Published online before print November 20, 2008, doi: 10.1161/CIRCRESAHA.108.180612
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© 2009 American Heart Association, Inc.
Cellular Biology |
Mitochondrial Regulation of Sarcoplasmic Reticulum Ca2+ Content in Vascular Smooth Muscle Cells
From the Department of Cell Physiology and Metabolism (D.P., C-H.L., N.D.), University of Geneva, Switzerland; and Department of Anesthesiology, Pharmacology & Therapeutics (C-H.L., C.v.B.), University of British Columbia, Vancouver, Canada.
Correspondence to Dr Damon Poburko, Department of Cell Physiology and Metabolism, University of Geneva, 1 Michel-Servet, CH-1211 Geneva 4, Switzerland. E-mail Damon.Poburko{at}medecine.unige.ch
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Abstract |
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Subplasmalemmal ion fluxes have global effects on Ca2+ signaling in vascular smooth muscle. Measuring cytoplasmic and mitochondrial [Ca2+]and [Na+], we previously showed that mitochondria buffer both subplasmalemmal cytosolic [Ca2+] and [Na+] in vascular smooth muscle cells. We have now directly measured sarcoplasmic reticulum [Ca2+] in aortic smooth muscle cells, revealing that mitochondrial Na+/Ca2+ exchanger inhibition with CGP-37157 impairs sarcoplasmic reticulum Ca2+ refilling during purinergic stimulation. By overexpressing hFis1 to remove mitochondria from the subplasmalemmal space, we show that the rate and extent of sarcoplasmic reticulum refilling is augmented by a subpopulation of peripheral mitochondria. In ATP-stimulated cells, hFis-1–mediated relocalization of mitochondria impaired the sarcoplasmic reticulum refilling process and reduced mitochondrial [Ca2+] elevations, despite increased cytosolic [Ca2+] elevations. Reversal of plasmalemmal Na+/Ca2+ exchange was the primary Ca2+ entry mechanism following ATP stimulation, based on the effects of KB-R7943. We propose that subplasmalemmal mitochondria ensure efficient sarcoplasmic reticulum refilling by cooperating with the plasmalemmal Na+/Ca2+ exchanger to funnel Ca2+ into the sarcoplasmic reticulum and minimize cytosolic [Ca2+] elevations that might otherwise contribute to hypertensive or proliferative vasculopathies.
Key Words: hFis1 • mitochondria • fragmentation • D1ER • sarcoplasmic reticulum
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Introduction |
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Mitochondria are known to modulate smooth muscle Ca2+ signaling,1,2 but their role in the refilling of sarcoplasmic reticulum (SR) Ca2+ stores has not been extensively studied in vascular smooth muscle (VSM). Refilling of intracellular Ca2+ stores is instrumental for prolonged activation of VSM, but the mechanisms mediating SR refilling are not completely understood. Activation of vascular contraction on stimulation of G protein–coupled receptors is almost invariably initiated by Ca2+ release from the SR and sustained by Ca2+ entry variably mediated by receptor-operated cation channels (ROCCs), store-operated cation channels (SOCCs), and voltage-gated Ca2+ channels.3 This classic model is based on highly reproducible observations that removal of extracellular Ca2+ or application of Ca2+ channel blockers leaves the initial Ca2+ transient and most of the peak contraction intact but aborts the maintained increase in free cytosolic Ca2+ ([Ca2+]i) and the tonic phase of vasoconstriction. Iino et al4 refined this model of VSM activation by discovering that agonists induce asynchronous Ca2+ waves, which, when summated over large cell populations, generate sustained plateaus of both [Ca2+]i and force development. Although these asynchronous Ca2+ waves in VSM are produced by regenerative inositol-1,4,5-trisphosphate receptor–mediated Ca2+ release, Ca2+ entry from the extracellular space is required to maintain excitatory Ca2+ waves and tonic contraction.5
Present evidence strongly suggests that SR refilling in several forms of smooth muscle requires reversal of the Na+/Ca2+ exchanger (NCX) coupled to Na+ entry.5–8 Smooth muscle ROCC and SOCC are nonselective cation channels, likely composed of TRPC proteins,3,9,10 mediating greater Na+ than Ca2+ influx. Na+ entry through these channels can cause NCX reversal at junctions of the plasmalemma (PM) and SR (PM-SR junctions), where clustering of TRPCs, NCX and Na+/K+-APTase-
211–14 combined with limited ionic diffusion15 generates localized high [Na+] elevations (LNats).16 Quantitative modeling suggests that extensive (
500 nm diameter) but narrow (20 nm) cytoplasmic nanodomains within the PM-SR junctions facilitate capture of NCX-mediated Ca2+ entry (NCE) by the sarco-/endoplasmic reticular Ca2+ ATPase (SERCA) before Ca2+ influx diffuses into the bulk cytosol.15 Consistent with this model, separation of PM-SR junctions abolishes agonist-induced [Ca2+]i oscillations and increases global [Na+]i elevations.7,17,18 Hellstrand and colleagues demonstrated that mitochondrial inhibition profoundly reduces the amplitude and increases the frequency of agonist-induced Ca2+ oscillations, consistent with a mitochondrial role in SR refilling during VSM activation.19 We and others have proposed that a subplasmalemmal population of mitochondria facilitates the linkage between Ca2+ entry and SR refilling.16,20–22 In aortic smooth muscle cells, mitochondria cooperate with the SR to buffer NCE,16,22 and purinergically stimulated Ca2+ entry enhances mitochondrial Ca2+ flux and mitochondrial NCX (mNCX) activity.22 In endothelial cells, such mitochondrial Ca2+ "funneling" promotes endoplasmic reticulum (ER) refilling in part by preventing Ca2+-dependent inactivation of store-operated channels.20 However, it is currently unclear whether a similar mechanism supports SR Ca2+ uptake of NCE in VSM, which is primarily driven by Na+-entry through ROCC.
