane) and subplasmalemmal (≤0.5 μm) mitochondrial volumes (% total mitochondrial volume; n=5 cells).

Figure 2. Active L-type Ca²⁺ channels associate with subplasmalemmal mitochondria

A, Representative TIRF images showing subplasmalemmal mitochondria (MitoTracker fluorescence, panel 1; thresholded MitoTracker fluorescence, panel 2), L-type Ca²⁺ channel-mediated Ca²⁺ influx (fluo-5F fluorescence; panel 3), and an overlay of panels 2 & 3 (panel 4). Yellow circles in panels 3 and 4 indicate sites of bone fide L-type Ca²⁺ channel sparklet activity (see Online Methods). B, Euclidean distance mapping showing cumulative distribution functions representing the distance of observed Ca²⁺ sparklet site peaks from mitochondria (solid black line; n=7 cells) and from 100 randomly distributed points within visible TIRF footprint (dashed black line). Solid red lines are best fits of the cumulative distributions with a single exponential function as described in the Online Methods. The vertical dashed grey line marks the distance separating mitochondrial associated (≤0.5 μm) and non-associated (>0.5 μm) Ca²⁺ sparklet sites. C, Plot of Ca²⁺ sparklet site activities (nP_s, where n is the number of quantal levels detected and P_s is the probability that the site is active) at sites >0.5 μm and ≤0.5 μm from the nearest thresholded MitoTracker signal (n=5 cells). The horizontal dashed grey line marks the threshold for high-activity Ca²⁺ sparklet sites (nP_s≥0.2; see Online Methods). Bracketed values indicate effect size (r). *P<0.05

Figure 3. ROS generation by subplasmalemmal mitochondria is punctate and stimulates L-type Ca²⁺ channels

A, Representative TIRF images showing subplasmalemmal DCF fluorescence (indicating intracellular oxidation) in a control cell (left) and in a cell incubated with the mitochondrial electron transport chain complex III inhibitor antimycin (500 nmol/L; right) at the times indicated. Yellow circles indicate sites of bone fide ROS puncta formation (see Online Methods). B, Plot of individual ROS puncta densities in control cells (open circles; n=5 cells) and cells treated with antimycin (filled red circles; n=5 cells) at 0 min and at 10 min (left) and plot of mean±SEM ROS puncta densities at 0 min (all values) and at 10 min in control and antimycin-treated cells (right). C, Representative TIRF images showing Ca²⁺ influx in a cell before and after application of antimycin (500 nmol/L). Traces show the time course of Ca²⁺ influx at the 3 circled sites. D, Plot of Ca²⁺ sparklet site activities (nP_s) and plot of mean±SEM Ca²⁺ sparklet densities (Ca²⁺ sparklet sites/μm²) before and after antimycin (n=5 cells). Bracketed values indicate effect size (r). *P<0.05

Figure 4. Mitochondria contribute to angiotensin II-dependent ROS and Ca²⁺ microdomain signaling

A, Representative TIRF images showing subplasmalemmal DCF fluorescence in a cell before and after application of Ang II (100 nmol/L; left) and in a cell before and after Ang II in the presence of the mitochondrial targeted antioxidant mitoTEMPO (25 nmol/L; right). Yellow circles indicate sites of ROS puncta formation. B, Plot of individual ROS puncta densities in cells exposed to Ang II (open circles; n=5 cells) and cells exposed to Ang II in the presence of mitoTEMPO (filled red circles; n=5 cells) at 0 min and at 10 min (left) and the plot of mean±SEM ROS puncta densities at 0 min (all values) and at 10 min in Ang II and Ang II in the presence of mitoTEMPO (right). C, Representative traces showing time courses of Ca²⁺ influx in cells before and after application of Ang II (100 nmol/L) in control cells and in the presence of the mitochondrial targeted antioxidant mitoTEMPO (25 nmol/L). D, Plots showing Ca²⁺ sparklet site activities (nP_s) and mean±SEM Ca²⁺ sparklet densities (Ca²⁺ sparklet sites/μm²) before after Ang II in control cells and in the presence of mitoTEMPO (n=5 cells each). Bracketed values indicate effect size (r). *P<0.05

