Sorcin expression in heart, aorta and cerebral artery tissues
Western blots of heart, aorta and cerebral artery rat homogenates (30 μg of protein per well) probed with sorcin antibody revealed a single discrete band (Fig. 1A, lane 2–4) of relative mobility comparable to purified recombinant sorcin (Fig. 1A, lane 5). The molecular weight (assessed by migration distance, Rf) for sorcin in SDS-PAGE was calculated with molecular weight standards (Bio-Rad) using LabWorks software. Heart and smooth muscle sorcin show a molecular weight of ∼19.5 kDa, which is in agreement with previous reports (Meyers et al. 1985, 1987) and differs slightly from the molecular weight calculated by mass spectrometry (21.6 kDa). Sorcin antibody specificity was demonstrated by preadsorbing the immune serum with purified recombinant sorcin. Sorcin expression in heart homogenates has been reported before (Meyers et al. 1995b; Lokuta et al. 1997; Farrell et al. 2003) and we used it here as an index of relative density to assess sorcin expression in vascular tissue. Western blot analysis showed that the expression of sorcin in rat aorta was ∼2-fold higher than that found in heart (1.91 ± 0.1 versus 1.00 ± 0.1 normalized optical density n= 3; Fig. 1C), and in cerebral artery (0.85 ± 0.1 normalized optical density). Therefore, sorcin appears to be more abundant in aorta than in heart and cerebral arteries, perhaps reflecting a more prominent role in this tissue.
Figure 1. Sorcin expression in cardiac and vascular tissues Western blot of rat tissue homogenates probed with sorcin antibody. A, samples containing 30 μg of protein from whole homogenates of heart (lane 2), aorta (lane 3) and cerebral arteries (lane 4) or 30 ng of purified recombinant sorcin (lane 5, positive control) were run on 10% acrylamide gels, blotted onto nitrocellulose membranes and probed with sorcin antibody (1: 6000). Smooth muscle sorcin was detected at the same position as purified sorcin. B, Western blot done as in A except that the sorcin antibody was preadsorbed with 300 μg of purified sorcin for 30 min before incubation with the proteins. The secondary antibody was peroxidase-conjugated donkey anti-rabbit and detection was by chemiluminiscence. Lane 1 contains molecular weight standards detected with Precision Streptactin-HRP conjugate (Bio-Rad). C, bar graph showing the relative amount of sorcin on the blots (n= 3) determined by densitometry using LabWorks software. Signal intensity was normalized with respect to heart levels.
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Ca2+ dependence of endogenous sorcin translocation
Quantitative Western blotting of aortic subcellular fractions shows that the relative density of sorcin is 3.7-fold higher in microsomal particulates than in whole homogenates (0.22 ± 0.05 versus 0.06 ± 0.03 ng sorcin per μg of protein loaded in the gel, n= 5; not shown), suggesting that smooth muscle sorcin, like cardiac muscle sorcin (Farrell et al. 2003), translocates from soluble to membranous compartments to interact with target proteins. Cardiac sorcin translocation is Ca2+ dependent (Farrell et al. 2003; Matsumoto et al. 2005); we thus tested whether smooth muscle sorcin was able to translocate from cytosolic to membranous compartments depending on free [Ca2+]. Figure 3 shows that smooth muscle sorcin gradually associates with microsomal fractions in a Ca2+-dependent manner. Aorta homogenates were incubated in medium containing the free [Ca2+] indicated in Fig. 3A and B and then centrifuged at high speed to separate soluble from membranous components. At 0 μm[Ca2+] (∼4 nm, calculated free [Ca2+]), the vast majority of sorcin is found in the supernatant (Fig. 3A); conversely, only traces of sorcin are found in the membranous component (pellet, Fig. 3B). This relationship is gradually inverted with increasing [Ca2+] so that at 1000 μm[Ca2+], the highest concentration tested, only ∼20% of sorcin is soluble. The Ca2+ dependence of sorcin translocation was sigmoidal and could be fitted with a Hill equation that yields a half-maximal effective [Ca2+] (EC50) of 1.5 ± 0.4 μm (n= 3) and cooperativity coefficient (nH) of 1.4 ± 0.2 (Fig. 3C). The EC50 is close to previous estimations of the sorcin–Ca2+ high affinity site (∼1 μm, Zamparelli et al. 1997; Mella et al. 2003) determined in vitro but differs substantially from the EC50 for Ca2+-dependent translocation of sorcin in cardiac cells (∼200 μm, Farrell et al. 2003). None of these EC50 values, however, is similar to the EC50 needed for Ca2+-dependent precipitation of sorcin (incapacity to remain soluble in the absence of membrane-embedded targets). Figure 3C, open circles, shows normalized sorcin absorbance (A280 nm) versus[Ca2+] in the absence of membrane proteins. The curve shows biphasic decay. The steepest decrease in absorbance (83%) may be fitted with a sigmoidal function with an EC50= 750 μm. This absorbance reduction most likely reflects sorcin precipitation due to exposure of hydrophobic domains induced by Ca2+ binding. A much smaller drop in absorbance appears at very low [Ca2+] (EC50= 200 nm) and probably reflects dimerization of sorcin (with concomitant shift in absorbance) or other conformational changes. In neither case, however, are these changes comparable to those observed when sorcin is exposed to similar [Ca2+]in the presence of aorta homogenates (this study, EC50= 1.5 μm) or permeabilized cardiomyocytes (Farrell et al. 2003, EC50≈ 200 μm), clearly indicating that they correspond to different phenomena.
