Arterial Smooth Muscle Mitochondria Amplify Hydrogen Peroxide Microdomains Functionally Coupled to L-Type Calcium ChannelsNovelty and Significance
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.
Mitochondria are central to eukaryotic aerobic metabolism. One consequence arising from the shuttling of electrons onto molecular oxygen during mitochondrial respiration is the formation of reactive oxygen species (ROS). Given the resultant necessity and subsequent ubiquity of ROS formation, it is not surprising then that these generally toxic products of metabolism also function as purposeful second messenger molecules.1,2
Editorial, see p 984
Advances in cellular pathophysiology implicate mitochondrial dysfunction in the development and progression of illnesses including cancer,3 neurodegeneration,4 diabetes mellitus,5 and cardiovascular disease.6,7 As such, mitochondria are being evaluated as potential therapeutic targets for novel disease prevention and management strategies.8,9 If mitochondria are to be viable therapeutic targets, then a mechanistic understanding of mitochondrial function in multiple cell types is necessary to rationally predict and account for pharmacological responses to mitochondrial perturbations.
Here, we investigated mitochondrial ROS signaling-dependent regulation of Ca2+ influx in arterial smooth muscle. These cells are an ideal experimental model for this work because of their large surface area and because their geometry and morphological simplicity minimizes confounding variables possible in cells with more complex structural features. Furthermore, mitochondria, ROS, and Ca2+ are each integral to arterial smooth muscle function and are thought to be involved in the development of cardiovascular disease.
Ca2+, as with ROS, is a ubiquitous signaling molecule that influences many processes ranging from contraction to gene expression. Ca2+ and ROS as second messenger signaling molecules share at least 4 key attributes relevant to this study: first, Ca2+ and ROS signaling events are known to be functionally coupled10,11; second, Ca2+ and ROS signaling cascades attain specificity, in part, by subcellular compartmentalization12,13; third, Ca2+ and ROS signaling events are influenced by mitochondria10; and fourth, Ca2+ and ROS signaling cascades are implicated in the development of disease.11,14
In accordance with the first 2 attributes, our previous works showed that formation of punctate sites of ROS generation involving nicotinamide adenine dinucleotide phosphate (NADPH) oxidase leads to colocalized Ca2+ influx through L-type Ca2+ channels.15–17 We found that oxidative activation of protein kinase C (PKC),18 which promotes localized Ca2+ influx through L-type Ca2+ channels,19–21 functionally links Ca2+ and ROS microdomain signaling in this context. Here, we addressed the second pair of Ca2+ and ROS signaling attributes listed above by investigating mitochondrial ROS–dependent regulation of arterial smooth muscle L-type Ca2+ channels in relation to the development of hypertension-associated arterial dysfunction.
Experiments were performed on single cells, on excised arterial segments, and in living animals. Using this progressive subcellular in vitro-to-organismal in vivo approach, we conclude that amplification of hydrogen peroxide (H2O2) microdomain signaling by subplasmalemmal mitochondria promotes the opening of adjacent L-type Ca2+ channels and subsequently the development of hypertension-associated arterial dysfunction.
Male Sprague–Dawley rats were euthanized with sodium pentobarbital (200 mg/kg IP) as approved by the Institutional Animal Care and Use Committee of Colorado State University. Smooth muscle cells were isolated from basilar and cerebral arteries. Confocal microscopy was used to image the plasma membrane and mitochondria of single cells. L-type Ca2+ channel sparklets and subplasmalemmal ROS were imaged with total internal reflection fluorescence (TIRF) microscopy.15,16 Arterial diameters were recorded from middle cerebral arterial segments. Hypertension was induced in rats by including Nω-nitro-l-arginine methyl ester (L-NAME) in their drinking water,22–24 and arterial blood pressure was monitored by telemetry as approved by the Institutional Animal Care and Use Committee of the University of California, Davis.
