Mitochondria Control Functional CaV1.2 Expression in Smooth Muscle Cells of Cerebral ArteriesNovelty and Significance
Rationale: Physiological functions of mitochondria in contractile arterial myocytes are poorly understood. Mitochondria can uptake calcium (Ca2+), but intracellular Ca2+ signals that regulate mitochondrial Ca2+ concentration ([Ca2+]mito) and physiological functions of changes in [Ca2+]mito in arterial myocytes are unclear.
Objective: To identify Ca2+ signals that regulate [Ca2+]mito, examine the significance of changes in [Ca2+]mito, and test the hypothesis that [Ca2+]mito controls functional ion channel transcription in myocytes of resistance-size cerebral arteries.
Methods and Results: Endothelin (ET)-1 activated Ca2+ waves and elevated global Ca2+ concentration ([Ca2+]i) via inositol 1,4,5-trisphosphate receptor (IP3R) activation. IP3R-mediated sarcoplasmic reticulum (SR) Ca2+ release increased [Ca2+]mito and induced mitochondrial depolarization, which stimulated mitochondrial reactive oxygen species (mitoROS) generation that elevated cytosolic ROS. In contrast, a global [Ca2+]i elevation did not alter [Ca2+]mito, mitochondrial potential, or mitoROS generation. ET-1 stimulated nuclear translocation of nuclear factor (NF)-κB p50 subunit and ET-1–induced IP3R-mediated mitoROS elevated NF-κB–dependent transcriptional activity. ET-1 elevated voltage-dependent Ca2+ (CaV1.2) channel expression, leading to an increase in both pressure (myogenic tone)– and depolarization-induced vasoconstriction. Baseline CaV1.2 expression and the ET-1–induced elevation in CaV1.2 expression were both reduced by IP3R inhibition, mitochondrial electron transport chain block, antioxidant treatment, and NF-κB subunit knockdown, leading to vasodilation.
Conclusions: IP3R-mediated SR Ca2+ release elevates [Ca2+]mito, which induces mitoROS generation. MitoROS activate NF-κB, which stimulates CaV1.2 channel transcription. Thus, mitochondria sense IP3R-mediated SR Ca2+ release to control NF-κB–dependent CaV1.2 channel expression in arterial myocytes, thereby modulating arterial contractility.
Intracellular calcium (Ca2+) signals modulate a wide variety of physiological functions in arterial myocytes, including contractility and gene expression.1 The spatial location of mitochondria nearby Ca2+ channels can expose these organelles to domains of elevated intracellular Ca2+ concentration ([Ca2+]i), leading to mitochondrial Ca2+ uptake.2,3 Mitochondrial Ca2+ sequestration can reduce the elevation and diffusion of cytosolic Ca2+ signals and feedback to alter the activity of Ca2+ channels that generate the signal.2,–,4 Mitochondria can also generate signaling molecules, including reactive oxygen species (ROS), which modulate local and global intracellular Ca2+ signals, leading to changes in arterial contractility.2,3,5 Although it is recognized that mitochondria can sequester Ca2+, intracellular Ca2+ signals that regulate mitochondrial Ca2+ concentration ([Ca2+]mito) and physiological functions of changes in [Ca2+]mito in arterial myocytes are poorly understood.
Arterial myocytes generate several distinct local and global Ca2+ signals.1,6 An elevation in global [Ca2+]i occurs in response to plasma membrane Ca2+ influx and sarcoplasmic reticulum (SR) Ca2+ release and directly regulates vascular contractility.1 Voltage-dependent Ca2+ (CaV1.2) channels are the major contributor to global [Ca2+]i in arterial myocytes and are essential for diameter regulation in resistance-size arteries that regulate blood pressure and regional blood flow.1,7,8 Ca2+ sparklets are subsarcolemmal Ca2+ signals generated by Ca2+ influx through CaV1.2 channels.6 Ca2+ sparklets contribute directly to global [Ca2+]i, thereby regulating contractility.6 Ca2+ sparks are local Ca2+ transients generated by SR ryanodine-sensitive Ca2+ release channels.1 Ca2+ sparks activate large-conductance Ca2+-activated potassium channels, leading to membrane hyperpolarization and vasodilation.1 Ca2+ waves are propagating Ca2+ transients that can occur because of the activation of SR inositol 1,4,5-trisphosphate receptor (IP3R) channels and ryanodine-sensitive Ca2+ channels.9 Physiological functions of Ca2+ waves are less clear, with studies reporting that these Ca2+ signals either directly stimulate contraction or do not alter contractility.10,–,12
Here, we investigated Ca2+ signaling mechanisms that regulate [Ca2+]mito in myocytes of resistance-size cerebral arteries and tested the hypothesis that changes in [Ca2+]mito control the expression of CaV1.2, an ion channel whose transcriptional regulation in arterial myocytes is unclear. Our data indicate that mitochondria sense IP3R-mediated SR Ca2+ release to control mitochondrial ROS (mitoROS) generation, nuclear factor (NF)-κB activity, and functional CaV1.2 expression in arterial myocytes.