Here we used Ca2+-sensitive proteins targeted to the SR, mitochondria and cytoplasm to investigate the role of subplasmalemmal mitochondria in NCX-dependent SR refilling during agonist stimulation. To study the role of the subplasmalemmal mitochondrial subpopulation, mitochondria were drawn away from the PM by overexpressing hFis1, a protein mediating mitochondrial fission23 and causing mitochondria to migrate toward the nucleus without changing their innate ability to take up Ca2+.24 Based on the ultrastructural effects of hFis1 and pharmacological manipulation of ion transport, we present a novel and more complete model for SR refilling in agonist-stimulated VSM, in which subplasmalemmal mitochondria increase the efficiency of Ca2+ "funneling" into the SR.
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Materials and Methods |
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Reagents
Reagents were purchased from Sigma, except KB-R7943 (Tocris, Bristol, UK), CGP-37157 and oligomycin (Calbiochem), Transfectin (Bio-Rad), pmitoDsRed (Clontech). YC3.6 (Kd
250 nmol/L),25 4mitD3-CPV (Kd, 1.2 µmol/L),26 and D1ER (Kd, 60 to 180 µmol/L)27 constructs were provided by Drs Amy Palmer and Roger Tsien (University of California, San Diego). hFis1 was provided by Dr Jean-Claude Martinou (University of Geneva, Geneva, Switzerland).23 For plasmid details, see the online data supplement, available at http://circres.ahajournals.org. Experiments were performed in HEPES-buffered saline solution (mmol/L): 145 NaCl, 5 KCl, 1 MgCl2, 10 glucose, 5 HEPES, 1.2 CaCl2, pH 7.6 at room temperature. N-Methyl-D-glucamine (NMDG) isoosmotically replaced Na+ in "0Na+" solutions.
Cell Culture and Transfection
Cultured rat aortic smooth muscle cells (RASMCs) from Dr Urs Ruegg (University of Geneva, Geneva, Switzerland)28 were used between passage 9 to 1316 and were grown on Matrigel-coated (BD Sciences) 25-mm glass coverslips for experiments. For morphological studies, cells were transfected with YC3.6 (2 µg, 4 µL of Transfectin) 1 day postplating and then mitoDsRed (1 µg) and hFis1 or pcDNA3 (2 µg, 6 µL of Transfectin) the next day. For Ca2+ measurements cells were transfected 2 days postplating (4 µL of Transfectin, 0.5 µg of calcium probe, 1.5 µg of pcDNA3 or hFis).
Calcium Measurements
Cytosolic, mitochondrial, and SR Ca2+ were measured with the FRET-based probes YC3.6cyto, 4mitD3-CPV and D1ER, respectively. Ratiometric FRET-imaging (x40, 1.3 NA, Zeiss Axiovert s100TV) was previously described (details in supplement).29 D1ER was calibrated in situ in semipermeabilized cells in intracellular solution (mmol/L: NaCl 10, KCl 135, MgCl2 1, HEPES 20, sucrose 20, digitonin 0.01, ionomycin 0.01, CCCP 0.005, pH 7.4) with [Ca2+]free from 0.003 to 10 mmol/L using HEDTA (0.4 mmol/L) to buffer 3 µmol/L and 10 µmol/L [Ca2+]free (Max Chelator v2.40, C. Patton, Stanford University, Calif). Normalized ratio values in 18 cells were fitted to: equation
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using GraphPad Prism 5.02 (GraphPad). Rmin and Rmax are fitted parameters, K'd the apparent dissociation constant and h the Hill slope. [Ca2+]SR was calculated by calibrating normalized D1ER ratios against a standard curve (Figure 1).
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Mitochondrial Localization and Fragmentation
Detailed methods are available in the online data supplement. Briefly, mitochondria were imaged in cells expressing cytosolic YC3.6 and mitoDsRed that were cotransfected with hFis1 or pcDNA. Confocal images of fixed cells were acquired and analyzed under blinded conditions.
Data Analysis
Unless otherwise stated, data are means±SE. Differences was considered significant for P<0.05. Two-population comparisons were made with Student t test (with Welch correction if nonparametric). Three-way comparisons were made with ANOVA and Bonferroni post hoc comparisons.