Normally distributed data are presented as the mean± SEM. To account for potential variability associated with cell isolation, all single myocyte experimental groups comprised cells obtained from ≥4 rats. Effect sizes, represented in the figures as bracketed values, are reported as Pearson r (range, −1.0 to 1.0; 0 indicates no effect)25,26; P values <0.05 were considered significant. An expanded Materials and Methods are available in the Online Data Supplement
Subpopulation of Mitochondria Associates With the Arterial Smooth Muscle Cell Plasma Membrane at Sites of Elevated L-type Ca2+ Channel Activity
To assess the subcellular distribution of mitochondria in arterial smooth muscle, we marked the plasma membrane of cells with a wheat germ agglutinin conjugate (Alexa 555-wheat germ agglutinin [WGA], red), labeled mitochondria with MitoTracker Green, and visualized the fluorescence with confocal microscopy (Figure 1A). We found that 8.64±0.42% of the total cell volume was occupied by mitochondria; this volume is consistent with previous report.27 Although the majority of the MitoTracker signal was located centrally (>0.5 µm from the center of the Alexa 555-WGA signal), 7.50±0.77% of the mitochondrial volume was peripheral (≤0.5 µm; yellow signal in Figure 1A, panel 3).
Next, we examined MitoTracker-loaded cells with TIRF microscopy. Our images showed regions of MitoTracker fluorescence indicating the presence of subplasmalemmal mitochondria (Figure 2A, panel 1). Analysis of thresholded MitoTracker images revealed that only 3.29±0.26% (n=7 cells) of the visible plasma membrane was associated with mitochondria (Figure 2A, panel 2). We imaged Ca2+ influx with a combination of TIRF microscopy and voltage-clamp electrophysiology.15,19,21 This approach permits visualization of Ca2+ influx through L-type Ca2+ channels (Ca2+ sparklets). To evoke Ca2+ sparklets, we exposed cells to angiotensin II (Ang II; 100 nmol/L), which is known to promote mitochondrial ROS production.6 Similar to our previous reports,15–17,28 Ang II induced L-type Ca2+ channel sparklet sites as revealed by localized changes in fluorescence of the Ca2+ indicator fluo-5F (Figure 2A, panel 3). The L-type Ca2+ channel sparklets induced by Ang II in this study were not different from the L-type Ca2+ channel sparklets observed previously in terms of quantal amplitude (36.2±3.2 nmol/L [Ca2+]) and site densities and activities (see below).15–17
To visualize the spatial relationship between L-type Ca2+ channel activity and subplasmalemmal mitochondria, we overlaid our fluo-5F and thresholded MitoTracker images (Figure 2A, panel 4). We then measured the distance from L-type Ca2+ channel sparklet site peaks (pixels of highest intensity) to the edge of the nearest MitoTracker signal and plotted the cumulative values (Figure 2B). Mitochondrial-associated L-type Ca2+ channel sparklet sites were defined a priori as those sites with peaks ≤0.5 µm from the edge of the nearest thresholded MitoTracker signal; this is represented by the vertical dashed gray line in Figure 2B. We found that L-type Ca2+ channel function was associated with subplasmalemmal mitochondria (Figure 2B; n=5 cells). The half distance of the observed L-type Ca2+ channel sparklet sites (n=41 sites) to the nearest mitochondria was 0.43 (95% confidence interval, 0.39–0.47) µm, whereas the half distance of 100 random points within the visible plasma membrane to the nearest mitochondria was 2.10 (95% confidence interval, 1.99–2.19) µm.
Next, we compared the activity of mitochondrial-associated (distance, ≤0.5 µm) and nonassociated L-type Ca2+ channel sparklet sites. L-type Ca2+ channel sparklet site activity can be expressed by the descriptor nPs, where n is the number of quanta detected (ie, number of functional channels) and Ps is the probability that the site is active.21 Ang II–induced L-type Ca2+ channel sparklet sites associated with mitochondria were more active than those not associated with mitochondria (Figure 2C; P<0.05, r=0.59; n=5 cells). Indeed, mitochondrial-associated L-type Ca2+ channel sparklet sites accounted for ≈73% of the total Ca2+ sparklet activity observed. From these data, we conclude that the spatial distribution of L-type Ca2+ channel activity is highly correlated with subplasmalemmal mitochondria.