Figure 3. Ca2+-dependent translocation of sorcin in smooth muscle tissue Western blots of aortic supernatants (A) and corresponding pellets (B) with sorcin antibody. Whole dog aorta homogenates were incubated with specified free [Ca2+] for 10 min at 37°C (pH = 7.2) and centrifuged at room temperature, for 40 min at 100 000 g to separate sorcin from cytosolic and membranous compartments. Proteins in supernatant and pellet fractions were separated by gel electrophoresis (20 μg per well), transferred to a nitrocellulose membrane and probed with the sorcin antibody. Smooth muscle sorcin was detected in the same position as purified recombinant sorcin (S). C, sorcin bands in B were normalized with respect to the sorcin band signal at free [Ca2+]= 1000 μm and the data are plotted as filled circles. Continuous line is a Hill equation fitting of these data with EC50= 1.5 μm and n= 1.4 (n= 3). Open circles are normalized O.D. (A280) nm readings of a solution containing 1 μm sorcin, 1 mm EGTA, various CaCl2 that yield the specified free [Ca2+]. Membrane fractions were omitted.
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Sorcin and RyRs colocalize in isolated myocytes
To determine the precise intracellular localization of sorcin and whether it interacts with RyRs within the same myocyte, freshly isolated cerebral artery cells and ventricular cardiomyocytes were labelled with antisorcin serum in combination with RyR antibodies. Images from these double-labelling experiments are presented in Fig. 5. Confocal images of a cerebral artery myocyte (0.7 μm thick) show a disperse distribution of sorcin in the cytosol combined with increased sorcin fluorescence signal at regions underneath the plasmatic membrane (Fig. 5Aa). The RyR spot-like distribution is more restricted and follows mainly the peripheral regions that are in close apposition to the plasmatic membrane (Fig. 5Ab). In the merged image (Fig. 5Ac) the orange/yellow labelling indicates sorcin–RyR overlap, which is only apparent in the SR regions close to the plasmatic membrane or junctional gaps. By contrast, sorcin and RyR2 distributions in cardiac cells run specifically on top of the cross-sectional striations of the cell (Fig. 5B). Within each transverse band in the merged image (Fig. 5Bc), sorcin and RyR fluorescence intensify at discrete spots, suggesting that the proteins are organized in clusters rather than being uniformly distributed.
The colocalization images of Fig. 6Aa and Ba display only the pixels that include sorcin and RyR labelling from the merged images of Fig. 5. The crosshair thresholds for identification of positive structures in the scattergram was set above the mean pixel intensity + 2 standard deviations for each Alexa derivative and represented at least ∼23% of the total fluorescence, thus ensuring that only the pixels with significantly strong fluorescence overlap were displayed. On an intensity scale of 0–4095, the RyR and sorcin channels threshold was 944 and 938 for vascular (Fig. 6Ab) and 1741 and 1050 for ventricular (Fig. 6Bb) myocytes, respectively (i.e. region 3 of their respective scattergrams). Figure 6Aa shows that the punctuate pattern seen in vascular myocytes is prevalent at regions close to the plasmatic membrane and where the SR is found. Using the automated weighted colocalization coefficients method (see Methods) to set the colocalization threshold, typically ∼57% of pixels labelled with sorcin colocalized with ∼61% of RyR-labelled pixels (Fig. 6Ab); the remaining sorcin was predominantly cytosolic, where few RyR-labelled pixels were found. When the colocalization threshold was higher, such as that shown in the scattergram of ventricular cardiomyocytes (≥ 42% of total fluorescence), only ∼30% of sorcin pixels colocalize with 34% of RyR-labelled pixels (not shown). These results contrast with those obtained with ventricular myocytes (Fig. 6Ca), where a pool of sorcin closely follows the orderly pattern of RyR2 distribution, brilliantly decorating the transverse tubules at regularly spaced intervals of ∼1.7 μm (Fig. 6Cb), i.e. the mean sarcomere length in a fixed cardiac myocyte (Powell et al. 2004). The scattergram of Fig. 6Bb shows that this pool of RyR2-colocalized sorcin pixels comprises ∼47% of total sorcin pixels. The remaining sorcin staining appears mostly in patches underneath the sarcolemma (Fig. 6Ba). Hence, our examination of VSM cells suggests that a small but significant fraction of sorcin, predominantly that of peripheral location, colocalizes with a pool of RyRs comprising less than half of the total population.
