Physical Coupling Supports the Local Ca2+ Transfer between Sarcoplasmic Reticulum Subdomains and the Mitochondria in Heart Muscle*

In many cell types, transfer of Ca2+ released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca2+] 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 [Ca2+] ([Ca2+]m) responses to caffeine-induced Ca2+ release that were prevented when thapsigargin was added to predeplete the SR or by mitochondrial Ca2+ uptake inhibitors. Importantly, caffeine failed to increase [Ca2+] in the large volume of the incubation medium, suggesting that local Ca2+ transfer between the SR particles and mitochondria mediated the [Ca2+]m signal. Despite the substantially reduced SR presence, pRHM still displayed a caffeine-induced [Ca2+]m rise comparable with the one recorded in cRHM. Thus, a relatively small fraction of the total SR is physically coupled and transfers Ca2+ locally to the mitochondria in cardiac muscle. The transferred Ca2+ stimulates dehydrogenase activity and affects mitochondrial membrane permeabilization, indicating the broad significance of the physical coupling in mitochondrial function.

SR Storage Buffer-SR storage buffer was 250 mM sucrose, 20 mM Tris/HCl, pH 7.0. All of the procedures were done at 4°C. Male Sprague-Dailey rat(s) (350 -400 g) were sacrificed by pentobarbital (75 mg) injection. The heart(s) were immediately removed and minced in isolation buffer with enhanced Ca 2ϩ chelation (containing 500 M EGTA/Tris). After elimination of visually distinguishable nonventricular parts (large blood vessels, thin atrial wall fragments), the pieces were washed three times into regular isolation buffer and then homogenized in ϳ8ϫ volume/volume using a Dounce glass/Teflon homogenizer (ϳ30 strokes at 1300 rpm). The homogenate was centrifuged at 500 ϫ g for 10 min to sediment unbroken tissue parts and nuclei. The 500 ϫ g supernatant was filtered through a 100-m nylon sieve (Falcon Cell Strainer) and centrifuged at 8,500 ϫ g for 10 min. The resulting pellet was called the crude mitochondrial fraction (cRHM) and was resuspended in mito storage buffer at ϳ20 -25 mg/ml final protein concentration if no further purification was desired.
Percoll Purification-For Percoll purification 400 mg of mannitol ϩ 250 mg of sucrose was dissolved in 20 ml of isolation buffer, and 17.5 ml of this solution was mixed with 10 ml of Percoll in a 30-ml centrifuge tube. cRHM pellet was resuspended in ϳ1-2 ml of isolation buffer, and 90% of this suspension was transferred to the Percoll mix in the 30-ml centrifuge tube and in turn spun at 50,000 ϫ g for 40 min. The resulting mitochondrial bands (lower/darker "heavy mito" or pRHM and the upper "light mito" or pRHM-light; see Fig. 1) were carefully separated and washed with isolation buffer (30 ml each) by centrifugation at 12,000 ϫ g for 10 min twice. After washing, the pellets were resuspended in mito storage buffer (ϳ20 -25 mg/ml protein). To obtain SR fraction (RHSR), the 8,500 ϫ g supernatant was centrifuged at 40,000 ϫ g for 45 min. The pellet was taken up in SR storage buffer, aliquoted, and stored on ice under N 2 until use.

Preparation for Microscopy
30 l of mitochondrial suspension was transferred to a Cell-Tak TM -coated (BD Biosciences, Bedford, MA) round glass coverslip (25-mm diameter) mounted to the bottom of a silicon incubation chamber designed for the microscope stage heater (Harvard Apparatus, Holliston, MA) and incubated for 10 -25 min at 37°C in a humidified enclosure. For fluorescence [Ca 2ϩ ] m imaging mitochondria were loaded with 4 M rhod2/AM during the attachment period, in the presence of 0.003% Pluronic F-127 (Molecular Probes).