Mitochondrial-Derived H2O2 Stimulates L-Type Ca2+ Channels
We reported previously that localized H2O2 microdomains stimulate L-type Ca2+ channels in arterial smooth muscle via a PKC-dependent mechanism.15–17,20 As mitochondria are a major source of ROS, we tested the hypothesis that localized H2O2 generated by subplasmalemmal mitochondria stimulate nearby L-type Ca2+ channels.
To promote mitochondrial ROS generation, we exposed cells to the electron transport chain complex III inhibitor antimycin (500 nmol/L).29,30 First, we investigated the effect of antimycin on subplasmalemmal ROS production with the fluorescent ROS indicator 5-(and-6)-chloromethyl-2′7 ′-dichlorodihydrofluorescein diacetate acetyl ester (DCF; 1 µmol/L) and monitored for changes in subplasmalemmal fluorescence (Figure 3A). Similar to Ang II,15,16 antimycin induced localized sites of elevated DCF fluorescence (ROS puncta15; Figure 3A and 3B; P<0.05; r=0.65; n=5 cells); comparably ROS puncta formation was not apparent in time-matched controls. In contrast to the ≈6-fold increase in DCF fluorescence associated with ROS puncta, the spatially averaged DCF fluorescence in cells treated with antimycin did not increase (Online Figure IA; P>0.05; n=5 cells). This observation is consistent with the concept of localized ROS generation by subplasmalemmal mitochondria.
Antimycin also induced localized L-type Ca2+ channel sparklets (Figure 3C and 3D). The L-type Ca2+ channel sparklet site activity (nPs; r=0.43) and density (sites/µm2; r=0.75) induced by antimycin was not different from that observed with Ang II (Figures 4C and 6D; P>0.05; n=5 cells). Ca2+ sparklet activity was not observed after antimycin treatment in cells pretreated with nicardipine (10 µmol/L; P>0.05; n=5 cells, data not shown) or the PKC inhibitor Gö6976 (100 nmol/L; Online Figure IB and IC). Similarly, dialyzing cells with catalase (500 U/mL) prevented the stimulatory effect of antimycin on L-type Ca2+ channels (Online Figure IB and IC). These data are consistent with the hypothesis that antimycin-dependent promotion of localized mitochondrial H2O2 generation stimulates neighboring L-type Ca2+ channels via a PKC-dependent mechanism.
Our previous work and results using the NADPH oxidase inhibitor ML171 (Online Figure III) indicate that NADPH oxidase activity is necessary for local regulation of L-type Ca2+ channels by Ang II–induced H2O2 microdomain signaling.15,16 Ang II also induces mitochondrial ROS generation.6,31 Therefore, we tested the effect of inhibiting mitochondrial ROS generation on Ang II–dependent stimulation of L-type Ca2+ channels.
The mitochondrial-targeted antioxidant mitoTEMPO attenuates the production of H2O2 by mitochondria in response to Ang II.6,8,31,32 We found that Ang II did not promote ROS puncta formation in cells pretreated with mitoTEMPO (25 nmol/L for 15 minutes; Figure 4A and 4B; P>0.05; r=−0.80; n=5 cells). MitoTEMPO also attenuated the stimulatory effect of Ang II on L-type Ca2+ channel sparklet site activity (Figure 4C and 4D; P>0.05; r=−0.39; n=5 cells). Interestingly, and in contrast to NADPH oxidase inhibition or catalase,15,16 mitoTEMPO did not prevent the increase in L-type Ca2+ channel sparklet site density (sites/μm2) induced by Ang II (Figure 4D; P<0.05; r=−0.09; n=5 cells). MitoTEMPO did not have apparent nonspecific effects on macroscopic L-type Ca2+ channel currents (Online Data Supplement i). These data suggest that in the presence of mitoTEMPO, Ang II–induced Ca2+ influx is minimal, despite increasing the number of observed L-type Ca2+ channel sparklet sites. Similar results were observed when mitochondrial ROS production was limited with rotenone (Online Data Supplement ii).