Animal protocols were reviewed and approved by the Animal Care and Use Committee of the University of Tennessee Health Science Center. All experiments were performed using Sprague–Dawley rat (≈250 g) resistance-size (≈50 to 200 μm diameter) cerebral arteries or myocytes isolated from these arteries as described previously.7
Laser-Scanning Confocal Ca2+ Imaging
Intracellular Ca2+ signals were imaged in myocytes of cerebral arteries using fluo-4 AM and a Noran Oz laser-scanning confocal microscope, as described previously.13
Imaging of Genetically Encoded Fluorescent Indicators
Vectors encoding 2mt8CG2, mt-cpYFP, or HyPer-CYTO were inserted into myocytes of intact cerebral arteries using reverse permeabilization. Expressed fluorescent indicators were imaged using a Zeiss LSM5 confocal microscope. Indicator localization was determined in myocytes isolated from arteries through colocalization with MitoTracker Orange CMTMRos using weighted colocalization.
Tetramethylrhodamine Methyl Ester Imaging
Isolated arterial myocytes were loaded with tetramethylrhodamine methyl ester (TMRM) and excited with 535-nm light. Background corrected TMRM fluorescence was collected at 610 nm using a Dage MTI iCCD camera.
Paraformaldehyde-fixed arteries were incubated with antibodies against NF-κB p50 subunit (p50), followed by Cy3-conjugated secondary antibody. YOYO-1 was used to counterstain nuclei. Images were obtained using a Zeiss LSM5 confocal microscope. Weighted colocalization was used to quantify p50 nuclear localization.
NF-κB–Dependent Luciferase Reporter Gene Activity
Vectors that express firefly luciferase under the control of an NF-κB promoter (NF-κB-p-Luc) were inserted into myocytes of intact arteries using reverse permeabilization. Promoter-deficient pGL3 Basic control vector containing a luciferase reporter gene was used as a control. Luciferase activity was quantified using a luminometer.
NF-κB p105 Subunit Knockdown
Two small interfering (si)RNAs directed against NF-κB p105 subunit (p105) (p105siRNA1 and p105siRNA2) were used, with scrambled siRNA (p105scrm) as control. siRNA was introduced into myocytes of intact arteries using reverse permeabilization.
Pressurized Artery Diameter Measurements
Diameter was measured in endothelial-denuded arteries using edge-detection myography. Myogenic tone (percentage) was calculated as 100×(1−active diameter/passive diameter).
OriginLab and GraphPad InStat software were used for statistical analyses. Values are expressed as mean±SEM. Student t test was used for comparing paired and unpaired data from two populations, and ANOVA with Student–Newman–Keuls post hoc test used for multiple group comparisons. P<0.05 was considered significant. Power analysis was performed where P>0.05 to verify that sample size was sufficient to give a power value >0.8.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
ET-1 Modifies Local and Global Ca2+ Signals in Cerebral Artery Myocytes
The regulation of local and global Ca2+ signals by endothelin (ET)-1, a phospholipase C–coupled receptor agonist and vasoconstrictor, was studied in myocytes of endothelium-denuded cerebral artery segments. ET-1 reduced mean Ca2+ spark frequency to ≈32% of control, elevated mean Ca2+ wave frequency to ≈135% of control, and increased mean global [Ca2+]i to ≈127% of control (Figure 1 A through 1D). Xestospongin C (XeC), an IP3R inhibitor, blocked ET-1–induced Ca2+ wave activation and reduced ET-1–induced global [Ca2+]i elevation, but did not alter ET-1–induced Ca2+ spark inhibition (Figure 1D).
