Favero, Christopher. W. Ward, and Jay H. Williams. Adaptation of the skeletal muscle calcium-release mecha- nism to weight-bearing condition. Am. J. PhysioZ.
Adaptation mechanism
of the skeletal muscle calcium-release to weight-bearing condition
SUSAN C. KANDARIAN, DAVID G. PETERS, TERENCE G. FAVERO, CHRISTOPHER W. WARD, AND JAY H. WILLIAMS Department of Health Sciences, Boston University, Boston, Massachusetts 02215; Department of Biology, University of Portland, Portland, Oregon 97203; and Department of Human Nutrition and Foods, Virginia Tech, Blacksburg, Virginia 24061 Kandarian, Susan C., David G. Peters, Terence G. Favero, Christopher W. Ward, and Jay H. Williams. Adaptation of the skeletal muscle calcium-release mechanism to weight-bearing condition. Am. J. PhysioZ. 270 (CeZZ PhysioL. 39): C1588-C1594, 1996.-In the present study, we examined whether weight-bearing condition can regulate the sarcoplasmic reticulum (SR) Ca2+-release mechanism. Measurements of cq-subunit dihydropyridine (q-DHP) and ryanodine receptor levels were made in hypertrophied fast-twitch plantaris muscles 5 wk after surgical removal of synergist muscles (increased weight bearing) and in atrophied slowtwitch soleus muscles (14 days of non-weight bearing) of the rat. Rates ofAgN03-induced SR Ca2+ release were measured with fura 2 as the Ca2+ indicator and pyrophosphate as the precipitating ion during vesicular Ca2+ loading. Ca2+-release rates were 38, 49, and 58% lower in vesicles from hypertrophied vs. control muscles at AgN03 concentrations of 0.05, 0.5, and 5 uM, respectively (control = 18.2 2 1.4 uM*rng-l. min). Western blots showed no differences in the relative expression of (x1-DHP or ryanodine receptor when IIID5 (monoclonal) or GP3 (polyclonal) antibodies were used. There was also no difference in ryanodine (10 nM> binding in Ca2+-incubated SR vesicles from hypertrophied muscles, suggesting no difference in the number of channels. In contrast, expression of q-DHP and ryanodine receptors was increased by 144 and 157% in non-weight-bearing soleus muscles, respectively. Scatchard analysis of DHP binding showed a 40% increase in maximum binding capacity and no change in the dissociation constant with non-weight-bearing muscles. The direction of modification of the SR Ca2+-release mechanism is opposite with increased and decreased weight bearing, but the mechanism by which this is achieved appears to be different .
COUPLING is the multistep process whereby T tubule depolarization results in the release of stored CaZ+ from the sarcoplasmic reticulum (SR), which subsequently binds to troponin to activate cross-bridge cycling. The mechanism of excitationcontraction coupling in skeletal muscle involves an interaction between the T tubule Ca2+ channel [dihydropyridine (DHP) receptor], which functions as a voltage sensor, and the SR Ca2+-release channel (ryanodine receptor) (for review, see Ref. 25). In the original working model, charge movement at the T tubule membrane was proposed to evoke a conformational change in the voltage sensor, which in turn causes the
SR Ca2+ channels to open (31), but the detailed mechanism of SR Ca2+ channel activation is still incomplete. Despite the variety of protein isoforms that distinguish fast- and slow-twitch muscle, the same isoform of the DHP receptor and ryanodine receptor is expressed in both slow- and fast-twitch mammalian skeletal muscles. However, the level of expression is considered a determinant of muscle phenotype because both proteins are present in lo-fold-greater levels in fastcompared with slow-twitch whole muscle and in 3-foldgreater levels in fast vs. slow SR (17, 18, 21). The level of protein expression is consistent with differences in contractile parameters between fiber types because fast-twitch muscle has considerably faster SR Ca2+release rates as well as more rapid twitch times (18, 29,30). Alterations in the activity pattern of skeletal muscle by electrical stimulation, exercise, and biomechanical loading condition induce changes in protein isoform expression and functional parameters (for recent reviews, seeRefs. 2 and 23). Besides alterations in metabolic and contractile protein expression, at least one protein having a Ca2+-regulatory role, the SR Ca2+-transporting ATPase, can be modulated by activity. In fast-twitch ! muscles, both chronic electrical stimulation (3, 4) and increased weight-bearing (15) activity induce a fast to slow shift in isoform expression, whereas chronic decreases in weight bearing activity induce a slow to fast isoform transition in slow-twitch muscles (32). Less is known about the modulation of proteins involved in SR Ca2+ release by chronic changes in activity. Levels of the ryanodine receptor and the cxI-subunit DHP (QL~DHP) receptor protein are significantly depressed after chronic electrical stimulation of fast-twitch dog muscle (21), whereas DHP receptor levels in fast- and slowtwitch leg skeletal muscles are