Mitochondrial ROS Production Contributes to Angiotensin II–Mediated Arterial Contraction
L-type Ca2+ channels are the primary conduit for contractile Ca2+ in arterial smooth muscle.33 As mitoTEMPO reduced Ang II–dependent stimulation of L-type Ca2+ channels, we reasoned that inhibiting mitochondrial H2O2 production with mitoTEMPO should decrease contractile responses to Ang II. To test this, we applied Ang II to excised arterial segments pressurized to 60 mm Hg. These experiments were performed in the presence of the nitric oxide synthase inhibitor Nω-nitro-l-arginine (300 µmol/L) to prevent vasodilatory influences of endothelial-derived nitric oxide.
In control experiments, Ang II constricted arteries to 14.86±0.99% below their baseline diameter (Figure 5A). In arteries incubated with mitoTEMPO (1 µmol/L for 15 minutes), Ang II induced a smaller contraction to only 3.98±0.99% below baseline (Figure 5B; P<0.05; r=−0.93; n=5 arteries). MitoTEMPO by itself had no apparent effect on baseline arterial constriction or the contractile response to 60 mmol/L KCl (P>0.05; n=3 arteries, data not shown). We reported previously that the nontargeted antioxidant TEMPOL does not suppress Ang II–dependent stimulation of L-type Ca2+ channels.16 Therefore, we replicated our contractile experiments with Ang II on arteries incubated with TEMPOL (10 µmol/L for 15 minutes). In contrast to mitoTEMPO, even at 10-fold higher concentration, TEMPOL did not reduce Ang II–dependent arterial constriction (Figure 5C and 5D; P>0.05; r=−0.29; n=5 arteries). From these data, we conclude that mitochondrial-derived ROS contribute to Ang II–dependent constriction of pressurized intact arterial segments.
Reducing Mitochondrial ROS Production In Vivo Attenuates Hypertensive Arterial Responses to Endothelial Dysfunction
To establish the importance of smooth muscle mitochondrial ROS production on arterial function in vivo, we induced hypertension in rats by including the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester (L-NAME; 0.75 mg/mL) in their drinking water (dose ≈50 mg/kg per day).22–24 A key feature of L-NAME–induced hypertension is frank endothelial dysfunction and increased arterial constriction.34 To examine the involvement of mitochondrial-derived ROS, we infused L-NAME–treated animals with mitoTEMPO at a rate of 150 µg/kg per day8 via subcutaneous osmotic minipumps; rats implanted with saline minipumps served as control. Arterial blood pressure was monitored by telemetry.
L-NAME induced a time-dependent increase in mean arterial pressure (Figure 6A; P<0.05; r=0.98; n=5 rats). Similar to Ang II–induced hypertension,8 coadministration of mitoTEMPO blunted (r=-0.72) the hypertensive response to L-NAME (Figure 6B; P<0.05; r=0.82; n=5 rats). The effect of mitoTEMPO on mean arterial pressure was evident on the third day of L-NAME treatment (Figure 6C; P<0.05; r=-0.96). Thus, despite continued disruption of endothelial function, mitoTEMPO produced a modest decrease in blood pressure. This observation suggests that mitoTEMPO could be working in vivo, at least in part, by reducing mitochondrial ROS generation and subsequent stimulation of arterial smooth muscle L-type Ca2+ channels.
To investigate this, we excised arterial segments from L-NAME and L-NAME plus mitoTEMPO-treated animals and compared their myogenic set points (ie, observed levels of myogenic tone). First, we pressurized arteries to 20 mm Hg and allowed them to equilibrate. Once a stable diameter was reached, we increased the pressure to 60 mm Hg and waited for an active myogenic contractile response to occur after passive dilation (Figure 6C). Experiments were terminated by superfusing the arteries with a nominally Ca2+-free solution.