ET-1–Induced IP3R-Mediated SR Ca2+ Release Elevates [Ca2+]mito and Depolarizes Mitochondria in Cerebral Artery Myocytes
Regulation of [Ca2+]mito by ET-1–induced Ca2+ signals was measured in myocytes of intact arteries using 2mt8CG2 (Kd for Ca2+, ≈5 μmol/L), a genetically encoded, mitochondria-targeted, fluorescent Ca2+ indicator.14,15 Confocal imaging revealed punctate 2mt8CG2 fluorescence that exhibited ≈95% pixel colocalization with MitoTracker Orange, indicating mitochondrial localization (Figure 2A). In myocytes of intact arteries, ionomycin, a Ca2+ ionophore, and carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a protonophore that disrupts mitochondrial potential, caused fluorescence changes in 2mt8CG2 consistent with the range of this indicator (Figure 2C ).15 ET-1 increased mean 2mt8CG2 fluorescence to ≈140% of control, and this elevation was blocked by thapsigargin, a SR Ca2+ ATPase inhibitor, XeC, or Ru360, a mitochondrial Ca2+ uniporter blocker (Figure 2B and 2C). Thapsigargin, XeC, or Ru360 alone, or membrane depolarization (60 mmol/L K+), which elevates global [Ca2+]i, did not alter 2mt8CG2 fluorescence (Figure 2C).
Mitochondria potential regulation by ET-1 was studied in isolated myocytes using TMRM. ET-1 caused reproducible concentration-dependent mitochondrial depolarization with an EC50 of 29 nmol/L (Figure 3 A through 3C). Thapsigargin and XeC abolished ET-1–induced mitochondrial depolarization, but did not alter TMRM fluorescence when applied alone (Figure 3A and 3C). CCCP depolarized mitochondria, whereas 60 mmol/L K+ did not alter mitochondrial potential (Figure 3A and 3C). These data indicate that ET-1–induced IP3R-mediated SR Ca2+ release elevates [Ca2+]mito and depolarizes mitochondria in arterial myocytes. In contrast, a global [Ca2+]i elevation does not alter [Ca2+]mito or mitochondrial potential.
ET-1–Induced IP3R-Mediated SR Ca2+ Release Stimulates mitoROS Generation
We tested the hypothesis that a [Ca2+]mito elevation may alter mitoROS generation. MitoROS was measured using mt-cpYFP, a genetically encoded mitochondria-targeted fluorescent O2−· indicator.16 mt-cpYFP exhibited ≈95% pixel colocalization with MitoTracker Orange, indicating mitochondrial localization (Figure 4A). In myocytes of intact arteries, ET-1 elevated mt-cpYFP fluorescence to ≈199% of control, and this was blocked by XeC, Ru360, rotenone, a mitochondrial electron transport chain (ETC) complex I inhibitor, and micromolar CCCP (Figure 4B and Online Figure I). Rotenone and micromolar CCCP reduced mt-cpYFP fluorescence when applied alone (Figure 4B ). In contrast, Ru360, XeC, and 60 mmol/L K+ did not alter mt-cpYFP fluorescence (Figure 4B ). CCCP (1 nmol/L), which induces a small mitochondrial depolarization in cerebral artery myocytes,5 elevated mt-cpYFP fluorescence (Figure 4B).
Cytosolic ROS was measured using HyPer-CYTO, a genetically encoded fluorescent cytosolic hydrogen peroxide (H2O2) indicator (Online Figure II, A).17 Exogenous H2O2 elevated HyPer-CYTO fluorescence (Figure 4C ; Online Figure II, B). ET-1 similarly increased HyPer-CYTO fluorescence in myocytes of endothelium-intact and -denuded arteries (Figure 4C ; Online Figure II, C). Rotenone and manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a superoxide dismutase and catalase mimetic, reduced HyPer-CYTO fluorescence when applied alone, and blocked ET-1–induced elevations in HyPer-CYTO fluorescence (Figure 4C ).
Similar results were also obtained using CM-H2DCFDA, an inorganic fluorescent ROS indicator (Online Figure III, A through D). Oxypurinol, a xanthine oxidase inhibitor, 17-octadecanoic acid, a cytochrome P450 blocker, or gp91ds-tat, a NADPH oxidase inhibitor, did not alter baseline dichlorofluorescein (DCF) fluorescence or ET-1–induced DCF fluorescence elevations (Online Figure III, C through D). gp91ds-tat inhibited DCF fluorescence elevations induced by tumor necrosis factor (TNF)-α, which stimulates NADPH oxidase-derived ROS in cerebral artery myocytes18 (Online Figure III, D). Scrambled gp91-tat did not alter baseline DCF fluorescence, or ET-1– or TNF-α–induced DCF fluorescence elevations (Online Figure III, D).