Immuno-organelle Chemistry
For immuno-organelle chemistry samples were fixed after the attachment period in 4% paraformaldehyde (10 min, room temperature), washed with phosphate-buffered saline, and labeled with primary and secondary antibodies (the latter ones as fluorescent conjugates) according to standard immunocytochemistry protocols. For blocking nonspecific binding of the applied antibodies, the samples were pretreated with either 8% bovine serum albumin in phosphate-buffered saline (for mouse monoclonal primary antibodies, where the secondary antibodies were from rabbit) or 10% goat serum in phosphate-buffered saline (for rabbit polyclonal antibodies, where the secondary antibodies were from goat), according to the suggestions of Affinity Bioreagents. To assess nonspecific binding of secondary antibodies, negative controls with no primary antibodies were used. Apparently, blocking the nonspecific binding was more effective in the case of the polyclonal primary antibodies, perhaps because the serum of the same species (goat) from which the secondary antibody derived was used. As shown in Fig. 2, negative control images were homogenous black in the case of the polyclonal primary antibodies, whereas in the case of the monoclonal ones (pRHM and cRHM), some bright labeling always occurred in the negative control images too, although their relative frequency (to the number of mitochondria) was much smaller than in the samples treated with the primary monoclonal antibodies. Fluorescent DNA probes (green fluorescent YO-PRO1 0.5-1 M iodide and far-red fluorescent SYTO TM 63 10 M) used as mitochondrial counter staining were applied during the attachment period for 12-15 min. Western blotting was carried out using a Bio-Rad system according to standard protocols and using pre-cast gels from Bio-Rad.

Fluorescence Wide Field Imaging
Fluorescence wide field imaging of [Ca 2ϩ ] was carried out using high quantum efficiency cooled CCD cameras attached to Olympus or Leica inverted epifluorescence microscopes fitted with the appropriate excitation/emission filter and beam splitter combinations (from Chroma Technology, Rockingham, VT) for simultaneous recordings of fura2 and rhodamine (rhod2, tetramethyl rhodamine ethyl ester (TMRE)) or fluorescein (YO-PRO1 iodide) fluorescence.
[Ca 2ϩ ] in the cytosol surrogate intracellular buffer ([Ca 2ϩ ] c ) in the imaging experiments was calibrated by sequential addition of saturating CaCl 2 (1.5 mM) and EGTA/ Tris, pH 8.7 (10 mM) and then translated to nMs using the Grynkiewicz formula: (17). NAD(P)H (auto) fluorescence was recorded without loading any other fluorophores to avoid possible cross-talk to the weak signal. At the end of the recording, either the reducing equivalents in the mitochondrial matrix were brought to a fully reduced state by the electron transport chain complex I inhibitor rotenone (2 M) (bringing NAD(P)H fluorescence to the maximum) or an uncoupler (5 M FCCP) was added to bring the reducing equivalents to the fully oxidized state (minimum fluorescence) (see Fig. 8). A small fraction of the mitochondria did not show Ca 2ϩ -related NAD(P)H response at all, even though they responded to rotenone and FCCP (not shown). These mitochondria were excluded from the evaluation of the Ca 2ϩ -dependent responses.

Confocal Imaging
Confocal imaging of immunofluorescence was carried out using a Bio-Rad Radiance 2100 system connected to an Olympus IX70 inverted microscope. The adherent particles were imaged via a 60ϫ oil objective with a numeric aperture of 1.45. The scanned field was 512 ϫ 512 pixels. The variable confocal aperture (with a range of 0.8 -12 mm) was set to 4 mm, which is wider than the theoretical optimum (1.5 mm) for maximum confocality, but we needed to do this compromise to obtain sufficient fluorescent signal. On the other hand, considering a single layer of organelles attached to the coverglass and that the average diameter of the organelles is close to (mitochondria) or less than (SR) the axial resolution of the lens (1.1 and 1.3 m at 488-and 568-nm excitation, respectively), calculation errors because of out of focus light are less likely. The axial resolution was calculated from the formula R ax ϭ 2/n(sin2⍜), where is the wavelength of the light, n is the refractive index of the immersion medium, and ⍜ is the semi-angle of the included cone (sin⍜ ϭ numeric aperture/n).

Image Analysis
Most of the collected wide field and confocal images and image sequences were analyzed using the Spectralyzer software. To determine the mean diameter of the SR (anti-RyR2labeled) and mitochondria (labeled by DNA probe) in the confocal images the Image J (National Institutes of Health) software was used. For the SR after smoothing, areas with particles of relatively similar intensities were selected, threshold masks were drawn to cover the particles, and then the average Feret's diameters for the masks was determined. Because the staining of mitochondria with the DNA probes was not sufficiently homogenous for thresholding, the individual particles were masked manually. Because of the nonoptimal confocality as discussed above, the calculated size measures should be considered as rough estimates.

