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First published online 10 June 2008
doi: 10.1242/jcs.032003
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Research Article |
1 School of Medical Sciences, Discipline of Physiology (F13), Bosch Institute, The University of Sydney, NSW 2006, Australia
2 Department of Rehabilitation Sciences, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong
* Author for correspondence (e-mail: davida{at}physiol.usyd.edu.au)
Accepted 22 April 2008
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Summary |
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Key words: Duchenne muscular dystrophy, Src, TRPC1, Caveolin-3, mdx
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Introduction |
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; Rao et al., 2006; Sours et al., 2006), including in cardiac (Williams and Allen, 2007a; Ju et al., 2007) and skeletal (Vandebrouck et al., 2002; Venkatachalam and Montell, 2007) muscles. TRPC1 can be activated by protein kinase C (Ahmmed et al., 2004), STIM1 (Huang et al., 2006), membrane stretch (Maroto et al., 2005) and depletion of Ca2+ stores (Sinkins et al., 1998). Even though many studies point to TRPC1 as being a store- and stretch-activated channel (SOC and SAC, respectively) (Ducret et al., 2006), this topic remains controversial, with tissue-specific investigations of TRPC1 function often providing negative results (Brereton et al., 2000; Dietrich et al., 2007; Gottlieb et al., 2008).
Regulatory proteins, such as caveolins, could also be involved in the regulation of TRPC1. In cells that express caveolin-1 (salivary gland and kidney), caveolin-1 binds to TRPC1, promotes its assembly into channels and regulates its activity (Brazer et al., 2003). Caveolins are the scaffolding proteins of caveolae – 20-50 nm invaginations of the cell surface enriched with specific lipids (glycosphingolipids, sphingomyelin and cholesterol) and lipid-modified signalling molecules (Engelman et al., 1998). Caveolins play an important role in signalling pathways because they bind to and regulate the activity of many proteins (for a review, see Williams and Lisanti, 2004). Caveolin-3, the muscle-specific caveolin isoform, seems to play an important role in muscle physiology. Mutations that reduce expression of the caveolin-3 gene cause limb-girdle muscular dystrophy (Minetti et al., 1998). Transgenic overexpression of caveolin-3 induces a phenotype similar to Duchenne muscular dystrophy (DMD) (Galbiati et al., 2000). DMD is a degenerative muscle disease caused by the absence of dystrophin, a membrane-anchoring protein that provides a mechanical link from the intracellular cytoskeleton to the extracellular matrix (Hoffman et al., 1987). Vaghy and colleagues also found higher levels of caveolin-3 in muscles of mdx mice, an animal model of DMD (Vaghy et al., 1998).
Both caveolin-3 and TRPC1 bind to Src kinase (Li et al., 1996; Kawasaki et al., 2006). The Src-kinase family is expressed in many cell types, including muscle (Abram and Courtneidge, 2000). Src kinase regulates many cellular events such as cell proliferation, cytoskeletal organisation, differentiation, survival, adhesion and migration (Thomas and Brugge, 1997). Members of the Src-kinase family can phosphorylate TRPC3 (Kawasaki et al., 2006) and TRPC6 (Hisatsune et al., 2004); however, regulation of TRPC1 activity by Src has not been investigated. Src kinase has been shown to be activated by reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) (Chen et al., 2005), and TRPC3 activity is increased by ROS (Poteser et al., 2006). Thus, it is possible that ROS-induced Src activation, leads to phosphorylation and activation of transient receptor potential canonical (TRPC) channels.
TRPC1 has been associated with many pathologies in which Ca2+ homeostasis is disturbed (Sweeney et al., 2002; Kumar et al., 2006; Pani et al., 2006), including DMD (Vandebrouck et al., 2002; Vandebrouck et al., 2007; Williams and Allen, 2007a). Reports of increased intracellular Ca2+ concentration ([Ca2+]i) in dystrophin-deficient cells have led to the hypothesis that the muscle damage and degeneration in DMD are a consequence of activation of Ca2+-dependent proteases, particularly calpains (Spencer et al., 1995; Mariol and Ségalat, 2001). TRPC1 has been proposed as one of the possible causes of increased [Ca2+]i in mdx muscle. Expression of TRPC1 is increased in mdx cardiac muscle (Williams and Allen, 2007a), and repression of TRPC1 by siRNA decreases both SOC and SAC occurrence and activity in mdx skeletal muscle cells (Vandebrouck et al., 2002). We have shown that SACs cause increased Ca2+ influx into mdx fibres when the fibres are stretched during active contractions (Yeung et al., 2005), and it is therefore plausible that TRPC1 channels mediate this Ca2+ entry. We have also recently shown that ROS contribute to stretch-induced muscle damage in mdx mice (Whitehead et al., 2008). Therefore, it is possible that ROS increase SAC- and/or TRPC1-mediated Ca2+ influx in mdx muscle, an idea supported by recent findings in mdx cardiac cells (Jung et al., 2008).