These data indicate that mitochondria generate ROS in the absence of ET-1, and that ET-1–induced IP3R-mediated SR Ca2+ release elevates mitoROS generation, leading to an increase in cytosolic ROS in cerebral artery myocytes.
ET-1–Induced IP3R-Mediated mitoROS Stimulate NF-κB
To examine physiological functions of an IP3R-mediated mitoROS elevation, we tested the hypothesis that mitochondria regulate the expression of genes that modulate myocyte contractility. Therefore, immunofluorescence was performed to study cellular localization of the p50 subunit of NF-κB, a ROS-sensitive transcription factor,19 in arterial myocytes. ET-1 increased nuclear translocation of p50, measured as an increase in mean pixel colocalization with YOYO-1 from ≈12% to 22% (Figure 5A through 5C).
To investigate the regulation of NF-κB–dependent transcriptional activity, assays were performed using vectors that express firefly luciferase under the control of an NF-κB promoter. As a control, regulation of luciferase activity by TNF-α, which activates NF-κB was measured. TNF-α increased mean luciferase activity to ≈547% of control (Figure 5D ). ET-1 increased luciferase activity to ≈372% of control, and this was blocked by thapsigargin and XeC (Figure 5D ). Rotenone and MnTMPyP reduced the ET-1–induced elevation in luciferase activity (Figure 5D ). Rotenone also blocked the ET-1–induced ROS elevation over the 24-hour period used for these transcriptional measurements (Online Figure IV). Exogenous H2O2 elevated luciferase activity, and this effect was not altered by rotenone, indicating that rotenone did not cause general inhibition of luciferase expression (Figure 5D ). When applied alone, thapsigargin, XeC, rotenone, and MnTMPyP reduced luciferase activity by ≈16, 22, 23, and 20%, respectively (Figure 5D ). TNF-α, ET-1, thapsigargin, XeC, rotenone, MnTMPyP, and H2O2 did not alter luciferase activity in arteries in which a promoter-deficient control vector was inserted (Online Figure V). These data indicate that IP3R-mediated SR Ca2+ release stimulates NF-κB primarily by elevating mitoROS generation, but also via a secondary mitochondria- and ROS-independent pathway in arterial myocytes.
IP3R-Mediated SR Ca2+ Release Stimulates NF-κB and CaV1.2 Expression
We sought to examine the functional significance of IP3R-mediated, mitochondrial-dependent NF-κB activation. CaV1.2 channels are the principal Ca2+ influx pathway in myocytes of resistance-size arteries and are essential for contractility regulation by a wide variety of stimuli, including intravascular pressure and membrane potential.8 However, mechanisms that regulate CaV1.2 gene (CACNA1C) transcription in arterial myocytes are unclear. Real-time PCR data indicated that ET-1 (6 hours) increased CaV1.2 channel mRNA expression to ≈244% of control in cerebral arteries (Figure 6A and Online Figure VI). Western blotting indicated that ET-1 (24 hours) increased CaV1.2 channel (190 and 240 kDa bands) protein to ≈141% of control (Figure 6B and 6C). XeC, rotenone, and MnTMPyP alone reduced basal CaV1.2 expression to ≈87, 89 and 80% of control, respectively (Figure 6B and 6C). XeC blocked, and rotenone and MnTMPyP reduced the ET-1–induced elevation in CaV1.2 expression (Figure 6B and 6C). Exogenous H2O2 increased CaV1.2 expression, and this elevation was not altered by rotenone, indicating that rotenone did not induce nonspecific inhibition of CaV1.2 expression (Figure 6C ). These data indicate that ET-1–induced IP3R-mediated SR Ca2+ release and mitoROS elevation stimulate CaV1.2 expression in cerebral arteries.