Arteries isolated from L-NAME–treated animals infused with saline constricted robustly when the intraluminal pressure was increased from 20 to 60 mm Hg (48.80±2.06% below passive diameter; n=5 arteries). Arteries from L-NAME–treated animals infused with mitoTEMPO constricted substantially less after the same increase in pressure (30.43±1.46% below passive diameter; P<0.05; r=-0.92; n=5 arteries). Note that arteries isolated from normotensive rats pressurized to 60 mm Hg constricted to 38.29±3.20% below their passive diameter (n=5 arteries), which was smaller than the constriction seen in arteries from L-NAME–treated animals infused with saline (P<0.05), but not significantly different from the constriction seen in arteries from L-NAME–treated animals infused with mitoTEMPO (P>0.05). From these data, we conclude that systemic inhibition of mitochondrial ROS generation with mitoTEMPO reduces L-NAME–induced arterial dysfunction in vivo (lowers systemic arterial pressure) and ex vivo (lowers arterial myogenic tone).
To investigate the mechanisms underlying mitoTEMPO-dependent preservation of arterial function, we examined baseline ROS and Ca2+ microdomain signaling activity in cells isolated from these animals. Using DCF fluorescence as an indicator of ROS generation with TIRF microscopy, we found that ROS puncta density (in the absence of acute stimulation) was elevated in smooth muscle cells from hypertensive L-NAME–treated rats when compared with those from non–L-NAME-treated controls (Figure 7A and 7B; P<0.05; r=0.70; n=5 cells). Importantly, ROS puncta densities in cells isolated from rats receiving L-NAME and mitoTEMPO were lower than those receiving L-NAME alone (P<0.05; r=−0.74; n=5 cells). The ROS puncta density in these cells was negligible and not different from unstimulated control cells (P>0.05; n=5 cells).
Paralleling our ROS puncta data and consistent with previous observations in Ang II–induced and genetic hypertension,28 basal L-type Ca2+ channel function was enhanced in cells isolated from hypertensive L-NAME–treated rats as evidenced by elevated Ca2+ sparklet site activities and densities (Figure 7C and 7D; P<0.05; nPs r=0.75; density r=0.64; n=5 cells). Notably, as with ROS puncta, mitoTEMPO infusion abolished L-NAME enhancement of L-type Ca2+ channel activity (P<0.05; r=−0.51; n=5 cells) with levels not different from unstimulated cells from normotensive animals (P>0.05; n=5 cells). Taken altogether, we conclude that arterial smooth muscle mitochondrial-amplified H2O2 microdomain signaling promotes Ca2+ influx through neighboring L-type Ca2+ channels, and that this process contributes mechanistically to the contractile action of Ang II and the development of hypertension-associated arterial dysfunction.
In this study, we tested the hypothesis that mitochondrial amplification of H2O2 microdomain signaling stimulates L-type Ca2+ channels in arterial smooth muscle. The major findings supporting this hypothesis are (1) subplasmalemmal mitochondria associate with sites of elevated L-type Ca2+ channel activity; (2) mitochondrial-derived H2O2 stimulates Ca2+ influx through L-type channels; (3) inhibiting mitochondrial H2O2 production reduces Ang II–mediated arterial contraction; and (4) inhibiting mitochondrial H2O2 production attenuates the development of hypertension-associated arterial smooth muscle dysfunction ex vivo and in vivo. From these data, we conclude that mitochondrial amplification of H2O2 microdomain signaling is an important regulator of L-type Ca2+ channels in arterial smooth muscle and contributes to the development of hypertension-associated arterial dysfunction.
Our imaging of subplasmalemmal fluo-5F and MitoTracker fluorescence with TIRF microscopy provides compelling evidence that L-type Ca2+ channel activity is enriched at areas of the plasma membrane associated with mitochondria (Figure 2). Consistent with this observation, the small amount of plasma membrane associated with mitochondria (≈5%) is sufficient to influence the surface area necessary (≈4%) to accommodate the L-type Ca2+ channel sparklet activity observed. This logic supports the conclusion that subcellular localization is a key factor in determining the stimulatory influence of mitochondria on L-type Ca2+ channels (Online Data Supplement iii).