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Results |
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Kinetics of Agonist-Induced SR Ca2+ Depletion and Refilling
To quantify [Ca2+]SR changes in RASMCs, we used the SR-targeted ratiometric Ca2+ indicator D1ER because it responds to changes in [Ca2+] between
100 nmol/L and 1 mmol/L and has a Kd optimally tuned for measuring [Ca2+]SR (see the online data supplement).27 D1ER exhibited the typical reticular pattern expected for a SR-resident protein (Figure 1A). For each cell, D1ER ratios were normalized to the average ratio in the first 4 image pairs (R/R0). Addition of the Ca2+-mobilizing agonist ATP induced a transient decrease in D1ER ratio (Figure 1C). The maximal SR depletion occurred 50±2 seconds after ATP addition (92 cells), and R/R0 recovered to, or slightly overshot, prestimulatory levels in the sustained presence of ATP (Figure 1C). In contrast, SERCA inhibition with cyclopiazonic acid (CPA) (20 µmol/L) monophasically decreased R/R0 by 0.25±0.02 
R/R0 (18 cells), similar to observations in mouse Tibialis muscle.30 R/R0 values were converted into [Ca2+]SR against a standard curve for D1ER in situ (see Materials and Methods) fitted with a Kd of 179±13 µmol/L and Hill coefficient of 0.73±0.11 (Figure 1B), consistent with values reported in HeLa and CHO cells.30 Notably, [Ca2+]SR calibration became increasingly imprecise above 1 mmol/L Ca2+ because of probe saturation (Figure 1B). Calibration of a random selection of D1ER recordings showed that ATP transiently decreased [Ca2+]SR from 0.9 to 2.0 mmol/L at rest to 100 to 200 µmol/L, whereas passive SR depletion with CPA decreased [Ca2+]SR to 40 to 50 µmol/L (Figure 1C).
Calcium Influx Refills SR Ca2+ Stores During Agonist Stimulation
Rapid [Ca2+]SR recovery to resting levels in the presence of ATP suggested that the SR efficiently refills despite the continuous engagement of P2Y receptors by ATP. To test whether SR Ca2+ refilling depends on Ca2+ influx or internal Ca2+ recycling, we stimulated RASMCs with ATP in Ca2+-free solution, in which ATP reduced D1ER ratios by 0.138±0.009 R/R0, corresponding to a [Ca2+]SR of 198±23 µmol/L (19 cells) (Figure 2A). This degree of depletion was comparable to depletion in Ca2+-containing media (Figure 1C) but less extensive than CPA-mediated depletion. [Ca2+]SR remained depleted until extracellular Ca2+ was restored, demonstrating that SR refilling required Ca2+ entry. Previous experiments with targeted aequorins illustrated that the SR and mitochondria cooperatively buffer NCE stimulated by extracellular Na+ removal.22 To delineate further the path of Ca2+ entry, we imaged changes in mitochondrial [Ca2+] ([Ca2+]mito) with 4mitD3-CPV.
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Removing external Ca2+ shortly before stimulation did not reduce peak [Ca2+]mito elevations evoked by ATP (Figure 2B and 2C). However, steady-state [Ca2+]mito elevations were reduced by 75%. Thus, sustained [Ca2+]mito elevations were largely dependent on Ca2+ influx and remained elevated for several minutes after [Ca2+]SR had returned to basal level, consistent with increased mitochondrial Ca2+ flux during agonist stimulation.
NCX-Mediated Ca2+ Entry and Transmitochondrial Ca2+ Flux Facilitate Rapid SR Ca2+ Refilling
To test whether NCE contributes to SR refilling, we monitored ATP-mediated [Ca2+]SR depletion in the presence of KB-R9743 (10 µmol/L). Despite several nonspecific effects reported for KB-R7943, our previous experience with this compound under similar conditions strongly indicate that its effects reported here can be attributed to selective inhibition of NCE (see the expanded Discussion section in the online data supplement and Poburko et al16). KB-R7943 increased maximal [Ca2+]SR depletion from 202±31 to 115±10 µmol/L (–0.136±0.015 to –0.178±0.008R/R0, P=0.015) and delayed the time to maximal [Ca2+]SR depletion by 10 seconds (from 36.6±3.0 to 47.3±4.0 seconds, P=0.02) (Figure 3A). Importantly, KB-R7943 delayed the onset of [Ca2+]SR recovery by 35±5 seconds (P<0.001) and slowed the half-time of [Ca2+]SR recovery (24±4 seconds, control; 35±4 seconds, KB-R7943; P=0.038). Despite enhanced depletion and delayed recovery, [Ca2+]SR eventually fully recovered in the presence of KB-R7943, suggesting that alternative Ca2+ entry mechanisms were active when NCE was inhibited. To examine the role of mitochondrial Ca2+ flux in SR refilling, we used CGP-37157 (20 µmol/L) to inhibit mitochondrial Ca2+ extrusion by the mNCX, and oligomycin (5 µg/mL) to inhibit the mitochondrial F1F0-ATP synthase. Neither CGP-37157 nor oligomycin affected maximal ATP-mediated [Ca2+]SR depletion or the time to onset of recovery (Figure 3B). However, CGP-37157, but not oligomycin, reduced the extent and slowed the kinetics of [Ca2+]SR recovery (Figure 3B, i). These findings are consistent with reports that mitochondria contribute to SR/ER Ca2+ refilling by funneling Ca2+ ions extruded by the mNCX into the SR.20,31
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P<0.05 Bonferroni post hoc test against control. ND indicates no difference.