Two different siRNAs were used to knockdown expression of p105, the p50 precursor. p105siRNA1, p105siRNA2, and a combination of both siRNAs (p105siRNAs) reduced mean p105 protein to ≈73, 80, and 57%, respectively, of that in arteries treated with scrambled siRNA (p105scrm) (Online Figure VII, A and B). p105siRNAs also reduced p50 expression to ≈53% of p105scrm (Online Figure VIII, C and D). p105siRNAs reduced CaV1.2 expression to ≈64% of p105scrm (Figure 7 A and 7B). In p105scrm-treated arteries, ET-1 increased CaV1.2 protein to ≈136% of p105scrm (Figure 7A and 7B). p105siRNAs reduced the ET-1–induced elevation in CaV1.2 expression by ≈71% (Figure 7A and 7B). These data indicate that NF-κB controls basal CaV1.2 expression and that IP3R-mediated SR Ca2+ release stimulates CaV1.2 expression via mitochondria-dependent NF-κB activation.
The p105 gene promoter contains an NF-κB–binding sequence, and p50 activation can elevate p105 expression.20 Therefore, mechanisms that regulate NF-κB subunit expression in cerebral arteries were investigated. XeC reduced basal p105 and p50 expression, whereas rotenone or MnTMPyP did not alter basal p105 or p50 expression (Online Figure VIII, A and B). ET-1 elevated p105 and p50 expression and XeC blocked this effect (Online Figure VIII, A and B). In contrast, rotenone or MnTMPyP had no effect on the ET-1–induced elevation in p105 and p50 subunit expression (Online Figure VIII, B). Exogenous H2O2 applied alone or in the presence of rotenone did not change p105 and p50 expression (Online Figure VIII, B). p105siRNAs did not alter the ET-1–induced relative increase in p105 and p50 expression (Online Figure VIII, C and D). These data indicate that IP3R-mediated SR Ca2+ release activates p105/p50 expression via a mitochondria-, ROS-, and NF-κB–independent pathway. Data also indicate that rotenone, MnTMPyP, and p105siRNAs do not cause general inhibition of gene transcription.
NF-κB Stimulates Functional CaV1.2 Expression in Cerebral Artery Myocytes
NF-κB regulation of functional CaV1.2 expression was examined in pressurized cerebral arteries. Arteries were treated with either p105scrm or p105siRNAs and then exposed to either no further treatment or to ET-1 for 24 hours. At 60 mm Hg, control arteries (p105scrm) developed ≈21% myogenic tone and membrane depolarization with 60 mmol/L K+ elevated tone to ≈45%. p105 knockdown (p105siRNAs) reduced myogenic tone and depolarization-induced vasoconstriction (Figure 7C and 7D). A 24 hour exposure to ET-1 elevated mean myogenic tone and depolarization-induced tone in control arteries (Figure 7C and 7D). p105 knockdown attenuated the ET-1–induced elevation in myogenic tone and depolarization-induced vasoconstriction (Figure 7C and 7D). Nimodipine fully dilated arteries regardless of treatment, indicating that vasoconstriction occurred because of CaV1.2 channel activation (Figure 7C and 7D). These data indicate that NF-κB is essential for functional basal CaV1.2 expression in cerebral artery myocytes and that ET-1–induced NF-κB activation elevates CaV1.2-dependent vasoconstriction.
Here, we investigated physiological signaling mechanisms that regulate [Ca2+]mito and functional consequences of changes in [Ca2+]mito in myocytes of resistance-size cerebral arteries. A schematic diagram summarizing major findings of this study is provided in Figure 8. Our data indicate that ET-1–induced IP3R-mediated SR Ca2+ release increases Ca2+ wave frequency, elevates [Ca2+]mito and depolarizes mitochondria, leading to an increase in mitoROS generation. ET-1–induced IP3R-mediated mitoROS elevate cytosolic ROS that increase NF-κB nuclear translocation and transcriptional activity. ET-1–induced NF-κB activation leads to an increase in CaV1.2 transcription and CaV1.2-dependent pressure- and depolarization-induced vasoconstriction. IP3R-mediated SR Ca2+ release, mitoROS, and NF-κB control basal CaV1.2 expression and NF-κB knockdown reduces CaV1.2 expression, leading to vasodilation. Collectively, these data indicate that mitochondria sense IP3R-mediated SR Ca2+ release to control functional CaV1.2 channel transcription in arterial myocytes, thereby regulating arterial contractility.