On the basis of the high degree of homology between caveolin-1 and caveolin-3 (Tang et al., 1996) and previous reports that caveolin-1 binds to TPRC1 (Lockwich et al., 2000; Brazer et al., 2003), we investigated the interaction of caveolin-3 and TRPC1 and how this regulates TRPC1 activity. The role of Src kinase, another TRPC1 binding partner, and its possible activation by ROS was also investigated. Here, we provide evidence that a Ca2+-influx pathway involving ROS, Src, TRPC1 and caveolin-3 might contribute to the elevated [Ca2+]i that is characteristic of mdx/DMD skeletal muscle.
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Results |
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) and caveolin-3 in mdx skeletal muscle (Vaghy et al., 1998). Caveolin-3 and TRPC1 showed a similar sarcolemma labelling pattern, consistent with the hypothesis that these proteins can interact. Double labelling of cryosections of mdx and WT muscle were then used to compare the degree of colocalisation (Fig. 3A-F). Binary images of each labelled protein were generated and overlaid (see Materials and Methods; Fig. 3F,G). White pixels in the overlaid image represent colocalisation, independent of fluorescence intensity (Gervásio and Phillips, 2005) (Fig. 3G). Quantification showed that over 50% of caveolin-3 and TRPC1 colocalised in the sarcolemma, in both WT and mdx groups (Fig. 3H).
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TRPC1 and caveolin-3 interact directly in skeletal muscle
Image analysis revealed a close spatial relationship between caveolin-3 and TRPC1 (Fig. 3A-H). To further investigate the interaction between caveolin-3 and TRPC1, we used co-immunoprecipitation. TA muscles were homogenised in immunoprecipitation (IP) buffer and the lysate was incubated with anti-TRPC1. Immunocomplexes were resolved by SDS-PAGE and the membranes were then probed for caveolin-3. Immunoblots showed that TRPC1 and caveolin-3 co-immunoprecipitated (Fig. 3I,J). Experiments using muscles from WT and mdx mice showed that caveolin-3 and TRPC1 co-immunoprecipitated in each case (Fig. 3J). In order to confirm that the detected band represented caveolin-3 and not a background signal from the immunoprecipitating antibody (Kelly et al., 2007), negative controls with no anti-TRPC1 antibody or no lysate were used (Fig. 3I). The reverse IP (IP with anti-caveolin-3 and immunoblot probed with anti-TRPC1) also showed that caveolin-3 and TRPC1 co-immunoprecipitated (Fig. 3K).
To test whether TRPC1 and caveolin-3 interact directly, we transfected TRPC1-CFP (cyan fluorescent protein) and caveolin-3–YFP (yellow fluorescent protein) into C2 myoblasts. Caveolin-3–YFP was expressed both in the cytoplasm and at the cell surface (Fig. 4A). Cells transfected with TRPC1-CFP only showed diffuse cytoplasmic expression of the protein, even though these cells express low levels of caveolin-1 (Galbiati et al., 1999). The levels of endogenous caveolin-1 might be insufficient to target the overexpressed TRPC1 to the plasma membrane in this system. When co-transfected with caveolin-3–YFP, TRPC1-CFP re-distributed to the plasma membrane. In myoblasts transfected with both proteins, a high degree of colocalisation was found between caveolin-3–YFP and TRPC1-CFP, both intracellularly and at the plasma membrane (Fig. 4A).