Negative Staining of Mitochondria for Transmission Electron Microscopy
On a 400 mesh copper electron microscopy grid with a carbon-coated Formvar support film (Electron Microscopy Sciences, Hatfield, PA), a drop of cRHM or pRHM suspensions was mixed with bacitracin at 25-50 g/ml final concentration and with either 4% ammonium-molybdate or 2% potassiumphosphotungstate. Bacitracin was used to enhance the hydrophilicity of the grid to promote better attachment of the membrane particles. After 1 min the mix was blotted away using the edge of a filter paper wedge followed by two additional 1-min washings with the molybdate or potassium-phosphotungstate solution. After washing, the grids were allowed to air dry and kept in a desiccator until the electron microscopy. The negatively stained samples were examined and imaged in a Tecnai 12 transmission electron microscope fitted with a high resolution CCD camera (Hamamastu ORCA-HR).
The data are represented as the means Ϯ S.E. unless specified otherwise and have been collected from at least two to five independent experiments. In the case of fluorescence imaging of adherent membrane particles, one data point used for statistic evaluation represents a mean value of 30 -200 individual particles or groups of particles on the imaged field from a single recording, unless it is specified otherwise.

Retention of SR Vesicles in Mitochondrial Fractions of Rat
Heart Homogenate-Crude and Percoll-purified mitochondria (cRHM 8,500 -12,000 ϫ g pellet and pRHM, respectively; see diagram in Fig. 1) were isolated from rat heart homogenate to determine whether fragments of the relatively light SR (usually sedimented at ϳ40,000 ϫ g (18 -20)) were copurified with the mitochondria, resisting the extensive separation forces. In the cRHM fraction, Western blot analysis revealed protein bands corresponding to the SR-resident calsequestrin (CSQ) and SERCA along with the outer mitochondrial membrane protein VDAC (Fig. 1). Conversely, pRHM showed hardly detectable SR marker proteins but displayed a strong band labeled with an anti-VDAC antibody (Fig. 1).
Transmission electron micrographs of negatively stained (using ammonium-molybdate or phosphotungstic acid) pRHM The crude 10,000 ϫ g mitochondrial pellet (cRHM) from the rat heart homogenate was further purified on a Percoll gradient (30% Percoll, 40 min at 50,000 ϫ 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 ϫ g 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.
or cRHM revealed relatively small membrane vesicles closely associated (10 -50-nm gap) with mitochondria, which were consistent in their appearance with SR fragments attached to the outer mitochondrial membrane (supplemental Fig. S1). Immunostaining of adherent membrane particles (immuno-organelle chemistry) detected RyR2-positive (Fig. 2, both polyclonal and monoclonal antibodies) and CSQ-positive ( Fig. 3) structures in association with the mitochondria (visualized by fluorescent DNA stains YO-PRO1 iodide and SYTO 63) in both cRHM and pRHM. However, the RyR2-and CSQ-positive SR particles were fewer (2.9 Ϯ 0.3 versus 5.1 Ϯ 0.2 polyclonal anti-RyR2-positive particles/mitochondria counted in three 21 ϫ 21-m fields) and somewhat fainter in pRHM (Figs. 2 and 3). In parallel, an SR-enriched fraction of the heart homogenate was also prepared that showed intense RyR and CSQ immunostaining but no distinct labeling of membrane particles with the DNA probe (Figs. 2 and 3). The mitochondria were roughly four times larger on average than the particles displaying SR markers In the cRHM, the RyR-or CSQ-positive SR particles frequently appeared in contact with the mitochondria visualized by DNA stains (Figs. 2 and 3). Colocalization of numerous CSQ-positive particles with larger structures positively labeled with anti-cytochrome c oxidase antibodies was also observed in cRHM (supplemental Fig. S2). However, association of the SR particles with the mitochondria was even more striking in pRHM (Figs. 2 and 3 and supplemental Fig. S3). Essentially, every SR particle showed the overlap with a mitochondrion (supplemental Fig. S3). Thus, SR fragments were present in both cRHM and pRHM in close association with mitochondria. The presence of mitochondria-associated SR in the pRHM suggested that the SR vesicles were physically coupled to heart mitochondria. Notably, the immunofluorescence approach showed more SR present in the pRHM than was detected by Western blot. This discrepancy between the two methods might be because the cRHM contained more SR fragments not connected to the mitochondria that settled slower to the attachment surface and got washed away at the end of the attachment period. After Percoll purification of cRHM, most of these non-mitochondria-associated SR fragments presumably moved to pRHM-light (Fig. 1).