Phospholipase C–coupled receptor agonists elevate IP3, which stimulates IP3Rs, and diacylglycerol, which activates protein kinase C. Vasoconstrictor-induced IP3R activation elevates [Ca2+]i in arterial myocytes via 2 distinct mechanisms: (1) SR Ca2+ release, which produces Ca2+ waves9,13; and (2) SR Ca2+ release–independent plasma membrane TRPC3 channel activation, which leads to depolarization-induced CaV1.2 channel activation and a global [Ca2+]i elevation.13,21,22 In contrast, vasoconstrictor-activated protein kinase C inhibits Ca2+ sparks, leading to membrane depolarization, CaV1.2 channel activation, and a global [Ca2+]i elevation.9 The regulation of arterial myocyte [Ca2+]mito by these different intracellular Ca2+ signals was unclear. Electron microscopy indicates that mitochondria can be located in close proximity (≈20 nm) to the SR membrane in many cell types, including cultured arterial myocytes.23,24 Such localization could place mitochondria within the vicinity of SR Ca2+ release channels that generate local micromolar Ca2+ transients necessary for mitochondrial Ca2+ uptake via the uniporter.25 Inorganic Ca2+ indicators, which cannot be targeted specifically to organelles, have been used to measure [Ca2+]mito in cultured vascular myocytes. In these studies, SR Ca2+ release elicited a [Ca2+]mito elevation.24,26 Ca2+ influx via the plasma membrane Na+/Ca2+ exchanger also elevated [Ca2+]mito in cultured vascular myocytes.27 In noncultured colonic myocytes, uncaging IP3 leads to mitochondrial Ca2+ uptake that feeds back to regulate IP3R activity.28 To our knowledge, our study is the first to use a genetically encoded mitochondria-targeted fluorescent indicator to measure [Ca2+]mito in contractile arterial myocytes. Our data indicate that in resting arterial myocytes, mitochondria contain Ca2+ and generate low levels of ROS through mechanisms that are independent of IP3R-mediated SR Ca2+ release. ET-1–induced IP3R-mediated SR Ca2+ release stimulated Ca2+ waves and elevated [Ca2+]mito, leading to mitochondrial depolarization and mitoROS generation. In contrast, a global [Ca2+]i elevation did not alter [Ca2+]mito, mitochondrial potential, or mitoROS generation. These findings also raise the possibility that Ca2+ waves specifically regulate [Ca2+]mito in arterial myocytes. Given that Ca2+ waves are propagating Ca2+ signals, [Ca2+]mito may also oscillate. Here, [Ca2+]mito was simultaneously measured within multiple myocytes in the arterial wall. Asynchronous Ca2+ oscillations within individual mitochondria would have been averaged out by the imaging protocol. Future studies should be designed to examine spatial and temporal relationships between Ca2+ waves and mitochondrial Ca2+ signals within individual mitochondria in arterial myocytes.
The mechanism by which mitochondrial Ca2+ uptake induces mitochondrial depolarization and elevates mitochondrial electron transport chain–generated mitoROS was not determined, but several possibilities exist. Mitochondrial depolarization has been demonstrated to increase or decrease ROS in different cell types, including vascular myocytes.5,16,17,29 Here, a small mitochondrial depolarization increased mitoROS generation, whereas a large mitochondrial depolarization inhibited mitoROS production, consistent with an earlier report in cerebral artery myocytes.5 A [Ca2+]mito elevation may stimulate mitoROS generation through multiple mechanisms, including stimulation of the tricarboxylic acid cycle.29 In addition, a [Ca2+]mito elevation and mitochondrial depolarization can open the mitochondrial permeability transition pore,29,30 which in turn can enhance depolarization.29 Mitochondrial permeability transition pore opening elevates mitoROS generation via several pathways, including dissipation of chemical gradients across the mitochondrial membrane and diversion of electrons in the mitochondrial electron transport chain to ROS generation.16 Therefore, a [Ca2+]mito elevation and mitochondrial depolarization may stimulate mitoROS generation by opening the mitochondrial permeability transition pore. MitoROS production is also regulated by multiple additional factors, including redox status of respiratory substrates, and proton pumping by mitochondrial electron transport chain complexes.31 Future investigations should examine the mechanisms by which a vasoconstrictor-induced [Ca2+]mito elevation and mitochondrial depolarization stimulate mitoROS generation.