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It has been shown that immunoprecipitation of caveolae-rich microdomains (using anti-caveolin-1 antibody) does not necessarily only reveal caveolin binding partners but can also reveal other proteins present in this lipid raft environment (Zheng and Bollinger Bollag, 2003). To test whether the interaction was likely to be a direct one, we performed fluorescence resonance energy transfer (FRET) using two independent approaches: photobleaching of acceptor, and fluorescence lifetime imaging microscopy (FLIM; see Materials and Methods). C2 myoblasts were transfected with either TPRC1-CFP or caveolin-3–YFP, or both. In cells transfected with both expression plasmids, photobleaching of the acceptor fluorophor (caveolin-3–YFP) increased the fluorescence intensity of the donor (TRPC1-CFP). Because the Förster distance of this FRET pair is 4.9 nm (and defines the distance of a FRET pair in which transfer is 50% efficient) (Patterson et al., 2000), the FRET efficiency calculated here (21%±0.01, n=10 cells) suggests that the two tag proteins were 6.1 nm from each other (Bastiaens et al., 1996; Gervásio et al., 2007) (Fig. 4B). Photobleaching using the 514 nm laser line of samples transfected either with TRPC1-CFP or caveolin-3–YFP alone resulted in no increase of the fluorescence in the CFP channel (supplementary material Fig. S1). In FLIM, the lifetime of the donor (CFP) is reduced when in close proximity to its FRET partner (YFP, acceptor) (Cox et al., 2007). The lifetime of CFP in C2 cells transfected with TRPC1-CFP alone was 2.7±0.03 nanoseconds (ns) (Fig. 5A-C). When cells were co-transfected with both TRPC1-CFP and caveolin-3–YFP plasmids, the lifetime of the CFP was reduced to 2.17±0.03 ns (P<0.001). This equates to a FRET efficiency of 19.6%. Thus, both photobleaching of acceptor and FLIM support the idea that TRPC1 and caveolin-3 bind directly.
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), (2) Src binds to caveolin-3 and to members of the TRPC family (Song et al., 1996; Kawasaki et al., 2006) and (3) Src activates TRPC3 (Vazquez et al., 2004). These findings led us to investigate the effect of caveolin-3 and Src kinase on TRPC1 activity. Given that Src kinase is activated by H2O2 in various cell types (Sato et al., 2001; Chen et al., 2001; Suzaki et al., 2002), we first tested whether H2O2 would activate endogenous Src in cultured C2 cells, and whether PP2 (a Src-kinase inhibitor) would prevent such activation. C2 cells were incubated with 10 µM H2O2 for 15 minutes followed by western blot analysis. The membranes were probed with antibodies against Src and pY418-Src, the active form of Src. H2O2 induced close to a twofold increase of the active form of Src (P<0.01; Fig. 6). This increase was completely blocked when cells were pre-incubated with 10 µm PP2 (P<0.05).
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The activity of TRPC1 upon Src activation was assessed by Ca2+ imaging. Fura Red was chosen because of its emission spectrum (far red), which does not interfere with the fluorescence of CFP and YFP. In addition, Fura Red being a ratiometric Ca2+ probe, estimation of Ca2+ concentration is not affected by probe loading, bleaching or illumination intensity. Myoblasts transfected with either TRPC1-CFP, caveolin-3–YFP or a combination of both plasmids were loaded with Fura Red and imaged with confocal microscopy. The resulting concentration of [Ca2+]i is shown as a ratio of the fluorescence emission (Fig. 7A, lower graph) upon excitation using 458 nm and 488 nm laser lines (Fig. 7A, upper graph). When H2O2 was applied to the cultures, an increase in the [Ca2+]i was detected, but only in cells transfected with TRPC1-CFP and caveolin-3–YFP (P<0.001; Fig. 7B,C). Cells transfected with caveolin-3–YFP (data not shown) or TRPC1-CFP (Fig. 7C) alone did not show a statistically significant [Ca2+]i increase with H2O2. In order to assess the participation of Src kinase on TRPC1 activity, cells co-transfected with both TRPC1-CFP and caveolin-3–YFP plasmids were incubated with PP2 1 hour prior to H2O2. PP2 abolished the rise in [Ca2+]i in such cells (P<0.001; Fig. 7C). These results suggest that activation of Src by ROS increases TRPC1–caveolin-3-dependent Ca2+ influx.