Figure 5. Smooth muscle mitochondrial ROS contribute to angiotensin II-dependent arterial contractions

A–C, Representative time courses showing luminal diameters (as % passive diameter) of pressurized (60 mmHg) middle cerebral arterial segments exposed to Ang II (10 nmol/L) in the absence (A) or presence of the mitochondrial-targeted antioxidant mitoTEMPO (1 μmol/L) (B) or the non-targeted antioxidant TEMPOL (10 μmol/L) (C). The horizontal dashed grey lines represent approximate points of measurement for analysis. D, Plot of the mean±SEM induced contraction (% passive diameter) by Ang II in the absence or presence of mitoTEMPO or TEMPOL (n=5 arteries each). Bracketed values indicate effect size (r). *P<0.05

Figure 6. Arterial smooth muscle mitochondrial ROS contribute to hypertension-associated arterial dysfunction

A, Time courses of mean arterial pressures in rats treated with the nitric oxide synthase inhibitor L-NAME and infused with saline (open circles) or infused with the mitochondrial targeted antioxidant mitoTEMPO (closed circles). B, Plot of the terminal mean arterial pressures (mean±SEM) for rats treated with L-NAME and infused with saline or with mitoTEMPO (n=5 rats each). C, Time courses of the change in mean arterial pressure in response to L-NAME for rats infused with saline or with mitoTEMPO (n=5 rats each). D, Representative time courses showing luminal diameters (as % passive diameter) of pressurized middle cerebral arterial segments (as indicated) isolated from rats treated with L-NAME and infused with saline (left) or with mitoTEMPO (right). The horizontal dashed grey lines represent approximate points of measurement for analysis. E, Plot of the mean±SEM contraction induced by increasing the intraluminal pressure (20 to 60 mmHg) of middle cerebral arterial segments isolated from rats treated with L-NAME and infused with saline or with mitoTEMPO (n=5 arteries each). Bracketed values indicate effect size (r). *P<0.05

Figure 7. Endothelial dysfunction increases arterial smooth muscle mitochondrial ROS and Ca²⁺ microdomain signaling

A, Representative TIRF images showing subplasmalemmal DCF fluorescence in a cell isolated from rats treated with the nitric oxide synthase inhibitor L-NAME and infused with saline (top) or infused with the mitochondrial targeted antioxidant mitoTEMPO (bottom). Yellow circles indicate sites of ROS puncta formation. B, Plot of mean±SEM ROS puncta densities in cells isolated from rats treated with L- NAME and infused with saline or infused with mitoTEMPO (n=5 cells). C, Representative TIRF images showing Ca²⁺ influx in cells isolated from rats treated with L-NAME and infused with saline (left) or infused with mitoTEMPO (right). Traces show the time course of Ca²⁺ influx at the 3 circled sites. D, Plot of Ca²⁺ sparklet site activities (nP_s) and plot of mean±SEM Ca²⁺ sparklet site densities (Ca²⁺ sparklet sites/μm²) in cells isolated from rats treated with L-NAME and infused with saline or infused with mitoTEMPO (n=5 cells). Black asterisks and effect sizes in panels B and D are in reference to non-L-NAME-treated controls. E, Proposed mechanism where mitochondrial ROS-induced ROS release (RIRR) amplifies H₂O₂ microdomain signaling leading to stimulation of colocalized Ca²⁺ influx through L-type Ca²⁺ channels resulting in changes (Δ) in cell function or induction of cell dysfunction. CaV=L-type Ca²⁺ channel; ETC=mitochondrial electron transport chain. Bracketed values indicate effect size (r). *P<0.05