Fragmentation and Redistribution of Subplasmalemmal Mitochondria by hFis1
To determine whether mitochondrial facilitation of [Ca2+]SR refilling requires close proximity between mitochondria and Ca2+ entry pathway(s), we removed mitochondria from the subplasmalemmal space by overexpressing hFis1, a strategy previously validated in HeLa cells.24 Mitochondria in VSM are typically rod-like and form tubular networks, readily visible with mitochondria-targeted DsRed (Figure 4A, i). As expected, hFis1 overexpression caused mitochondrial fragmentation (Figure 4A, ii). hFis1 decreased average mitochondrion cross-sectional area by 30% and decreased mitochondrial form factor indicating a shift toward more spherical morphology (Figure 4A, iii and iv). However, the total mitochondrial area per cell area, an indirect measure of mitochondrial mass, was not affected by hFis1 (Figure 4B, iii). Consistent with our previous study of mitochondrial fragmentation,24 hFis1 overexpression did not reduce mitochondrial membrane potential assessed by TMRM uptake, nor did it impair cell proliferation, cell size, or indicators of apoptosis (Annexin-V labeled and nuclear condensation) (supplemental Figure I). hFis1 did, however, increase the distance from the cell perimeter to the nearest mitochondrion by 35% (control, 1.75±0.18 µm; hFis1, 2.37±0.19 µm) (Figure 4B). The percentage of pixels along the cell perimeter within 0.5, 1.0 or 1.5 µm of a mitochondrion was also decreased by 30% by hFis1 (supplemental Table I). Thus, hFis1 moved mitochondria away from the PM in RASMCs.
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Effect of hFis1 on Agonist-Induced and NCX-Mediated [Ca2+]i and [Ca2+]mito Elevations
To determine whether the separation of PM and mitochondria altered Ca2+ signals, we measured the effect of hFis1 on [Ca2+]i elevations induced by ATP and by extracellular sodium removal. hFis1 overexpression increased the amplitude of the ATP-induced [Ca2+]i transient, which is dependent on SR Ca2+ release,7 by 30% and increased the amplitude of the [Ca2+]i plateau, which depends on Ca2+ influx, by 140% (Figure 5A and 5B, i and ii). Importantly, hFis1 increased the amplitude of NCX-mediated [Ca2+]i elevations directly induced by 0Na+ by 70% (Figure 5A and 5B, iii). In contrast with its effects on [Ca2+]i, hFis1 overexpression reduced the peak [Ca2+]mito responses (reflecting SR Ca2+ release) by 25% and reduced the ATP-mediated [Ca2+]mito plateau (reflecting Ca2+ influx) by 30% (Figure 5C and 5D, i and ii). Furthermore, hFis1 reduced the amplitude of 0Na+-stimulated [Ca2+]mito elevations by 50%, consistent with reduced mitochondrial Ca2+ buffering of NCE (Figure 5C and 5D, iii).
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Effect of hFis1 on ATP-Mediated SR Ca2+ Depletion and Refilling
Finally, to test whether removal of subplasmalemmal mitochondria impaired SR refilling, we measured [Ca2+]SR changes in cells overexpressing hFis1. hFis1 significantly enhanced ATP-mediated [Ca2+]SR depletion (minimum [Ca2+]SR 0.28±0.03 mmol/L versus 0.41±0.04 mmol/L for control cells, P=0.004) (Figure 6A and 6C, i). Time to maximum [Ca2+]SR depletion was not affected (51.8±4.5 seconds, control; 53.1±4.2s hFis1, P=0.96 Mann–Whitney). In contrast, hFis1 reduced the rate and extent of [Ca2+]SR recovery (Figure 6A (inset) and 6B, ii and iii). Moreover, following ATP removal, control cells exhibited a transient [Ca2+]SR overshoot that was absent in hFis1 overexpressing cells, further indicating that hFis1 impaired SR Ca2+ loading. These effects of hFis1 overexpression on the kinetics and extent of [Ca2+]SR refilling resembled those observed on pharmacological inhibition of the mNCX with CGP-37157.
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Discussion |
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The principal and novel finding of this study is that separation of mitochondria from the PM has a profound inhibitory effect on refilling of smooth muscle SR during agonist stimulation. The separation was achieved by overexpressing hFis1, a procedure that does not alter mitochondrial Ca2+ fluxes, and SR refilling was monitored directly using the modified cameleon D1ER. Data so obtained led to a Ca2+ transport model where peripheral mitochondria funnel NCX-mediated Ca2+ entry into SR to support rapid store refilling, a process essential for maintenance of cytoplasmic Ca2+ waves and vascular tone.5
These first quantitative measurements of [Ca2+]SR in agonist-stimulated VSM demonstrated a relatively high resting [Ca2+]SR in VSM. The high [Ca2+]SR values do not reflect abnormal behavior of the genetic probe, because D1ER is well tuned to measure physiological changes in [Ca2+]SR and our calibration curve was similar to values measured in HeLa and CHO cells that display lower resting [Ca2+]ER.27 Rather, the high [Ca2+]SR in VSM may be a common characteristic of muscle tissue, as similarly high values were reported in cardiac muscle.32,33 Purinergic stimulation induced only partial depletion of SR Ca2+, half as much as was depleted by blocking Ca2+ uptake by SERCA. Interestingly, contrary to common depictions of SR/ER refilling occurring after the removal of agonist, SR refilling was initiated within 20 to 30 seconds in the continued presence of ATP, such that [Ca2+]SR returned to basal levels within 90s (Figure 1).