Regulation and physiological functions of NF-κB in contractile arterial myocytes are poorly understood. Data here indicate that ET-1–induced NF-κB activation occurs primarily via IP3R-mediated mitoROS generation in cerebral artery myocytes. MitoROS activate NF-κB in several cell types, including cultured vascular myocytes.19,32 Alternate ROS-mediated mechanisms can also activate NF-κB. For example, following balloon injury, NAD(P)H oxidase–derived ROS stimulate NF-κB in arterial myocytes.33 The mechanisms by which ROS activate NF-κB are unclear, with reports suggesting that ROS stimulate IκB kinase, leading to phosphorylation and proteasomal degradation of IκB.32 Other studies indicate that ROS activate kinases other than IκB kinase to phosphorylate IκB.34 Additional studies will be necessary to identify the specific ROS involved and the mechanisms by which ROS activate NF-κB in contractile arterial myocytes. Data reported here indicate that IP3R-mediated SR Ca2+ release also activates NF-κB via a secondary mitoROS-independent pathway. Supporting bimodal activation, redox-dependent and -independent mechanisms activate NF-κB in U937 cells.35 Redox-independent NF-κB activation mechanisms may be mediated by calcineurin, PI3K/Akt, and/or protein kinase C, as demonstrated in neurons.36
CaV1.2 channels are the principal functional Ca2+ influx pathway in myocytes of resistance-size arteries.1,7,8 Our data indicate that basal CaV1.2 expression is controlled through both an IP3R-mediated mitochondria-independent pathway and an IP3R-independent mitoROS pathway acting through NF-κB. ET-1–induced IP3R activation stimulates an NF-κB–dependent elevation in CaV1.2 expression primarily via a mitoROS-dependent pathway and via a secondary mitochondria-independent pathway. In the absence of phospholipase C–coupled receptor ligands, intracellular IP3 concentration ([IP3]i) and thus, IP3R activity should be low. However, in the intact artery preparation studied here endothelial cell release of receptor ligands, including ET-1, may generate low levels of [IP3]i in myocytes. In our experiments, the degree of p105 knockdown and the reduction in basal CaV1.2 expression were similar, indicating that NF-κB is a major CaV1.2 gene transcription activator in arterial myocytes. In contrast, in colonic myocytes NF-κB p50 and p65 subunit activation reduced CaV1.2 expression.37 Opposing regulation by NF-κB in these different myocyte types may occur through interaction with different κB binding motifs, of which there are several upstream of the CaV1.2 gene.37 Furthermore, the presence or absence of additional transcriptional activators and/or repressors may explain differential regulation of CaV1.2 expression by NF-κB. Our data also indicate that ET-1–induced IP3R-mediated SR Ca2+ release stimulates NF-κB subunit expression through a mitochondria-, ROS-, and NF-κB–independent pathway. The ET-1–induced elevation in NF-κB expression may serve to amplify CaV1.2 expression. In rat renal arteries, membrane depolarization increased CaV1.2 protein,38 whereas, here, IP3Rs were necessary for ET-1–induced elevation in CaV1.2 expression. Data from these studies raise several possibilities, including that local and global Ca2+ signals regulate CaV1.2 expression by different mechanisms in cerebral and renal artery myocytes. Conceivably, this could occur at many levels, including that global Ca2+ may regulate [Ca2+]mito in renal artery myocytes, leading to ROS generation and NF-κB activation.
Data reported here and in previous studies suggest that vasoconstrictors cause a similar shift in local and global Ca2+ signals in myocytes of anatomically different arteries.9,39 Therefore, IP3R regulation of Ca2+ waves, [Ca2+]mito, and mitoROS generation may be a common mechanism by which vasoconstrictors regulate myocyte NF-κB activity and functional CaV1.2 expression. IP3R-mediated SR Ca2+ release and local Ca2+ influx through CaV1.2 channels also stimulates calcineurin-dependent nuclear translocation of NFATc3 (nuclear factor of activated T cell c3) in arterial myocytes.40,41 Data here not only indicate that IP3Rs control physiological CaV1.2 expression via a mitoROS/NF-κB pathway but also raise the possibility that disease-associated alterations in ion channel expression may also occur through activation of this pathway. Many vasoconstrictors, including ET-1, are elevated in cardiovascular diseases, including systemic hypertension.42 Hypertension is also associated with an increase in vascular ROS, NF-κB activity, and CaV1.2 protein and currents.43,–,45 Therefore, targeting this transcriptional pathway may be beneficial in treating cardiovascular diseases.
In summary, this study indicates that mitochondria sense IP3R-mediated SR Ca2+ release to control the activity of NF-κB, which stimulates functional CaV1.2 expression in cerebral artery myocytes.
Sources of Funding
This work was supported by NIH grants HL67061, HL77678, and HL094378 (to J.H.J.). D.N. is a recipient of a Predoctoral Fellowship from the American Heart Association Greater Southeast Affiliate (R079008156).