Evidence suggests that mitochondrial buffering of proximate Ca2+ flux events is an important mechanism by which these organelles influence Ca2+ signaling.35 With respect to L-type Ca2+ channels, localized Ca2+ buffering by mitochondria could reduce the amplitude of the Ca2+ microdomain formed near the pore when the channel is open.36 Lowering the effective Ca2+ concentration around the channel could alter Ca2+ signaling by reducing the Ca2+-dependent inactivation characteristic of L-type Ca2+ channels.37 Previous works in smooth muscle suggest that mitochondrial Ca2+ buffering does not influence Ca2+ influx through L-type Ca2+ channels.35,38 However, our TIRF images demonstrate that L-type Ca2+ channel activity is associated with subplasmalemmal mitochondria (Figure 2). To reconcile these apparently contradictory observations, we propose a mechanism of mitochondrial promotion of Ca2+ influx that does not rely on Ca2+ buffering per se. Rather, we propose that mitochondria promote Ca2+ influx by amplifying H2O2 microdomain signaling in the vicinity of L-type Ca2+ channels, which leads to oxidant-dependent stimulation of Ca2+ influx.15–17
Functionally compartmentalized submembranous accumulation of H2O2 is thought to bring about competent H2O2 signaling microdomains.39 We reported previously that H2O2 microdomains involving NADPH oxidase promote colocalized Ca2+ influx through L-type Ca2+ channels via oxidative activation of PKC.15–17 Experiments in this study provide evidence indicating that peripheral mitochondria also participate in H2O2 microdomain-dependent stimulation of L-type Ca2+ channels. Antimycin induced the formation of subplasmalemmal ROS puncta and induced L-type Ca2+ channel sparklet activity. Finally, antimycin-dependent stimulation of L-type Ca2+ channels was abolished by enhanced decomposition of H2O2 with intracellular catalase.
To specifically reduce the influence of mitochondrial ROS on L-type Ca2+ channels, we used the mitochondrial-targeted antioxidant mitoTEMPO at a concentration where cytosolic antioxidant effects are not apparent.6,8,31 MitoTEMPO prevented the induction of ROS puncta formation by Ang II and reduced the stimulatory effect on L-type Ca2+ channel sparklets. This suggests that mitochondrial ROS (visualized as ROS puncta) stimulate L-type Ca2+ channels. Intriguingly, unlike the effect on Ca2+ sparklet activity, mitoTEMPO did not reduce the effect of Ang II on L-type Ca2+ channel sparklet density (sites/µm2). This is in contrast to the effects of NADPH oxidase inhibition and catalase on Ang II–dependent L-type Ca2+ channel sparklets where activity and density are reduced.15,16 Although initially perplexing, we think that this subtle observation provides valuable mechanistic insight into the signaling events underlying oxidant-dependent regulation of L-type Ca2+ channels. Activation of Ang II type 1 receptors stimulates NADPH oxidase resulting in ROS formation.40,41 Evidence suggests that ROS produced by NADPH oxidase can induce subsequent ROS generation from mitochondria through the process of ROS-induced ROS release.8,31,32 We suggest that an ROS-induced ROS release mechanism, initiated by NADPH oxidase and performed by proximate mitochondria, is essential for Ang II–dependent stimulation of L-type Ca2+ channels in arterial smooth muscle.
This hypothesis is compatible with our data. Disrupting Ang II signaling by NADPH oxidase inhibition or enhancing H2O2 decomposition with catalase reduces the initial availability of signaling H2O2 and abolishes the stimulatory effect of Ang II on L-type Ca2+ channel sparklets (ie, activity and density).15,16 However, mitoTEMPO impairment of H2O2 generation occurs further along the signaling cascade by limiting ROS-induced ROS release by mitochondria. Thus, the initial production of signaling H2O2 by NADPH oxidase remains intact, which could trigger limited stimulation of L-type Ca2+ channels leading to an increase in low-activity L-type Ca2+ channel sparklet sites. From this, we suggest that mitochondria, as a consequence of providing a means of ROS-induced ROS release, function as amplifiers of NADPH oxidase–initiated H2O2 microdomain signaling, and that this mechanism is necessary for physiological stimulation of arterial smooth muscle cell L-type Ca2+ channels by Ang II (Figure 7E).