NCX-Mediated Ca2+ Entry Enables Rapid SR Refilling
NCE is increasingly reported to mediate Ca2+ influx in various cell types.5,6,8,18,34–36 We initially demonstrated that extracellular Ca2+ can be routed to the SR without altering bulk [Ca2+]i via preferential interaction of NCE with the superficial SR of smooth muscle.37 Subsequent observations by Blaustein and colleagues,38 Groschner and colleagues,13 Moore et al,14 and our group5,15 provided structural and functional evidence that reverse-mode NCX can be coupled to Na+ entry through ROCCs or SOCCs to mediate SR Ca2+ refilling. Observation that NCE sustains agonist-stimulated [Ca2+]i oscillations driven by SR Ca2+ release provides compelling indirect evidence for NCE-mediated SR refilling in vascular and airway smooth muscle.6,8,17 Critical support for this model came from our recent observation of the localized [Na+]i elevations (LNats) that were predicted to underlie NCE.16
Here, the conclusion that SR refilling is mediated by NCE is based on the observations that KB-R7943 (1) increased [Ca2+]SR depletion, (2) delayed the onset of refilling, and (3) slowed the rate of SR refilling. KB-R7943 has been reported to nonspecifically inhibit several ion transporters other than NCX, but such interactions are not compatible with the cumulative effects of KB-R7943 reported in smooth muscle (see the expanded Discussion section and Poburko et al16). The increase in maximal SR depletion by KB-R7943 further illustrates that SR refilling via ROCC-NCX coupling is rapidly initiated following ATP stimulation. Although the SR eventually refilled with KB-R7943 present, the 30- to 40-second delay in the onset of refilling likely represents the time required to activate SOCC. STIM1, the ER/SR Ca2+ sensor essential for SOCC,39 translocates to PM-SR junctions to activate SOCC 30 to 40 seconds after agonist stimulation.40 However, [Ca2+]SR normally fell to values reported to activate STIM1 for only a few seconds, suggesting that SOCC likely provides a backup SR-refilling mechanism during prolonged or extensive SR depletion. This underscores the likely importance of rapidly activated NCE versus SOCC for "physiological" SR refilling in intact VSM exhibiting rapid, SR-driven Ca2+ oscillations.
Subplasmalemmal Mitochondria and SR Refilling
Mitochondria often neighbor PM-SR junctions in VSM (reviewed by Poburko et al41), where they restrict the diffusion of NCX-mediated Ca2+ entry into the bulk cytosol.22 The critical mitochondrial role shown herein for efficient refilling of agonist-depleted SR may further explain how mitochondrial inhibition decreases the amplitude of SR-driven [Ca2+]i oscillations in VSM.19 The conclusion that mitochondria optimize SR refilling follows the results of inhibition of the mNCX with CGP-37157 and molecular relocalization of mitochondria by hFis1. Unlike KB-R7943, neither CGP-37157 nor hFis1 altered the time to onset of refilling, consistent with SR refilling being initiated by direct uptake of NCE by SERCA molecules within the PM-SR junctions. Nonetheless, both CGP-37157 and hFis1 slowed [Ca2+]SR recovery and reduced the extent of refilling, demonstrating that subplasmalemmal mitochondria facilitate the transfer of Ca2+ ions from the plasma membrane to the SR.
In blood and endothelial cells, subplasmalemmal mitochondria sustain Ca2+ influx by locally buffering Ca2+ to prevent Ca2+-dependent inactivation of store-operated CRAC channels.21 This mechanism is unlikely to explain our present observations because: (1) hFis1 impaired [Ca2+]SR recovery despite a parallel increase in the Ca2+ entry-dependent [Ca2+]i plateau (Figure 5A); (2) CGP-37157 impairs SR refilling despite enhancing global [Na+]i elevations mediated by TRPC67,16; and (3) ROCCs and SOCCs in VSM are likely composed of TRPC proteins exhibiting less Ca2+-dependent inactivation than CRAC channels.3,9,42 TRPCs and NCX can physically couple and are concentrated in PM-SR junctions,11,13–15 and constrained Ca2+ diffusion within the PM-SR junction15 makes it unlikely that mitochondria could reduce the [Ca2+] within the junction sufficiently to relieve potential Ca2+ inhibition of ROCCs. As in HeLa cells,24,43 hFis1 did not increase basal apoptosis rates, and, as discussed in supplemental text, hFis1 does not directly impair mitochondrial Ca2+ up-take.24 Therefore, the hFis1-mediated changes in Ca2+ signaling likely reflected the loss of local Ca2+ transfer between subplasmalemmal mitochondria and the SR. We propose that subplasmalemmal mitochondria in VSM sustain SR refilling by sequestering Ca2+ ions spilling from PM-SR junctions and transferring them to the SR (Figure 7, no. 3) before they diffuse into the bulk cytosol or are extruded by PM Ca2+ ATPases located outside of the junctions (reviewed by Poburko et al41). In this capacity, subplasmalemmal mitochondria increase the effective SR Ca2+ buffering capacity and cooperate with NCE and SERCA to ensure efficient SR refilling during agonist stimulation of VSM.
