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. 2008 Nov 21;283(47):32771-80.
doi: 10.1074/jbc.M803385200. Epub 2008 Sep 12.

Physical coupling supports the local Ca2+ transfer between sarcoplasmic reticulum subdomains and the mitochondria in heart muscle

Cecilia García-Pérez  1 , György Hajnóczky, György Csordás
Affiliations

Physical coupling supports the local Ca2+ transfer between sarcoplasmic reticulum subdomains and the mitochondria in heart muscle

Cecilia García-Pérez et al. J Biol Chem. .

Abstract

In many cell types, transfer of Ca(2+) released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca(2+)] microdomains at close contacts between the sarcoplasmic reticulum (SR) and mitochondria. Here we studied whether the close contacts were secured via direct physical coupling in cardiac muscle using isolated rat heart mitochondria (RHMs). "Immuno-organelle chemistry" revealed RyR2 and calsequestrin-positive SR particles associated with mitochondria in both crude and Percoll-purified "heavy" mitochondrial fractions (cRHM and pRHM), to a smaller extent in the latter one. Mitochondria-associated vesicles were also visualized by electron microscopy in the RHMs. Western blot analysis detected greatly reduced presence of SR markers (calsequestrin, SERCA2a, and phospholamban) in pRHM, suggesting that the mitochondria-associated particles represented a small subfraction of the SR. Fluorescence calcium imaging in rhod2-loaded cRHM revealed mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) responses to caffeine-induced Ca(2+) release that were prevented when thapsigargin was added to predeplete the SR or by mitochondrial Ca(2+) uptake inhibitors. Importantly, caffeine failed to increase [Ca(2+)] in the large volume of the incubation medium, suggesting that local Ca(2+) transfer between the SR particles and mitochondria mediated the [Ca(2+)](m) signal. Despite the substantially reduced SR presence, pRHM still displayed a caffeine-induced [Ca(2+)](m) rise comparable with the one recorded in cRHM. Thus, a relatively small fraction of the total SR is physically coupled and transfers Ca(2+) locally to the mitochondria in cardiac muscle. The transferred Ca(2+) stimulates dehydrogenase activity and affects mitochondrial membrane permeabilization, indicating the broad significance of the physical coupling in mitochondrial function.