Figure 6. Sorcin-RyR colocalization analysis A, sorcin and RyR colocalized pixels from the cerebral artery myocyte in Fig. 5 were extracted and superimposed in a; they correspond to region 3 of the scattergram (b). Same procedure was applied to the ventricular myocytes in B. Scattergrams show the pixel intensity distribution for the fluorescence of each Alexa Fluor (488 and 568). The crosshair lines in the scattergrams were positioned above the calculated background threshold for each Alexa. The crosshair lines define four regions: region 1 corresponds to RyR pixels only; region 2 corresponds to sorcin pixels only; region 3 contains the pixels where the sorcin–RyR overlap is the greatest; region 4 corresponds to sub-thoeshold pixels. Co-localization analysis was performed with Carl Zeiss LSM 5 software (version 3.2, SP2). Ca, image of a cardiomyocyte to show the fluorescence intensity distribution of sorcin and RyRs across several T-tubules (red line). Cb, intensity–distance plot showing sorcin (red) and RyR (black) fluorescence intensity peaks. The distance between T-tubules was ∼1.7 μm.
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Sorcin attenuates the amplitude, duration and spatial spread of spontaneous Ca2+ sparks in permeabilized smooth muscle cells. Ca2+ sparks in cerebral artery myocytes are, like in cardiac cells, localized and brief Ca2+ release events caused by the coordinated activity of clusters of RyRs (Nelson et al. 1995; Jaggar et al. 1998, 2000; Gollasch et al. 2000). Like in the heart, too, RyR2 in vascular smooth muscle appears to be the major RyR isoform involved in the generation of Ca2+ sparks (Gollasch et al. 2000; Ji et al. 2004), but there is no evidence yet that endogenous sorcin regulates Ca2+ spark activity in this tissue. We thus examined the spatio-temporal characteristics of spontaneous Ca2+ sparks in freshly isolated intact and permeabilized cerebral artery myocytes in the absence and presence of purified recombinant sorcin. Spontaneous Ca2+ sparks were recorded in Fluo3-loaded cells with a confocal microscope in line-scan mode. In the intact, non-permeabilized condition, Ca2+ sparks were observed with a frequency of 0.010 ± 0.001 events s−1μm−1 and amplitude of 1.75 ± 0.04 F/F0 (27 cells, 151 sparks, Fig. 7A, Table 1). The duration (measured as full duration at half-maximum amplitude) and size (measured as full width at half-maximum amplitude) of Ca2+ sparks were 55.5 ± 2.6 ms and 2.6 ± 0.1 μm, respectively, values which are close to those of Ca2+ sparks of adult rat cerebral artery myocytes (Gollasch et al. 2000). The addition of a submaximal concentration of caffeine (0.5 mm) into the recording chamber increased Ca2+ spark frequency 1.5-fold (0.015 ± 0.007 events s−1μm−1, n= 23 sparks), amplitude 1.3-fold (F/F0= 2.26 ± 0.12), duration ∼2-fold (FDHM = 115.3 ± 12.6 ms) and spatial spread 1.4-fold (FWHM = 3.6 ± 0.3 μm), indicating that these were bona fide RyR-originated Ca2+ sparks.
Figure 7. Spatio-temporal properties of Ca2+ sparks in intact and permeabilized rat cerebral artery myocytes A, normalized representative line scan images (acquired at 1.92 ms line−1) and corresponding intensity plots (below the images) of one release site per image (green bar) that show the spontaneous local Ca2+ events observed in Fluo-3 loaded intact or permeabilized myocytes under the specified conditions. The scan line was orientated in parallel with the long axis of the cell. The fluorescence intensity was normalized to the resting fluorescence intensity within the same cell. B, surface plot of a representative Ca2+ spark from the corresponding line scan indicating measures used for spark characteristics.