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Clinically, cultured VSM cells from hypertensive pulmonary arteries exhibit fragmented mitochondria with perinuclear clustering, resembling hFis1-overexpressing cells.44 Ca2+-dependent calcineurin activates Drp1, the cytosolic ligand of hFis1, and fragments mitochondria.45 Calcineurin inhibition can reverse the mitochondrial dysfunction that contributes to chronic [Ca2+]i elevations associated with VSM proliferation in pulmonary artery hypertension.46 Beyond impairing SR refilling, hFis1 caused a reciprocal increase in [Ca2+]i elevation and [Ca2+]SR depletion (Figures 5 and 6
), indicating that mitochondrial fragmentation not only impaired SR refilling but also effectively increased SR Ca2+ release. Although the impact of mitochondria on SR Ca2+ release remains controversial,1 we propose that perinuclear clustering of mitochondria will reduce the surface of SR in close contact with mitochondria, thereby removing a physical barrier to diffusion of released Ca2+ (Figure 7, no. 6). In addition, preliminary results showed that chronic stimulation (24 hours) of RASMCs with angiotensin II (10 to 100 nmol/L), a promoter of proliferation, induces mitochondrial fragmentation (supplemental Figure II). Causal and temporal links between proproliferative/hypertensive insults, mitochondrial morphology, and VSM Ca2+ handling merit further investigation, and we propose a positive feedback between cumulative, chronic insults to VSM, altered mitochondrial morphology and distribution, and the exacerbated tonic [Ca2+]i elevations contributing to VSM proliferation in diseases like pulmonary artery hypertension.
In conclusion, we demonstrate that NCX-mediated Ca2+ entry and subplasmalemmal mitochondria are both required for the rapid and efficient SR Ca2+ refilling in aorta smooth muscle. The coordinated interaction of subplasmalemmal mitochondria with the NCX and SR facilitates store refilling, while preventing excessive increases in bulk cytosolic [Ca2+]cyt during agonist activation of VSM. Demonstration that disruption of mitochondrial morphology alters VSM Ca2+ homeostasis represents novel avenues to explore in the etiology of proliferative vasculopathies.
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Acknowledgments |
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Sources of Funding
This work was supported by Swiss National Science Foundation grant 31-068317 (to N.D.). Partial support for D.P. came from a Natural Sciences and Engineering Research Council of Canada postdoctoral fellowship.
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received June 3, 2008; revision received October 15, 2008; accepted November 12, 2008.
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References |
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1. Chalmers S, Olson ML, MacMillan D, Rainbow RD, McCarron JG. Ion channels in smooth muscle: regulation by the sarcoplasmic reticulum and mitochondria. Cell Calcium. 2007; 42: 447–466.[CrossRef][Medline] [Order article via Infotrieve]
2. Poburko D, Lee CH, van Breemen C. Vascular smooth muscle mitochondria at the cross roads of Ca2+ signaling. Cell Calcium. 2004; 35: 509–521.[CrossRef][Medline] [Order article via Infotrieve]
3. McFadzean I, Gibson A. The developing relationship between receptor-operated and store-operated calcium channels in smooth muscle. Br J Pharmacol. 2002; 135: 1–13.[CrossRef][Medline] [Order article via Infotrieve]
4. Iino M, Kasai H, Yamazawa T. Visualization of neural control of intracellular Ca2+ concentration in single vascular smooth muscle cells in situ. EMBO J. 1994; 13: 5026–5031.[Medline] [Order article via Infotrieve]
5. Lee CH, Poburko D, Sahota P, Sandhu J, Ruehlmann DO, van Breemen C. The mechanism of phenylephrine-mediated [Ca(2+)](i) oscillations underlying tonic contraction in the rabbit inferior vena cava. J Physiol. 2001; 534: 641–650.
6. Dai JM, Kuo KH, Leo JM, Pare PD, van Breemen C, Lee CH. Acetylcholine-induced asynchronous calcium waves in intact human bronchial muscle bundle. Am J Respir Cell Mol Biol. 2007; 36: 600–608.
7. Lemos VS, Poburko D, Liao CH, Cole WC, van Breemen C. Na+ entry via TRPC6 causes Ca2+ entry via NCX reversal in ATP stimulated smooth muscle cells. Biochem Biophys Res Commun. 2007; 352: 130–134.[CrossRef][Medline] [Order article via Infotrieve]
8. Hirota S, Pertens E, Janssen LJ. The reverse mode of the Na(+)/Ca(2+) exchanger provides a source of Ca(2+) for store refilling following agonist-induced Ca(2+) mobilization. Am J Physiol Lung Cell Mol Physiol. 2007; 292: L438–L447.