If mitochondrial ROS–induced ROS release is necessary for physiological stimulation of L-type Ca2+ channels by Ang II, then disrupting this process with mitoTEMPO should reduce arterial contractile responses to Ang II. Consistent with this, mitoTEMPO reduced Ang II–dependent contraction of pressurized cerebral arteries by ≈70%. In contrast, MitoTEMPO had no effect on the baseline myogenic tone of these arteries. Similarly, we noted previously that inhibition of NADPH oxidase15 and application of cell-permeable catalase16 reduced contractile responses to Ang II without altering arterial tone. These observations suggest that, at least in cerebral arteries from healthy rats, H2O2 microdomain stimulation of smooth muscle L-type Ca2+ channels contributes to the vasoconstrictor capacity of Ang II but does not influence baseline myogenic function.
In contrast to our observations, work by others has shown that mitochondrial and NADPH oxidase–derived ROS reduce arterial contractility by enhancing activation of hyperpolarizing large-conductance, Ca2+-activated potassium (BK) channels.42,43 We contend that our H2O2 microdomain signaling hypothesis reconciles these seemingly diametrically opposed observations. In the case of our data, H2O2 microdomain signaling is coupled to the activation of L-type Ca2+ channels leading to contraction. For vasodilatory-associated ROS, we hypothesize that discrete ROS signaling events are selectively coupled to vasodilatory processes. In addition, we hypothesize that ROS microdomain signaling attains differential coupling specificity as a consequence of distinct subcellular distribution patterns inherent to the underlying ROS-generating mechanisms (Online Data Supplement iv).
Anomalous Ang II signaling is implicated in the development of arterial dysfunction and hypertension.6,32,44 We found that mitoTEMPO attenuated the vasoconstrictive capacity of Ang II, and, conversely, that stimulating mitochondrial ROS production with antimycin increased L-type Ca2+ channel activity. Therefore, we examined the efficacy of mitoTEMPO in mitigating arterial smooth muscle dysfunction in hypertension. Systemic administration of mitoTEMPO blunts the hypertensive response to Ang II infusion in mice, at least in part, by increasing the bioavailability of nitric oxide and preserving endothelial function.8 To investigate the effect of mitoTEMPO on arterial smooth muscle function, we used a nitric oxide–deficient model of hypertension where endothelial dysfunction is an causal hallmark.34,45 Similar to Ang II infusion,8 coadministration of mitoTEMPO reduced the hypertensive response to L-NAME. This observation suggests that the antihypertensive effect of mitoTEMPO resides, at least in part, at the level of arterial smooth muscle.
To examine the effect of systemically administered mitoTEMPO on arterial smooth muscle function, we isolated and pressurized cerebral arteries obtained from L-NAME–treated rats. Arteries from these rats contracted to ≈50% below their passive diameter when pressurized to 60 mm Hg. In contrast, arteries from L-NAME–treated rats infused with mitoTEMPO contracted to only ≈30% below their passive diameter. For perspective, the control arteries used in our Ang II/mitoTEMPO experiments contracted to ≈38% below their passive diameter, which was not significantly different from that seen with mitoTEMPO. Thus, systemically administered mitoTEMPO normalized the intrinsic myogenic set point of arteries isolated from L-NAME–treated rats.
To investigate the mechanisms by which mitoTEMPO normalized arterial function in L-NAME–treated rats, we examined L-type Ca2+ channel and H2O2 microdomain signaling in isolated cells. Similar to Ang II–dependent and genetic hypertension,28 basal L-type Ca2+ channel sparklet activity and ROS puncta density were elevated in cells from hypertensive L-NAME–treated rats. Strikingly, arterial smooth muscle cells isolated from rats infused with mitoTEMPO showed no increase in either ROS puncta density or L-type Ca2+ channel sparklet activity. From these data, we conclude that mitoTEMPO preserves arterial function and reduces arterial blood pressure, at least in part, by inhibiting mitochondrial amplified H2O2 microdomain-dependent stimulation of arterial smooth muscle L-type Ca2+ channels.