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Table 1. Spatio-temporal properties of Ca2+ sparks in intact and permeabilized cerebral artery myocytes under different conditions
|Treatment||Frequency (events s−1μm−1)||Amplitude (F/F0)||Duration (ms)||Width (μm)|
|Intact (151)||0.010 ± 0.001 ||1.75 ± 0.04 ||55.5 ± 2.6 ||2.6 ± 0.1|
| Low [EGTA] (61)||0.024 ± 0.004*||1.47 ± 0.02* ||84.0 ± 7.6 *||2.9 ± 0.2|
| Low [EGTA]+ sorcin (36)||0.020 ± 0.006*||1.32 ± 0.02*†||45.1 ± 4.4*†|| 2.3 ± 0.2†|
| Low [EGTA]+ F112L-sorcin (144)|| 0.025 ± 0.003*†||1.49 ± 0.01*†||58.2 ± 3.2*†||2.94 ± 0.1†|
| High [EGTA] (123)||0.019 ± 0.003*||1.71 ± 0.04 ||42.3 ± 2.2* || 2.2 ± 0.08 *|
| High [EGTA]+ sorcin (62)||0.017 ± 0.004*||1.46 ± 0.03*†||34.7 ± 2.0 *†|| 1.95 ± 0.11 *†|
Permeabilization of external membranes was achieved by perfusion of a solution containing 0.005% saponin, 0.5 mm EGTA, and 50 μm Fluo-3 (salt) for ∼60–90 s. Once the cell membrane was permeable (monitored by the presence of membrane-impermeant Fluo-3 into the cytosol), an internal solution with low (0.02 mm) or high (0.5 mm) [EGTA] was introduced and Ca2+ sparks were recorded after 2 min of equilibration (see Methods). Under low Ca2+ buffering conditions (low [EGTA]), Ca2+ sparks were observed with an increased frequency (0.024 ± 0.004 events s−1μm−1; 6 cells, 61 sparks; Fig. 7A and Table 1), duration (84.0 ± 7.6 ms) and spatial spread (2.9 ± 0.2 μm), but decreased amplitude (1.47 ± 0.02 F/F0) with respect to intact cell data. Thus, permeabilization modifies significantly the characteristics of Ca2+ sparks in patterns that appear unpredictable, but the increase of spark frequency in permeabilized cells suggests that the internal solution used to mimic the cytosolic milieu lacks components that appear to restrain RyR activity in intact cells.
To test the hypothesis that sorcin dissociation and washing from permeabilized cells may be responsible, at least in part, for the changes in Ca2+ spark properties observed upon permeabilization, we recorded Ca2+ sparks after equilibration (2 min perfusion) of permeabilized myocytes with 2 μm recombinant purified sorcin. While the physiological concentration of sorcin in cerebral artery myocytes has not been determined, it appears to be close to 1 μm in cardiac cells (Meyers et al. 1995b), thus providing a good estimate of the concentration of sorcin to be tested here. Table 1 and Fig. 7 show that 2 μm sorcin decreases the duration of Ca2+ sparks to levels comparable to those seen in intact cells (45.1 ± 4.4 ms, n= 6 cells, 36 sparks). However, sorcin exaggerated the reduction in Ca2+ spark amplitude (1.32 ± 0.02 F/F0) and spatial spread (2.3 ± 0.2 μm) seen upon permeabilization. Thus, direct perfusion of sorcin onto weakly buffered permeabilized cells restores some, but not all the Ca2+ spark parameters affected by permeabilization. These effects of sorcin appear to be dependent on its ability to interact with RyR2 more than on its bulk structure or electrical charge inasmuch as F112L-sorcin, a single-amino acid mutant that lacks the potency of wild-type sorcin to modulate RyR2 activity (Mohiddin S, Antaramian A, Gómez AM, Farrell EF & Lin J-P 2005, unpublished observations) had limited effect on Ca2+ sparks properties. Table 1 shows that, except for Ca2+ spark duration, all other parameters in cells perfused with F112L-sorcin were significantly different from those obtained in the presence of WT-sorcin; by contrast, they closely resembled those obtained in the absence of sorcin (Table 1).
We next increased the Ca2+ buffering capacity of the internal solution (with 0.5 mm EGTA) without modifying free [Ca2+] and determined Ca2+ spark properties in the absence and presence of sorcin. The increase in Ca2+ buffering capacity alone resulted in decreased duration (42.3 ± 2.2 ms, 14 cells, 123 events, Table 1) and size (2.2 ± 0.1 μm, 14 cells) of Ca2+ sparks with respect to intact cells, as expected. The frequency was again higher than that in intact cells (0.019 ± 0.003 events s−1μm−1), but lower than that obtained in low Ca2+ buffering strength (Table 1), also as expected. Remarkably, addition of 2 μm sorcin decreased even more the amplitude (1.46 ± 0.03 F/F0, 10 cells, n= 62 events), duration (34.7 ± 2.0 ms), and spatial spread (1.95 ± 0.11 μm) of Ca2+ sparks without producing notable effects on Ca2+ spark frequency (0.017 ± 0.004 events s−1μm−1). Thus, sorcin can substantially modify the behaviour of RyRs in permeabilized cells without substantially modifying Ca2+ spark frequency; rather, it appears to alter the time that RyRs remain open. This notion is supported by the fact that sorcin significantly decreases the amplitude and duration of Ca2+ sparks, especially when low Ca2+ buffering conditions were used but also when Ca2+ buffering was stronger.