9. Large WA. Receptor-operated Ca2(+)-permeable nonselective cation channels in vascular smooth muscle: a physiologic perspective. J Cardiovasc Electrophysiol. 2002; 13: 493–501.[CrossRef][Medline] [Order article via Infotrieve]
10. Dietrich A, Chubanov V, Kalwa H, Rost BR, Gudermann T. Cation channels of the transient receptor potential superfamily: their role in physiological and pathophysiological processes of smooth muscle cells. Pharmacol Ther. 2006; 112: 744–760.[CrossRef][Medline] [Order article via Infotrieve]
11. Golovina VA. Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum. J Physiol. 2005; 564: 737–749.
12. Lee MY, Song H, Nakai J, Ohkura M, Kotlikoff MI, Kinsey SP, Golovina VA, Blaustein MP. Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor. Proc Natl Acad Sci U S A. 2006; 103: 13232–13237.
13. Rosker C, Graziani A, Lukas M, Eder P, Zhu MX, Romanin C, Groschner K. Ca(2+) signaling by TRPC3 involves Na(+) entry and local coupling to the Na(+)/Ca(2+) exchanger. J Biol Chem. 2004; 279: 13696–13704.
14. Moore ED, Etter EF, Philipson KD, Carrington WA, Fogarty KE, Lifshitz LM, Fay FS. Coupling of the Na+/Ca2+ exchanger, Na+/K+ pump and sarcoplasmic reticulum in smooth muscle. Nature. 1993; 365: 657–660.[CrossRef][Medline] [Order article via Infotrieve]
15. Fameli N, van Breemen C, Kuo KH. A quantitative model for linking Na(+)/Ca(2+) exchanger to SERCA during refilling of the sarcoplasmic reticulum to sustain [Ca(2+)] oscillations in vascular smooth muscle. Cell Calcium. 2007; 42: 565–575.[CrossRef][Medline] [Order article via Infotrieve]
16. Poburko D, Liao CH, Lemos VS, Lin E, Maruyama Y, Cole WC, van Breemen C. Transient receptor potential channel 6 mediated, localized cytosolic [Na+] transients drive Na+/Ca2+ exchanger mediated Ca2+ entry in purinergically stimulated aorta smooth muscle cells. Circ Res. 2007; 101: 1030–1038.
17. Lee CH, Kuo KH, Dai J, Leo JM, Seow CY, van Breemen C. Calyculin-A disrupts subplasmalemmal junction and recurring Ca2+ waves in vascular smooth muscle. Cell Calcium. 2005; 37: 9–16.[CrossRef][Medline] [Order article via Infotrieve]
18. Harper AG, Sage SO. A key role for reverse Na+/Ca2+ exchange influenced by the actin cytoskeleton in store-operated Ca2+ entry in human platelets: evidence against the de novo conformational coupling hypothesis. Cell Calcium. 2007; 42: 606–617.[CrossRef][Medline] [Order article via Infotrieve]
19. Sward K, Dreja K, Lindqvist A, Persson E, Hellstrand P. Influence of mitochondrial inhibition on global and local [Ca(2+)](I) in rat tail artery. Circ Res. 2002; 90: 792–799.
20. Malli R, Frieden M, Osibow K, Zoratti C, Mayer M, Demaurex N, Graier WF. Sustained Ca2+ transfer across mitochondria is Essential for mitochondrial Ca2+ buffering, sore-operated Ca2+ entry, and Ca2+ store refilling. J Biol Chem. 2003; 278: 44769–44779.
21. Parekh AB. Mitochondrial regulation of store-operated CRAC channels. Cell Calcium. 2008; 44: 6–13.[CrossRef][Medline] [Order article via Infotrieve]
22. Poburko D, Potter K, van BE, Fameli N, Liao CH, Basset O, Ruegg UT, van Breemen C. Mitochondria buffer NCX-mediated Ca(2+)-entry and limit its diffusion into vascular smooth muscle cells. Cell Calcium. 2006; 40: 359–371.[CrossRef][Medline] [Order article via Infotrieve]
23. James DI, Parone PA, Mattenberger Y, Martinou JC. hFis1, a novel component of the mammalian mitochondrial fission machinery. J Biol Chem. 2003; 278: 36373–36379.
24. Frieden M, James D, Castelbou C, Danckaert A, Martinou JC, Demaurex N. Ca(2+) homeostasis during mitochondrial fragmentation and perinuclear clustering induced by hFis1. J Biol Chem. 2004; 279: 22704–22714.
25. Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A. Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A. 2004; 101: 10554–10559.
26. Palmer AE, Giacomello M, Kortemme T, Hires SA, Lev-Ram V, Baker D, Tsien RY. Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem Biol. 2006; 13: 521–530.[CrossRef][Medline] [Order article via Infotrieve]
27. Palmer AE, Jin C, Reed JC, Tsien RY. Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc Natl Acad Sci U S A. 2004; 101: 17404–17409.