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ROS cause increased Ca2+ influx through SACs in mdx fibres
We have recently shown that the stretch-induced rise of [Ca2+]i in mdx muscle fibres can be prevented by blockers of SACs (Yeung et al., 2005). Given that TRPC1 is a candidate protein for SACs in mdx muscle (Vandebrouck et al., 2002; Vandebrouck et al., 2007), we were interested to determine whether ROS could activate these channels in mdx muscle, as was the case for TRPC1 in our cultured C2 cells.
In the first series of experiments, we looked at the effect of the ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron) on resting [Ca2+]i following stretched contractions of mdx fibres. As shown in Fig. 8A, [Ca2+]i increased almost threefold over the 20 minutes after the stretched (eccentric) contractions for control mdx fibres. This rise in Ca2+ was inhibited by 5 mM Tiron. Tetanic force after the stretched contractions was also significantly greater (
25%; P<0.05) for fibres treated with Tiron compared with controls (data not shown).
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Src inhibition protects mdx muscle against stretch-induced damage
If SACs in mdx muscle are formed by TRPC1, is this channel activity regulated by Src kinase, as we had shown in the C2 cell culture experiments? To test this, we subjected isolated extensor digitorum longus (EDL) muscles from mdx mice to a series of stretched contractions, which we have recently shown leads to a large force deficit and ROS-dependent increase in membrane permeability in mdx muscle (Whitehead et al., 2008). Throughout the experiment, muscles were perfused with either the Src-kinase inhibitor PP2 or with its inactive analogue PP3. Muscle force was measured at three time points after the stretched contractions (0, 30 and 60 minutes) and compared with the pre-stretch value. At all three time points, force was significantly greater for muscles perfused with PP2 compared with PP3 (P<0.05 at 0 and 30 minutes; P<0.001 at 60 minutes). These results suggest that inhibition of Src-kinase activity provides protection against stretch-induced damage in mdx muscle.
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Discussion |
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; Liu et al., 2000; Zagranichnaya et al., 2005) and via different pathways (Sinkins et al., 1998; Vanden Abeele et al., 2004). Activation of TRPC1 by membrane stretch, however, has been a more controversial topic. Skeletal muscle is well-known to contain a mechanosensitive channel (Guharay and Sachs, 1984), and Maroto and colleagues proposed that TRPC1 encoded this channel (Maroto et al., 2005). In a more recent report, Gottlieb and colleagues were unable to confirm that TRPC1 expression produced a mechanosensitive channel in COS cells (Gottlieb et al., 2008). However, a weakness of these later experiments is that TRPC1 was not expressed in the plasma membrane. In our experiments, caveolin-3 expression was found to assist trafficking of TRPC1 to the plasma membrane, and TRPC1 contributed to Ca2+ influx only when expressed in the membrane.
In this study, we show that TRPC1 activity depends on Src phosphorylation. STIM1 and Src kinase both bind to TRPC1 (Huang et al., 2006; Kawasaki et al., 2006). Src has been shown to activate TRPC3 via phosphorylation of its N-terminus (Y226). Moreover, when TRPC3 was expressed in cells lacking Src, Fyn and Yes (Src-kinase family members), only the co-transfection of Src was able to rescue the activity of TRPC3 (Kawasaki et al., 2006). Tyrosine 226 of TRPC3, the target of Src phosphorylation, is not present in TRPC1. Nevertheless, TRPC1 has five other consensus sites for tyrosine phosphorylation in its N-terminus, including Y224 (www.cbs.dtu.dk/services/NetPhos). We have shown that the Ca2+ influx is six-times greater in TRPC1–caveolin-3-transfected myoblasts compared with control, and this influx was dependent on Src activation (by ROS). The FRET results might also be interpreted as evidence that Src induces the opening of TRPC1 channels (Fig. 5D). Activation of Src by H2O2 decreased the efficiency of FRET between TRPC1 and caveolin-3 from 20% to 14% (Fig. 5D). One possible explanation for the decrease in FRET efficiency is an increase in the distance between TPRC1 and caveolin-3, which could be either a cause or a consequence of channel opening. Caveolins generally inhibit proteins that they bind to (Williams and Lisanti, 2004); thus, reduced binding between TRPC1 and caveolin-3 might be expected to increase the activity of TRPC1. However, notwithstanding decrease in the FRET efficiency when Src became active, caveolin-3 and TRPC1 interaction was not abolished (FRET efficiency 14%; Fig. 5D). Another possibility is that conformation changes in the TRPC1 channels (between the open and closed states) lead to the change in FRET efficiency. Kobrinsky and colleagues tagged the C- and N-termini of a K+-gated channel with CFP and YFP, and observed a lower FRET efficiency between the subunits of the assembled channel when it became open (Kobrinsky et al., 2006). According to these authors, the decrease in FRET efficiency might reflect a conformational change in the intracellular termini that occurs during opening of the channel. Conceivably, some similar phenomenon might occur with TRPC1 upon Src activation. Thus, H2O2 activated Src and reduced the interaction between TRPC1 and caveolin-3, but the precise molecular rearrangement of the TRPC1–caveolin-3 complex remains uncertain.