Adherent Mitochondria Preserve Their Membrane Integrity-cRHM and pRHM attached to CellTak TM -coated coverglasses and energized by succinate readily accumulated the potentiometric dye, TMRE (Fig. 4). Upon the addition of an uncoupler, FCCP, TMRE was rapidly released (Fig. 4). The spatial distribution of the TMRE uptake was similar to the distribution of the DNA staining (Fig. 4), confirming the mitochondrial localization. Thus, isolated rat heart membrane particles attached to 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 TM 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 Ͼ cRHM Ͼ SR required to achieve similar fluorescence intensities with the monoclonal anti-RyR2 antibodies. coverglass represent a suitable model system to study functionally the individual mitochondria.
Preservation of SR Ca 2ϩ Uptake, RyR-dependent Ca 2ϩ Mobilization, and SR-Mitochondrial Local Ca 2ϩ Coupling in cRHM-Incubation of cRHM with rhod2/AM resulted in rhod2 compartmentalization in the mitochondrial particles (Fig. 5A) (Fig. 5B). Dissipation of the driving force of mitochondrial Ca 2ϩ uptake attained by the addition of the protonophore FCCP (2 M) prevented the caffeine-and Tg-induced rise in the rhod2 fluorescence (94 Ϯ 4% reduction of the peak [Ca 2ϩ ] m increase; n ϭ 5, not shown). Ru360 (10 M), a specific inhibitor of the mitochondrial Ca 2ϩ uniporter (MCU) also inhibited the [Ca 2ϩ ] m rise detected upon caffeine stimulation (85 Ϯ 6% inhibition on the peak [Ca 2ϩ ] m increase, means Ϯ S.D., n ϭ 2), confirming further the mitochondrial location of the dye and that the [Ca 2ϩ ] m response was a consequence of the activation of the MCU (Fig. 6, right panel). Thapsigargin pretreatment (5-10 M Tg for 10 -20 min) to predeplete of Ca 2ϩ the SR vesicles retained in the mitochondrial fraction suppressed the caffeine-induced [Ca 2ϩ ] m rise (Fig. 5C, 60 Ϯ 11% inhibition, means Ϯ S.E., n ϭ 6), suggesting that the caffeine effect was principally caused by RyR2-mediated Ca 2ϩ release from the SR. Interestingly, under the present conditions 10 M thapsigargin caused only partial inhibition of the SR 45 Ca uptake (not shown), in line with the results of Feher (20) in rat heart homogenates. When [Ca 2ϩ ] in the extravesicular bath medium ([Ca 2ϩ ] c ) was simultaneously recorded with [Ca 2ϩ ] m , it showed no increase upon caffeine stimulation, suggesting that the mitochondrial Ca 2ϩ uptake was activated by a local rather than global [Ca 2ϩ ] c change (Fig. 6, left panel). Collectively, these results show that the SR retained in the cRHM preserved its Ca 2ϩ -accumulating and -releasing function, as well as its ability to support mitochondrial Ca 2ϩ uptake via a local Ca 2ϩ transfer from RyR-dependent Ca 2ϩ release to the mitochondria. Because Ru360 inhibits only the  NOVEMBER (21), it was unlikely that the [Ca 2ϩ ] m rise was mediated by a recently proposed mitochondrial RyR (22).