28. Lo Russo A, Passaquin AC, Andre P, Skutella M, Ruegg UT. Effect of cyclosporin A and analogues on cytosolic calcium and vasoconstriction: possible lack of relationship to immunosuppressive activity. Br J Pharmacol. 1996; 118: 885–892.[Medline] [Order article via Infotrieve]
29. Jousset H, Malli R, Girardin N, Graier WF, Demaurex N, Frieden M. Evidence for a receptor-activated Ca(2+) entry pathway independent from Ca(2+) store depletion in endothelial cells. Cell Calcium. 2008; 43: 83–94.[CrossRef][Medline] [Order article via Infotrieve]
30. Rudolf R, Magalhaes PJ, Pozzan T. Direct in vivo monitoring of sarcoplasmic reticulum Ca2+ and cytosolic cAMP dynamics in mouse skeletal muscle. J Cell Biol. 2006; 173: 187–193.
31. Arnaudeau S, Kelley WL, Walsh JV Jr, Demaurex N. Mitochondria recycle Ca(2+) to the endoplasmic reticulum and prevent the depletion of neighboring endoplasmic reticulum regions. J Biol Chem. 2001; 276: 29430–29439.
32. Chen W, Steenbergen C, Levy LA, Vance J, London RE, Murphy E. Measurement of free Ca2+ in sarcoplasmic reticulum in perfused rabbit heart loaded with 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid by 19F NMR. J Biol Chem. 1996; 271: 7398–7403.
33. Launikonis BS, Zhou J, Royer L, Shannon TR, Brum G, Rios E. Confocal imaging of [Ca2+] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence. J Physiol. 2005; 567: 523–543.
34. Dong H, Sellers ZM, Smith A, Chow JY, Barrett KE. Na(+)/Ca(2+) exchange regulates Ca(2+)-dependent duodenal mucosal ion transport and HCO(3)(-) secretion in mice. Am J Physiol Gastrointest Liver Physiol. 2005; 288: G457–G465.
35. Eder P, Probst D, Rosker C, Poteser M, Wolinski H, Kohlwein SD, Romanin C, Groschner K. Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex. Cardiovasc Res. 2007; 73: 111–119.
36. Paluzzi S, Alloisio S, Zappettini S, Milanese M, Raiteri L, Nobile M, Bonanno G. Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization. J Neurochem. 2007; 103: 1196–1207.[CrossRef][Medline] [Order article via Infotrieve]
37. van Breemen C, Aaronson P, Loutzenhiser R. Sodium-calcium interactions in mammalian smooth muscle. Pharmacol Rev. 1978; 30: 167–208.[Medline] [Order article via Infotrieve]
38. Arnon A, Hamlyn JM, Blaustein MP. Na(+) entry via store-operated channels modulates Ca(2+) signaling in arterial myocytes. Am J Physiol Cell Physiol. 2000; 278: C163–C173.
39. Lewis RS. The molecular choreography of a store-operated calcium channel. Nature. 2007; 446: 284–287.[CrossRef][Medline] [Order article via Infotrieve]
40. Smyth JT, Dehaven WI, Bird GS, Putney JW Jr. Ca2+-store-dependent and -independent reversal of Stim1 localization and function. J Cell Sci. 2008; 121: 762–772.
41. Poburko D, Kuo KH, Dai J, Lee CH, van Breemen C. Organellar junctions promote targeted Ca2+ signaling in smooth muscle: why two membranes are better than one. Trends Pharmacol Sci. 2004; 25: 8–15.[CrossRef][Medline] [Order article via Infotrieve]
42. Shi J, Mori E, Mori Y, Mori M, Li J, Ito Y, Inoue R. Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells. J Physiol. 2004; 561: 415–432.
43. Szabadkai G, Simoni AM, Chami M, Wieckowski MR, Youle RJ, Rizzuto R. Drp-1-dependent division of the mitochondrial network blocks intraorganellar Ca2+ waves and protects against Ca2+-mediated apoptosis. Mol Cell. 2004; 16: 59–68.[CrossRef][Medline] [Order article via Infotrieve]
44. Bonnet S, Michelakis ED, Porter CJ, Andrade-Navarro MA, Thébaud B, Bonnet S, Haromy A, Harry G, Moudgil R, McMurtry MS, Weir EK, Archer SL. An abnormal mitochondrial-hypoxia inducible factor-1alpha-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: similarities to human pulmonary arterial hypertension. Circulation. 2006; 113: 2630–2641.
45. Cribbs JT, Strack S. Reversible phosphorylation of Drp1 by cyclic AMP-dependent protein kinase and calcineurin regulates mitochondrial fission and cell death. EMBO Rep. 2007; 8: 939–944.[CrossRef][Medline] [Order article via Infotrieve]
46. Bonnet S, Rochefort G, Sutendra G, Archer SL, Haromy A, Webster L, Hashimoto K, Bonnet SN, Michelakis ED. The nuclear factor of activated T cells in pulmonary arterial hypertension can be therapeutically targeted. Proc Natl Acad Sci U S A. 2007; 104: 11418–11423.
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