On the basis of the observations that Src activation induced Ca2+ influx through TRPC1 channels (Fig. 7), we then investigated whether Src could affect TRPC1 activity via direct phosphorylation. We carried out different approaches in order to detect phosphorylation of TRPC1 by Src by using phospho-immunoprecipitation and FRET (intramolecular FRET). Neither of the techniques provided evidence of a significant increase of TRPC1 phosphorylation upon Src activation (data not shown). It remains possible that the techniques we have used are not sufficiently sensitive to detect phosphorylation of TRPC1 by endogenous Src. Indeed, Kawasaki and colleagues only detected TRPC3 phosphorylation when exogenous Src was co-transfected with TRPC3 (Kawasaki et al., 2006). Alternatively, Src might regulate TRPC1 via an indirect path (activation of other kinases or cytoskeleton modifications) and our experiments do not rule out either possibility. Whether Src kinase acts directly or indirectly on TRPC1 remains to be further investigated.
We have shown here that activation of TRPC1 depends on Src-kinase phosphorylation, but a heteromeric channel containing TRPC1 might also be activated by phosphorylation of other subunits. It has been shown that TRPC1 binds to TRPC3 and that this heteromer forms a SOC (Liu et al., 2005). However, immunolabelling of TRPC3 in isolated adult muscle fibres showed an intracellular pattern that did not colocalise with TRPC1, which was present in the sarcolemma (Vandebrouck et al., 2002). TRPC4, which is also found in skeletal muscle, can bind to TRPC1 (Hofmann et al., 2002), is located in the sarcolemma (Vandebrouck et al., 2002) and is phosphorylated by Src (Odell et al., 2005). Therefore, we suspect that TRPC4 activation by Src might also participate in the activation of a TRPC1-TRPC4 heterologous channel in adult muscle fibres.
Role of caveolin-3 in TRPC1 activity
Interaction with caveolin-3 is important for the plasma membrane expression and function of TRPC1. Caveolin-1 binds to TRPC1, helping to target TRPC1 to lipid rafts and regulate its activity (Brazer et al., 2003). Caveolin-1-knockout mice showed impaired endothelial Ca2+ entry and a decreased interaction between TRPC1 and TRPC4, both of which were rescued by the reintroduction of caveolin-1 (Murata et al., 2007). Caveolin-1 can bind to both the N- and C-terminal ends of TRPC1, but the N-terminal interaction is responsible for targeting the channel to the plasma membrane (Brazer et al., 2003). Deletion of the N-terminus of TRPC1 prevented the surface expression of the channel and reduced store-operated Ca2+ entry in transfected cells. Kwiatek and colleagues reported that the caveolin-1 scaffolding domain bound to the TRPC1 C-terminus and markedly reduced Ca2+ influx (Kwiatek et al., 2006). Caveolin-3 is 85% similar to caveolin-1 (Tang et al., 1996) and we have shown evidence that, similar to caveolin-1 (Brazer et al., 2003), caveolin-3 also binds to TRPC1.