We thank Drs John Bannister and Adebowale Adebiyi for comments on the manuscript and Drs Lidia A. Gardner and John Cox for technical assistance with immunofluorescence analysis.
In May 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.6 days.
Non-standard Abbreviations and Acronyms
- intracellular Ca2+ concentration
- mitochondrial Ca2+ concentration
- voltage-dependent Ca2+ channel
- carbonyl cyanide 3-chlorophenylhydrazone
- baseline fluorescence
- gp91phox docking sequence peptide conjugated to tat
- scrambled gp91phox peptide conjugated to tat
- inositol 1,4,5-trisphosphate
- intracellular inositol 1,4,5-trisphosphate concentration
- inositol 1,4,5-trisphosphate receptor
- mitochondrial reactive oxygen species
- manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin
- nuclear factor κB
- vectors that express firefly luciferase under the control of an nuclear factor κB promoter
- NF-κB p105 subunit
- small interfering RNA directed against NF-κB p105 subunit
- scrambled small interfering RNA
- nuclear factor κB p50 subunit
- reactive oxygen species
- ribosomal protein S5
- small interfering RNA
- sarcoplasmic reticulum
- tetramethylrhodamine methyl ester
- tumor necrosis factor
- xestospongin C
- Received December 4, 2009.
- Revision received May 14, 2010.
- Revision received June 21, 2010.
- Accepted June 29, 2010.
- © 2010 American Heart Association, Inc.
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- Rusch NJ
- Mauban JR,
- Lamont C,
- Balke CW,
- Wier WG
- Gomez MF,
- Stevenson AS,
- Bonev AD,
- Hill-Eubanks DC,
- Nelson MT
- Nieves-Cintron M,
- Amberg GC,
- Navedo MF,
- Molkentin JD,
- Santana LF
- Abdel-Sayed S,
- Nussberger J,
- Aubert JF,
- Gohlke P,
- Brunner HR,
- Brakch N
- Wu L,
- Juurlink BH
- Wang WZ,
- Saada N,
- Dai B,
- Pang L,
- Palade P
Novelty and Significance
What Is Known?
Mitochondria can sequester Ca2+ when exposed to elevated levels of intracellular Ca2+ ([Ca2+]i).
Mitochondria generate reactive oxygen species (ROS), which can regulate the activity of a variety of downstream targets, including several transcription factors.
Voltage-dependent Ca2+ (CaV1.2) channels are a major contributor to arterial smooth muscle cell global [Ca2+]i, a Ca2+ signal that regulates blood pressure and regional blood flow.
What New Information Does This Article Contribute?
In contractile arterial smooth muscle cells, sarcoplasmic reticulum (SR) Ca2+ released by inositol 1,4,5-trisphosphate receptors (IP3R) specifically elevates mitochondrial Ca2+ concentration ([Ca2+]mito), leading to mitochondrial depolarization.
An IP3R-mediated elevation in [Ca2+]mito induces the generation of mitochondrial ROS (mitoROS), which activate nuclear factor (NF)-κB, a transcription factor.
NF-κB controls basal CaV1.2 expression and IP3R-mediated mitoROS-induced NF-κB activation elevates CaV1.2, leading to vasoconstriction.
Physiological functions of mitochondria in contractile arterial smooth muscle cells are poorly understood. Arterial smooth muscle cells generate several distinct local and global Ca2+ signals. Mitochondria can sequester Ca2+, but Ca2+ signals that regulate [Ca2+]mito and the significance of changes in [Ca2+]mito are unclear. We show that IP3R-mediated SR Ca2+ release, but not a global [Ca2+]i elevation, elevates [Ca2+]mito. An IP3R-mediated [Ca2+]mito elevation stimulates the generation of mitoROS, which activate NF-κB. IP3R-mediated mitoROS-induced NF-κB activation elevates functional CaV1.2 expression. This study indicates that mitochondria sense changes in IP3R-mediated SR Ca2+ release to alter NF-κB–mediated CaV1.2 expression, thereby modulating arterial contractility. Many cardiovascular diseases, including hypertension, are associated with altered ROS generation, NF-κB activity, CaV1.2 expression, and vascular contractility. The identification of this novel mitochondrial signaling pathway controlling arterial contractility may lead to the development of new approaches to treat cardiovascular diseases.