Local Ca 2ϩ Communication between SR and Mitochondria in pRHM-Mitochondria in rhod2-loaded adherent pRHM (and pRHM-light; not shown) displayed [Ca 2ϩ
] m rises evoked by caffeine stimulation, comparable with those recorded in cRHM (Fig. 7A, left panel). These signals were also inhibited by Ru360 (76 Ϯ 3% inhibition, n ϭ 5), confirming the primary role of the MCU in the Ca 2ϩ uptake mechanism mediating the recorded [Ca 2ϩ ] m signal (Fig. 7A, right panel). To ensure optimal Ca 2ϩ loading of the SR, in the beginning of these recordings, a small CaCl 2 pulse (0.5-1 M) was added. This initial CaCl 2 pulse, which raised [Ca 2ϩ ] c to ϳ400 -800 nM, frequently caused by itself a small [Ca 2ϩ ] m rise. By contrast, the [Ca 2ϩ ] m response evoked by the addition of caffeine and Tg appeared without a rise in [Ca 2ϩ ] c (Fig. 7A), and it persisted even when [Ca 2ϩ ] c was clamped by 50 M EGTA at ϳ600 nM prior to the caffeine stimulation (Fig. 7B), suggesting that the [Ca 2ϩ ] m response was activated by a high [Ca 2ϩ ] microdomain.
The data above shows that RyR-mediated Ca 2ϩ release from small, biochemically hardly detectable "SR appendices" connected to the mitochondria in pRHM locally supports the [Ca 2ϩ ] m signal activation. This suggests that a small subdomain of the SR physically coupled to the mitochondria bears particular relevance in mitochondrial calcium signaling in cardiac muscle (see scheme in Fig. 10). Notably, the Ca 2ϩ uptake by the mitochondria may utilize an interplay between the local SR Ca 2ϩ source and the global [Ca 2ϩ ] c signal. Elevation of the global [Ca 2ϩ ] c may sensitize the MCU to the Ca 2ϩ provided by the local SR Ca 2ϩ store, and vice versa the local [Ca 2ϩ ] c rise may sensitize the mitochondrial Ca 2ϩ uptake from a global [Ca 2ϩ ] c signal (23,24). Indeed, the most effective caffeine-induced Ca 2ϩ transfer to the mitochondria was observed when the [Ca 2ϩ ] c was elevated by the small Ca 2ϩ prepulse (Fig. 7). Thus, the global [Ca 2ϩ ] c may also have a contribution in the mitochondrial Ca 2ϩ uptake during Ca 2ϩ release from the mitochondrion-coupled SR Ca 2ϩ store.

NAD(P)H Increase Associated with Caffeine Stimulation in pRHM-[Ca 2ϩ
] m regulates the activity of the Ca 2ϩ -sensitive mitochondrial dehydrogenases, the activity of which can be monitored fluorometrically through changes in the redox state of their pyridine nucleotide cofactors (1-3, 25, 26). To evaluate whether the [Ca 2ϩ ] m signal evoked by RyR-dependent Ca 2ϩ release in pRHM was sufficient to stimulate the oxidative metabolism, NAD(P)H autofluorescence was monitored during stimulation of pRHM with caffeine and Tg (Fig. 8).
An increase in the NAD(P)H fluorescence followed the caffeine stimulation (see the difference image showing the fluorescence increase in blue and the time course traces in Fig. 8) in most of the mitochondrial areas that responded to the uncoupling agent FCCP (by a drop in fluorescence to the minimum, Fig. 8A) or to the electron transport chain complex I inhibitor rotenone (by a fluorescence increase to the maxi-  mum; Fig. 8B). Interestingly, the CaCl 2 pulse (20 M) following the caffeine stimulation caused only modest or no further increase in the NAD(P)H fluorescence (Fig. 8), although it evoked a substantial additional [Ca 2ϩ ] m rise (Fig. 7A), suggesting that the Ca 2ϩ -responsive NAD(P)H generation was close to saturation. There was notable heterogeneity in the NAD(P)H responses to caffeine stimulation, and in some instances mitochondria displayed a response even to the preloading Ca 2ϩ pulse (Fig. 8).
These data show that the [Ca 2ϩ ] m signal driven by the mitochondria-associated SR is competent to stimulate the oxidative metabolism that is a means to enhance mitochondrial ATP production (27). The available room for Ca 2ϩ regulation of the oxidative metabolism can be maximally utilized by the [Ca 2ϩ ] m signals generated upon caffeine stimulation, further supporting the possible physiological relevance of the "resilient" SR-mitochondrial complexes in the pRHM.