In addition to binding to TRPC1, caveolin-3 also assisted in the targeting of this protein to the plasma membrane when both proteins were co-expressed in myoblasts. Myoblasts transfected with only TRPC1 showed no clear plasma membrane expression, nor Ca2+ influx induced by Src activation. Thus, caveolins appear to play an important role in determining the plasma membrane residency (and perhaps assembly) of TRPC1 (Figs 4 and 7) (Brazer et al., 2003). Caveolin-3 might also be responsible for stabilisation of TRPC1 on the cell surface. Hernández-Deviez and colleagues (Hernández-Deviez et al., 2008) recently found that, in cells lacking caveolin-1, dysferlin on the plasma membrane was less stable. By analogy, caveolin-3 might well be important for the stabilisation of TRPC1. Once both TRPC1 and caveolin-3 are assembled in the membrane, caveolin-3 might exert a partial inhibitory effect on the activity of the TRPC1 channel, as has been observed in other systems (Kwiatek et al., 2006; Williams and Lisanti, 2004).
Role of caveolin-3, Src kinase and TRPC1 in DMD
Our data suggest that caveolin-3 expression is necessary for TRPC1 localisation. Src activation produced detectable changes in the interaction between TRCPC1 and caveolin-3 (by FRET) and stimulated opening of the TRPC1 channel. This might be important in muscular dystrophies, especially DMD, because levels of TRPC1, caveolin-3 and Src kinase were all increased in mdx muscle. Cell culture experiments showed that Src activation by ROS markedly increased Ca2+ influx in cells expressing TRPC1 and caveolin-3. Therefore, we carried out experiments on mdx muscle to investigate the role of ROS and Src on the activation of SACs, which might be formed by TRPC1 (Vandebrouck et al., 2002; Maroto et al., 2005; Vandebrouck et al., 2007). First, we showed that the stretch-induced Ca2+ influx in mdx fibres was prevented by the ROS scavenger Tiron, suggesting that ROS mediates SAC activity in mdx muscle. We then provided evidence that exogenous ROS also increased Ca2+ influx through SACs in mdx, but not WT, fibres in the absence of stretch. This result suggests that mdx muscle is more sensitive to ROS. This is consistent with the findings of Rando and colleagues (Rando et al., 1998), who demonstrated that mdx muscle is more susceptible to oxidative cell death than WT. To further investigate the possible role of Src kinase in mdx muscle, we carried out stretched contractions on isolated mdx muscles to assess the role of this protein in muscle damage. Our experiments show that Src inhibition during stretched contractions provided protection against muscle damage. Because we have previously shown that blockers of SACs prevented the rise in [Ca2+]i (Williams and Allen, 2007a) and significantly improved force after stretched contractions in mdx muscle (Yeung et al., 2005; Whitehead et al., 2006b), our current findings lead us to hypothesise that Src inhibition might provide protection against muscle damage in mdx muscle owing to a reduction in Ca2+ influx through SACs and/or TRPC1. In addition, given that ROS can activate Src and that ROS production is increased in dystrophic muscles (Williams and Allen, 2007b; Whitehead et al., 2008), this pathway could mediate increased activity of TRPC1 in mdx/DMD muscle. Based on the results presented here, we suggest that targeting the ROS-Src-TRPC1/caveolin-3 pathway could lead to the development of new therapeutic approaches for the treatment of DMD.
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Materials and Methods |
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).
Immunoblotting and immunoprecipitation
Animals were killed with an overdose of sodium pentobarbitone (163 mg/kg of body weight) diluted in heparin (Whitehead et al., 2006a). TA muscle lysates were immunoblotted as previously described (Whitehead et al., 2008) for anti-caveolin-3 (1:2000; BD Biosciences, San Jose, CA), anti-TRPC1 (1:1000; Alomone Labs, Jerusalem, Israel), anti-Src (1:1000; Chemicon/Millipore, Billerica, MA), anti-pY418-Src (Sigma) and anti-GAPDH (1:15,000; Chemicon/Millipore). Total protein was measured using a Bradford assay (Bio-Rad, Hercules, CA). Bands were visualised with HRP-conjugated anti-mouse IgG (for caveolin, anti-Src and GAPDH) or anti-rabbit IgG (for TRPC1 and anti-pY418-Src; 1:1000; Santa Cruz, CA) (Whitehead et al., 2008). Quantification of western blots was obtained by multiplying the area and intensity of each band using ImageJ software (Whitehead et al., 2008). The graphs shown in Fig. 1 were normalised to GAPDH as a loading control (probed on the same gel).