The implications of our observations are broad. First, we suggest that mitochondrial amplification of H2O2 microdomain signaling in arterial smooth muscle is a potentially viable therapeutic target for reducing hypertension-associated arterial dysfunction. Second, because mitochondria and L-type Ca2+ channels are widely distributed, we suggest that localized mitochondrial amplification of H2O2 microdomain signaling could be a general mechanism for promoting Ca2+ influx through L-type Ca2+ channels in many cell types, including cardiac myocytes as demonstrated by others.46–48 Indeed, we recently described a mechanism responsible for localized L-type Ca2+ channel function in neuroendocrine pituitary gonadotrope cells, which bears a striking resemblance to our observations in smooth muscle49 Finally, this mechanism could be an important factor contributing to the exaggerated ROS production and Ca2+ influx associated with the pathogenesis of numerous cardiovascular and noncardiovascular diseases.
We thank Dr Chao-Yin Chen for the use of the telemetry system.
Sources of Funding
This work was supported by the National Institute of Health grants 1R01HL111060 (to Dr Amberg) and 1R01HL098200 and 1R01HL121059 (to Dr Navedo), the American Heart Association (14GRNT18730054; to Dr Navedo), the Colorado State University College Research Council (to Dr Amberg), and the Pew Charitable Trusts (to Dr Amberg).
In August 2015, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.31 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.115.306996/-/DC1.
- Nonstandard Abbreviations and Acronyms
- Ang II
- angiotensin II
- 5-(and-6)-chloromethyl-2’7’-dichlorodihydrofluorescein diacetate acetyl ester
- Nω-nitro-l-arginine methyl ester
- nicotinamide adenine dinucleotide phosphate
- protein kinase C
- reactive oxygen species
- total internal reflection fluorescence
- Received June 9, 2015.
- Revision received September 14, 2015.
- Accepted September 18, 2015.
- © 2015 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Angiotensin II (Ang II) is a clinically targeted endogenous vasoconstrictor implicated in the development of cardiovascular diseases, including hypertension and congestive heart failure.
In arterial smooth muscle, Ang II receptor activation promotes localized reactive oxygen species (ROS) generation by nicotinamide adenine dinucleotide phosphate oxidase, which is associated with colocalized calcium (Ca2+) influx through L-type Ca2+ channels.
Mitochondria are also a source of ROS generation and are known to integrate Ca2+ and ROS signaling pathways in many cells.
What New Information Does This Article Contribute?
Mitochondria residing near the plasma membrane of rat cerebral arterial smooth muscle cells are associated with sites of elevated Ca2+ influx through L-type Ca2+ channels.
Following Ang II exposure, subplasmalemmal mitochondria amplify localized ROS (hydrogen peroxide) production initiated by nicotinamide adenine dinucleotide phosphate oxidase in signaling microdomains resulting in protein kinase C–dependent activation of neighboring L-type Ca2+ channels.
This mitochondrial ROS–dependent stimulation of L-type Ca2+ channels contributes to contraction of rat cerebral arteries by Ang II.
Disrupting mitochondrial ROS production in vivo normalizes arterial function and attenuates the hypertensive response to pharmacologically induced systemic endothelial dysfunction.
Mitochondria are a major source of cellular ROS. In addition, mitochondrial dysfunction is associated with cardiovascular disease. However, the importance of mitochondrial ROS in Ang II–dependent arterial contraction and in hypertension-associated arterial dysfunction is unclear. In this study, we tested the hypothesis that mitochondrial ROS generation regulates the activity of L-type Ca2+ channels in rat cerebral arterial smooth muscle in the context of Ang II signaling. Using an image-based approach, we found that mitochondrial amplification of ROS (hydrogen peroxide) signaling near the plasma membrane stimulates L-type Ca2+ channels. By providing a means of increased Ca2+ influx, this mechanism contributes to Ang II–dependent arterial contraction. Our data also show that disrupting this mitochondrial amplification mechanism in vivo normalizes arterial function and attenuates the hypertensive response to systemic endothelial dysfunction. From these observations, we conclude that mitochondrial amplification of hydrogen peroxide signaling leads to increased Ca2+ influx through L-type Ca2+ channels. We suggest that this mechanism plays an important role in arterial smooth muscle physiology and contributes to arterial dysfunction in hypertension. We also propose that mitochondrial amplification of ROS signaling in arterial smooth muscle is a potentially viable therapeutic target for reducing hypertension-associated arterial dysfunction.