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Figures

FIGURE 1.
FIGURE 1.
Mitochondrial fractions obtained from rat heart homogenate. The crude 10,000 × g mitochondrial pellet (cRHM) from the rat heart homogenate was further purified on a Percoll gradient (30% Percoll, 40 min at 50,000 × g), resulting in two characteristic (upper pRHM-light and the lower heavy pRHM) bands enriched in mitochondria (scheme on the left). WB analysis of the mitochondrial fractions is shown on the right. Anti-VDAC antibodies were used to evaluate the presence of mitochondria, and anti-SERCA and anti-phospholamban were used to detect SR. For reference, SR (RHSR, 40,000 μg pellet from the RHM supernatant) fraction was also loaded to the gel. Note the drastic reduction in the SR markers in the heavy mitochondrial band.
FIGURE 2.
FIGURE 2.
Visualization of RyR2 in the rat heart mitochondrial fractions using immuno-organelle chemistry. pRHM, cRHM, and RHSR “glued” to CellTak-coated coverslip were fixed with paraformaldehyde and labeled with mouse monoclonal or rabbit polyclonal anti-RyR2 (even and odd rows as labeled). The secondary antibodies were conjugated with Alexa Fluor 488 (Monoclonal ab, green) or 568 (Polyclonal ab, red). The mitochondria were counterstained with the fluorescent DNA probes YO-PRO1 iodide (green fluorescent, shown in green, for polyclonal antibody) and SYTO™ 63 (far red; shown in red, monoclonal antibody), and the slides were imaged using confocal microscopy. Mitochondria and the RyR2 images are overlaid on the right. Negative controls processed without primary antibodies are shown on the left (see also the discussion on the difference between the polyclonal and monoclonal antibodies under “Materials and Methods”). Note the progressively decreasing laser power in the order of pRHM > cRHM > SR required to achieve similar fluorescence intensities with the monoclonal anti-RyR2 antibodies.
FIGURE 3.
FIGURE 3.
Visualization of CSQ in the rat heart mitochondrial fractions using immuno-organelle chemistry. CellTak-attached pRHM, cRHM, and RHSR were labeled similarly as in Fig. 2. with polyclonal rabbit anti-CSQ antibodies. As a reference for distribution of mitochondrially loaded fluorophores, the image of TMRE-loaded pRHM is also shown (top row).
FIGURE 4.
FIGURE 4.
Well maintained mitochondrial membrane potential in CellTak-mounted RHM. Accumulation of the membrane potential probe TMRE was recorded in pRHM attached to CellTak-coated coverslip using wide field fluorescence CCD imaging. A, time course of TMRE accumulation. After reaching steady state, uncoupler was added. B, images of TMRE distribution (top row) right after (24 s, left panel) and 4 min (middle panel) following the addition of the dye and after exposure to mitochondrial uncoupler (FCCP/Oligomycin). Bottom row, distribution of YO-PRO1 iodide fluorescence (left panel) and its overlay with the TMRE fluorescence (right panel). As a reference for size comparison, a segment of the overlay image framed in red is magnified to the same frame size as the confocal images in Figs. 2 and 3.
FIGURE 5.
FIGURE 5.
Fluorescence imaging of [Ca2+]m responses associated with RyR-mediated Ca2+ release in cRHM. [Ca2+]m was recorded using rhod2/AM loaded to the mitochondria during the attachment period to the CellTak coverslip. A, distribution of rhod2 fluorescence imaged at rest (left), after stimulation with caffeine (middle panel, Caf+Tg, caffeine 10 mm + thapsigargin 5–10 μm, the latter to maximize Ca2+ release from and prevent Ca2+ reuptake to the SR), and after a test Ca2+ pulse (right panel, 10Ca, CaCl2 10 μm to evoke nearly saturating [Ca2+]m rise). B, time course traces corresponding to the fluorescence changes (normalized to the base line) recorded from the numbered rhod2-loaded particles (mitochondria) in the images above. Note the substantial heterogeneity in the caffeine response. C, mean traces of 35– 45 individual mitochondrial particles recorded under control condition (black) and after Tg predepletion (∼10–15 min) of the SR (red traces). To prevent mitochondrial Ca2+ preloading, Tg pretreatment was carried out in the presence of 20 μm EGTA that was washed out before recording.
FIGURE 6.
FIGURE 6.
Local delivery of RyR-mediated Ca2+ release to the mitochondria in cRHM. [Ca2+]m was recorded as described for Fig. 5, [Ca2+]c was followed using fura2 (1.5 μm) dissolved in the incubation buffer. Note the lack of increase in [Ca2+]c during the [Ca2+]m response to caffeine and Tg (left panel). To verify pharmacologically the participation of the mitochondrial uniporter in the [Ca2+]m response, the stimulation protocol on the left was repeated in the presence of a specific inhibitor of the MCU, Ru360 (10 μm, right panel).
FIGURE 7.
FIGURE 7.
Local delivery of RyR-mediated Ca2+ release to the mitochondria in pRHM. A, [Ca2+]c and [Ca2+]m responses to sequential caffeine stimulation and CaCl2 (10 μm) addition were recorded in CellTak-attached pRHM using similar setup as in Fig. 6. B, [Ca2+]c and [Ca2+]m responses to caffeine stimulation and subsequent 10Ca pulse at [Ca2+]c clamped to ∼600 nm by EGTA (50 μm EGTA/Tris and 24 μm CaCl2 were added to the running buffer).
FIGURE 8.
FIGURE 8.
NAD(P)H responses of Percoll-purified mitochondria to caffeine stimulation. NAD(P)H autofluorescence was recorded at 360-nm excitation without using any other fluorophore to avoid possible bleed-through to the weak fluorescence signal. Similar Ca2+ preloading and stimulation protocol was used as in Fig. 7A. To eliminate minor Ca2+ contamination, the intracellular buffer contained 3–5 μm EGTA; hence a larger initial Ca2+ pulse (loCa, 2–5 μm CaCl2) was needed to elevate [Ca2+]c to ∼5–600 nm, and the test CaCl2 pulse following the caffeine stimulation was 20 μm. At the end, either FCCP (5 μm) was added to maximize NAD(P)H oxidation (minimize fluorescence, A) or rotenone (Rot, 2 μm) was applied to prevent NAD(P)H oxidation by complex I (maximize fluorescence, B). A, images of NAD(P)H fluorescence distribution in the adherent mitochondria (in gray scale, left panel) overlaid by difference-images depicting the fluorescence increases caused by caffeine and Tg in blue (middle panel) and the decreases in the NAD(P)H fluorescence caused by FCCP in red (right panel). Below, time courses recorded from the mitochondrial areas labeled with the numbered squares (thin lines) and the mean trace of 30 randomly selected mitochondrial areas (bottom panel, thick line). B, time course traces from the experiment where rotenone was applied at the end; recordings from four individual mitochondrial areas (thin lines) and mean trace of 68 individual mitochondrial areas (thick line, bottom panel).
FIGURE 9.
FIGURE 9.
Sustained mitochondrial depolarization following caffeine stimulation in pRHM. To record ΔΨm, pRHM was loaded with 50 nm TMRE during the attachment period, and 5 nm TMRE was also present in the running buffer. Similar stimulation protocol was used as in the NAD(P)H measurements, except that the 20 μm CaCl2 test pulse was omitted because in the initial test runs it did not affect the time course. In the time control an equal volume of running buffer to that of the caffeine and Tg mix was applied as a “mock” stimulus. At the end of the run, ΔΨm was dissipated by the addition of FCCP (5 μm). The data points are normalized to the maximum range calculated as the difference between the time points right before the caffeine stimulation and after application of FCCP. The bar chart shows the cumulative ΔΨm values at the time of FCCP addition (∼8 min after caffeine stimulation) from three separate recordings.
FIGURE 10.
FIGURE 10.
RyR-dependent local Ca2+ signal propagation to the mitochondria from physically attached SR fragments. The schematic is showing the proposed proportions (in terms of SR presence) and suggested topology in the spatial relation between the mitochondria and the subfractions of SR relevant in the RyR2-dependent local Ca2+ signal transmission. In the cRHM there are SR subregions abundant in RyR2 (terminal cysternae-junctional SR) and physically linked to mitochondria as well as other SR domains lacking direct connection to mitochondria and/or with membrane surfaces less abundant (corbular SR) or mostly devoid (network SR) of RyR2. In pRHM the SR-mitochondrial associations comprise mainly those RyR2-rich subregions physically coupled to the mitochondria. Because the local [Ca2+] coupling depends on the adjacency of SR Ca2+ release sites (RyR2) with the mitochondrial surface, from this aspect only the closely connected RyR2-rich SR subregions will be relevant.
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