Progressive Mitochondrial Depolarization Following Caffeine Stimulation in pRHM-In the cell, Ca 2ϩ mobilization via RyRs is important for the stimulation of mitochondrial energy metabolism but may also induce mitochondrial membrane permeabilization and release of apoptosis-inducing factors to the cytoplasm if some stress factors (reactive oxygen species, ceramide) are also present or the Ca 2ϩ release is augmented (Refs. 28, 29; see also Ref. 30 for review). However, it remains elusive whether Ca 2ϩ release from the mitochondria-associated SR particles is sufficient to evoke mitochondrial membrane permeabilization. RyR-dependent mitochondrial Ca 2ϩ uptake causes a small and transient mitochondrial depolarization (10,31), whereas the Ca 2ϩ -induced membrane permeabilization leads to progressive loss of the ⌬⌿ m (28). When TMREloaded pRHM were exposed to a similar stimulation protocol that we used for the characterization of the caffeine-induced [Ca 2ϩ ] m and NAD(P)H responses, caffeine stimulation prompted a biphasic decrease in ⌬⌿ m , with a faster initial drop (23 Ϯ 7% in 2 min, from three recordings) followed by a slow continuous decay (42 Ϯ 5% decrease in 8 min after correction to time control; see time course and bar chart in Fig. 9). Notably, the progressive delayed depolarization was evoked by a short lasting Ca 2ϩ release event. The decay in ⌬⌿ m caused by caffeine stimulation was sensitive to cyclosporine A (2 M, not shown), suggesting involvement of the permeability transition pore opening.
Thus, the mitochondria-bound SR vesicles can provide a trigger for both Ca 2ϩ -mediated stimulation of ATP production and permeability transition pore opening. Membrane permeabilization by the small and short lasting Ca 2ϩ release could be promoted by the stress represented by the isolation and storage of the pRHM.

DISCUSSION
The present work reveals associations between SR and mitochondria in the rat heart, which are highly resistant to purification of the mitochondria, indicating the presence of a direct SR-mitochondrial physical coupling. By imaging single isolated mitochondria, we show that the mitochondria-bound SR vesicles preserve their ability to store and release Ca 2ϩ via the ryanodine receptors to locally activate generation of [Ca 2ϩ ] m signals and in turn to stimulate oxidative metabolism. Furthermore, the present evidence shows that the mitochondria-associated SR vesicles can provide sufficient Ca 2ϩ trigger for induction of mitochondrial membrane permeabilization, which is of relevance for engaging a main cell death pathway. To our knowledge, these results are the first evidence for the presence of direct SR-mitochondrial linkage in the heart and for the relevance of this coupling in mitochondrial metabolism and a possible role in mitochondrial injury.
Percoll purification of cRHM greatly reduced the presence of SR proteins according to the Western blot analysis; however, mitochondria-associated SR vesicles still could be detected by immuno-organelle chemistry. Furthermore, the SR vesicles associated with the pRHM mitochondria were able to locally activate [Ca 2ϩ ] m signals via RyR2-dependent (caffeine-stimu- lated) Ca 2ϩ release. Thus, the SR subdomains relevant in the local Ca 2ϩ coupling with the mitochondria apparently represent a relatively small fraction of the total SR that is physically coupled to mitochondria (Fig. 10). In line with our data, mitochondrial "contamination" has been observed in fractionated SR vesicles of skeletal muscle. Interestingly, those data showed 2.8-fold greater mitochondrial presence (verified by F 1 F 0 -ATPase quantitative Western blotting) in the heavy SR comprised predominantly of terminal cisternae than in the light SR fraction (19). Thus, the terminal cisternae, the SR subdomains where the bulk of the RyR reside (32,33), appeared to be preferred sites for SR-mitochondrial associations. In addition, Protasi and co-workers (14,15) were able to visualize tethering structures between the terminal cisternae of the SR and mitochondria of skeletal muscle using conventional transmission electron microscopy as well as electron tomography.