For the IP, muscles were lysed in IP buffer containing 50 mM Tris pH7.5, 150 mM NaCl, 25 mM EDTA, 25 mM EGTA and 1% NP-40, and protease and phosphatase inhibitors (Sigma, St Louis, MO). Lysate (200 µg of protein) was first incubated with 50 µl of protein-A-Sepharose beads (Amersham Biosciences-GE Healthcare, Piscataway, NJ) for 1 hour. The beads were precipitated (800 g, 3 minutes) and the supernatant (cleared lysate) was incubated with 2.5 µg of anti-TRPC1 antibody for 2.5 hours. Fresh beads (25 µl) were added to the lysate and incubated for 1 hour. The immunocomplex beads were precipitated and were washed four times with the same buffer described above containing 0.1% NP-40. The immunoprecipitate was resolved by 8% SDS-PAGE. The membranes were probed for caveolin-3 and bands were detected as described above. In a reverse IP, the lysate was incubated with anti-TRPC1 and the membrane was probed with anti-caveolin-3. All the steps of the IP were carried out at 4°C.
Immunofluorescence and image analysis
TA muscles were processed and cryosections stained for immunofluorescence as previously described (Gervásio el al., 2007) but using anti-caveolin-3 and anti-TRPC1 antibodies (overnight, 4°C) visualised with Alexa-Fluor-488-conjugated goat anti-mouse antibody (1:1000; Molecular Probes, Eugene, OR) and Cy3-conjugated goat anti-rabbit (1:250; Jackson ImmunoResearch Laboratories, West Grove, PA), respectively. Control sections were incubated with secondary antibody (no primary antibody) to confirm the absence of significant non-specific binding or fluorescence cross-bleed (data not shown). Sections were imaged with a Zeiss EC Plan-Neofluar 40x 0.75 NA dry objective by laser scanning confocal microscopy (Zeiss LSM 510 Meta). ImageJ software was used for fluorescence and colocalisation quantitation of caveolin-3 and TRPC1 (Gervásio and Phillips, 2005).
Fluorescence resonance energy transfer (FRET)
FRET between caveolin-3 and TRPC1 was tested using two independent approaches: photobleaching of acceptor (Bastiaens et al., 1996; Kenworthy, 2001) and FLIM (Cox et al., 2007). Plasmids were kindly donated by Robert Parton [caveolin-3–YFP (Pol et al., 2004)], and Thomas Hofmann and Thomas Gudermann [TRPC1-CFP (Hofmann et al., 2002)]. The identity of the plasmids was confirmed by sequencing. Immunocytochemistry of transfected cells also confirmed that the exogenously expressed proteins were indeed TRPC1 and caveolin-3 (supplementary material Fig. S2). C2 myoblasts were cultured on glass coverslips and transfected with either plasmid or a combination of both using Effectene transfecting reagent (Qiagen; Chatsworth, CA) according to manufacturer's instructions. At 48 hours after transfection, cells were washed in PBS and fixed with 2% paraformaldehyde in PBS (20 minutes, room temperature). Coverslips were mounted in DABCO anti-fade reagent (FUKA) and cells were visualised using either the Zeiss LSM 510 Meta confocal microscope (photobleaching of acceptor) or Nikon D-Eclipse C1 confocal microscope (Japan) attached to a Nikon TE2000 microscope (equipped with a High Speed Lifetime Imaging Module – LIMO/FLIM).
For photobleaching of acceptor, a region of interest (ROI) within transfected cells was selectively exposed to ten scans using the 514 nm laser line at 100% power. The whole cell was imaged at lower laser intensity (15%) for both donor and acceptor optical channels immediately before and after the bleaching of acceptor. FRET efficiency (E) was calculated from the increase of the fluorescence intensity of the donor after the acceptor was selectively photobleached (Bastiaens et al., 1996; Gervásio et al., 2007).
FLIM was also used to detect FRET between the tagged caveolin-3 and TRPC1. Experimentally, this can be calculated by measuring the fluorescence lifetime of the donor in the presence and absence of the acceptor using the following expression: E=1–(tDA/tD), where tDA and tD are the donor lifetimes in the presence and absence of acceptor, respectively (E=FRET efficiency) (Bastiaens and Squire, 1999). CFP was excited by a picosecond-pulsed 405 nm laser (PicoQuant GmbH, Berlin, Germany) and emission was collected using a band-pass barrier filter (470-490 nm, Nikon). The average lifetime of each pixel was calculated by the LIMO software from images of 100x100 pixels.