We have successfully applied limited proteolysis by trypsin or proteinase K to disrupt the physical coupling between the ER and mitochondria and to show its role in the ER-mitochondrial Ca 2ϩ transfer (8). Also, Saks et al. (16) were able to disrupt the local metabolic interplay between the sarcomere, the SERCA, and mitochondria (so called intracellular energetic units) by limited trypsinization of permeabilized cardiac and skeletal muscle cells, suggesting the participation of protein elements in that local spatial arrangement. However, in the present system of adherent RHM, trypsin started to trim integral membrane proteins of the SR (e.g. phospholamban; not shown) before an effect on the SR-mitochondrial Ca 2ϩ coupling was detected. Thus, limited trypsinization could not be used to dissociate SR from the mitochondria in isolated RHM. Nevertheless, the SR-mitochondrial physical coupling resisted purification procedures that cause demolition of the cytoskeletal structures and was also present in highly purified mitochondria, suggesting that some membrane/membrane-associated proteins may form the bridge between the cardiac SR and mitochondria. Recently, Szabadkai et al. (34) have reported that the ER-mitochondrial links can be formed by the inositol 1,4,5- trisphosphate receptors and VDAC1 through the molecular chaperone grp75. A similar complex may exist in cardiac muscle, because the RyRs show considerable structural homology with the inositol 1,4,5-trisphosphate receptors, and the VDAC1 is also present in cardiac mitochondria. However, because most of the RyR2s reside on the far side of the terminal cisternae (35), it is likely that some other SR membrane proteins are also involved in the interorganellar linkage.
The present single-organelle calcium imaging experiments provided firm evidence that the RyR2 can transfer Ca 2ϩ to the cardiac mitochondria through a local [Ca 2ϩ ] c elevation. Although the majority of the RyR2 does not seem to directly face the mitochondria in the intact cardiac muscle, the RyR2s are sufficiently close (27-200 nm (9)) to the mitochondrial surface to expose the Ca 2ϩ uptake sites to an estimated 10 -20 M [Ca 2ϩ ] c during Ca 2ϩ release (36). Regarding the molecular mechanism of the Ca 2ϩ uptake by cardiac mitochondria, an interesting candidate is the mitochondrial mRyR1, recently described by Beutner, Sheu, and co-workers (22,37,38). The ruthenium derivative Ru360 has been reported highly specific to the MCU with no inhibitory effect on RyR2 in heart (21) or on RyR1 in skeletal muscle (39) (as opposed to ruthenium red that inhibits both MCU and RyR). Because Ru360 blocked the caffeine-stimulated [Ca 2ϩ ] m signal almost as effectively as the mitochondrial uncoupler, major contribution of mRyR1 activity to that caffeine-induced [Ca 2ϩ ] m signal was unlikely.
One might wonder whether the dynamics of [Ca 2ϩ ] in the SR pools ([Ca 2ϩ ] SR ) associated with the mitochondria could also be monitored. Indeed, Shannon et al. (40) reported successful recordings of [Ca 2ϩ ] SR increases in association with ATP-dependent Ca 2ϩ accumulation or decreases evoked by caffeine in immobilized (in an agarose matrix) SR vesicles isolated from rabbit or rat heart ventricles using the low-affinity Ca 2ϩ tracer fluo-5N. Unfortunately, in the present RHM system mitochondria readily accumulated fluo-5N, and the bright fluorescence derived from the mitochondria even in the presence of FCCP prevented us from distinguishing the SR-loaded dye (not shown).
The local Ca 2ϩ communication between SR and mitochondria have been difficult to study until very recently, and therefore limited information is available about its physiological and possible pathological role(s) in the cardiac energy metabolism and excitation-contraction coupling (41). However, it has been discussed more and more that the SR-mitochondrial Ca 2ϩ transfer is a significant regulatory factor of excitation-contraction and excitation-oxidative metabolic coupling (recently reviewed in Refs. [42][43][44]. This direction is strengthened by our results that caffeine stimulation of RyR2-mediated Ca 2ϩ release also evoked a rapid increase in the NAD(P)H levels in the pRHM mitochondria, underlying the metabolic relevance of the SR-mitochondrial complexes. FIGURE 9. Sustained mitochondrial depolarization following caffeine stimulation in pRHM. To record ⌬⌿ m , 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 M CaCl 2 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, ⌬⌿ m was dissipated by the addition of FCCP (5 M). 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 ⌬⌿ m values at the time of FCCP addition (ϳ8 min after caffeine stimulation) from three separate recordings. FIGURE 10. RyR-dependent local Ca 2؉ 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 Ca 2ϩ 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 [Ca 2ϩ ] coupling depends on the adjacency of SR Ca 2ϩ release sites (RyR2) with the mitochondrial surface, from this aspect only the closely connected RyR2-rich SR subregions will be relevant.