Hydrogen peroxide incubations
H2O2 was used at 10 µM final concentration for 15 minutes in culture media for all experiments. Cells were washed once with cold PBS and lysed for immunoblot analysis of total Src and pY418-Src as described above. In a subset of dishes, the Src-kinase inhibitor PP2 (Calbiochem, San Diego, CA) was added 1 hour prior to H2O2 incubation.
Ratiometric Ca2+ imaging
Cells were grown on 35 mm glass bottom dishes (Matek, Homer, MA) and transfected with either caveolin-3–YFP, TRPC1-CFP or a combination of both plasmids as described above. Fura Red AM (Molecular Probes) was used at a final concentration of 10 µM. At 48 hours after transfection, cells were loaded with Fura Red for 45 minutes at 37°C and imaged using a Zeiss C-Apochromat 40x 1.2 NA water-immersion objective (Zeiss LSM 510 Meta). A sequential acquisition mode was set up using the 458 and 488 nm argon laser lines (four images/second; emission 636-750 nm using Meta detector). ROI were drawn around transfected and non-transfected cells, and fluorescence intensities were plotted against time using the Zeiss 510 Meta Software version 4.2. [Ca2+]i is shown as a ratio of fluorescence intensities of each ROI [458 nm/488 nm (Ippolito et al., 2006)].
Ex vivo experiments on mdx single fibres
mdx and WT mice (8-12 weeks of age) were killed by cervical dislocation and single fibres were dissected, as described previously (Yeung et al., 2005). Single fibres were then transferred to a perspex chamber and perfused with a standard solution containing (mM): NaCl (121), KCl (5), CaCl2 (1.8), MgCl2 (0.5), NaH2PO4 (0.4), NaHCO3 (24) and glucose (5.5). The solution was continuously bubbled with 95% O2 – 5% CO2 (pH 7.4). Experiments were performed at room temperature. Aluminium clips clamped to the tendons enabled the fibre to be attached to a force transducer and the lever of a motorised length controller. Fibres were loaded with 10 µm Fluo-4-AM (Molecular Probes), to measure the resting [Ca2+]i.
In the first experiments, we investigated the effect of the ROS scavenger Tiron on stretch-induced [Ca2+]i. mdx fibres underwent a series of ten stretched (eccentric) contractions, using the same stimulation and stretch protocol as described previously (Yeung et al., 2005). In some experiments, fibres were perfused with 5 mM Tiron from 20 minutes before the stretched contractions until the conclusion of the experiment (30 minutes after the stretched contractions).
In the second series of experiments, we investigated whether application of an exogenous ROS could increase [Ca2+]i via SACs, independent of muscle stretch. Here, WT and mdx fibres were incubated with 10 µM H2O2 for a total of 70 minutes while [Ca2+]i was measured. Fibres were initially exposed to H2O2 for 30 minutes and then 200 µM streptomycin, a SAC blocker, was also added to the perfusate (see Fig. 8B).
Ex vivo experiments on mdx muscle
EDL muscles were dissected from mdx mice and transferred to a perspex chamber as described above for the single-fibre experiments (for details, see Whitehead et al., 2008). The muscles were subjected to a series of ten stretched contractions, using the same protocol as described in Whitehead et al. (Whitehead et al., 2008). Before, during and after the stretched contractions, muscles were perfused with either 10 µM PP2 (a Src-kinase inhibitor) or 10 µM PP3 (an inactive analogue of PP2), which was used as a control. Maximum tetanic force (120 Hz stimulation) was measured at 0, 30 and 60 minutes after the stretched contractions and compared with pre-stretch values.
Statistics
Student's t-test (two-tailed) was used to compare means of two different groups. Analysis of variance (ANOVA) was used in situations of more than two groups (one-way ANOVA: Fig. 5D, Fig. 6 and Fig. 7C) or two variables (two-way ANOVA: Fig. 8). Mean and standard error of the mean (s.e.m.) are shown for each group.
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