increased after run training exercise (26). The purpose of the present study was to determine if chronic changes in weight-bearing condition can regulate the SR Ca2+-release mechanism. We have previously shown that as little as 24 h of non-weight bearing induces significant increases (80%) in the (xi-DHP receptor mRNA levels in slow-twitch muscles that plateau by 14 days of non-weight bearing at 200% greater than control (14). In the present study, we test whether these changes are apparent at the protein level for the rxl-DHP receptor as well as for the ryanodine receptor in non-weight-bearing limb slowtwitch muscle. We also examine whether the opposite condition, chronic increased weight bearing in fasttwitch muscle, elicits decreases in DHP and ryanodine
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the American
dihydropyridine receptor; nels; mechanical loading; phy; muscle hypertrophy;
ryanodine receptor; calcium chanhindlimb suspension; muscle atrosarcoplasmic reticulum
EXCITATION-CONTRACTION
0363-6143/96
$5.00
Copyright
o 1996
Physiological
Society
ADAPTATION
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receptor protein expression as well as decreases in the rate of SR Ca2+ release in actively loaded SR vesicles. IMETHODS
ChemicaZs. All chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Surgical muscle ablation to effect increased weight bearing. Anesthetized (pentobarbital sodium, 50 mg/kg) rats underwent bilateral ablation of the gastrocnemius and soleus muscles to effect chronic increased muscle loading, leading to hypertrophy as detailed previously (15). Five weeks later, plantaris muscles were removed from both legs and SR vesicles were immediately prepared. Non-weight bearing. Female Wistar rats (3 mo old) were tail suspended to effect a non-weight-bearing condition, using an elastic tail cast that was detailed previously (32). Soleus muscles were collected from the right and left legs after 14 days, and SR vesicles or whole muscle homogenates were prepared. Some soleus muscles from 7 days of non-weight bearing were used for DHP-binding experiments in crude membranes. Preparation of SR vesicles. Muscles were removed, and SR were vesicles prepared as described by Luckin et al. (19) and employed in our laboratory (15). Muscles were homogenized in an isolation buffer containing (in mM) 20 N-2-hydroxyethylpiperazineJV’-2-ethanesulfonic acid (HEPES, pH 7.5), 250 sucrose, 0.2% sodium azide, and 0.2 phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 1,600 g (10 min). The supernatant was passed through cheesecloth and centrifuged twice at 10,000 g (20 min) and 48,000 g (90 min). The pellet was resuspended in isolation buffer, washed in 0.6 M KCl, and centrifuged at 48,000 g (60 min). The final pellet was homogenized in isolation buffer, and protein concentration was determined with Bradford protocol as described in the Bio-Rad kit. Protein concentration was the same when either a modified Lowry assay to solubilize membrane proteins was used or the Bio-Rad kit was used. Preparation of crude membranes. Membranes were isolated as previously described (32). Briefly, muscles were homogenized in (in mM) 10 tris(hydroxymethyl)aminomethane (Tris), 250 sucrose, 1 ethylene glycol-bis( P-aminoethyl ether&N& N’,N’-tetraacetic acid (EGTA), and 0.5 PMSF and centrifuged 2.5 l
at 10,OOOg (20 min). The supernatant was brought to 0.6 M KCl, allowed to stand on ice for 1 h, and centrifuged at 150,000 g (45 min). The pellet was resuspended in Trissucrose, and protein concentration was determined with the use of the Bradford protocol as described in the Bio-Rad kit. Antibodies. The antibodies used for Western analysis have been described by previous investigators. A guinea pig polyclonal anti-rabbit antibody (GP3) was used to assess ryanodine receptor levels (16), and a mouse monoclonal anti-rabbit antibody (IIID5, specific to the (wi-DHP) was used to assess (xi-DHP receptor levels (13). These antibodies do not recognize cardiac isoforms. Western blot analysis. SR vesicle protein was electrophoretitally separated on a 5 or 7% polyacrylamide gel for analysis of ryanodine or DHP receptor, respectively, essentially as we have described (15). Protein in the gel was then transferred to nitrocellulose or polyvinylidene difluoride membrane in transfer buffer (20% methanol in electrode buffer with 0.01% sodium dodecyl sulfate) in a high-intensity electrical field (65 V) for 1 h, using plate electrodes (Bio-Rad) at 5°C. Blots were blocked for 2 h in 3% bovine serum albumin in phosphatebuffered saline or 5% Blotto and then incubated overnight (5°C) with primary antibody. An alkaline phosphataseconjugated secondary antibody was used for color development, using 5-bromo-4-chloro-3-indolyphosphate p-toluidine salt-nitro blue tetrazolium as the substrate. Signals on blots were quantitated with an LKB Pharmacia UltroScan densitometer. Preliminary experiments showed that the signals vs. protein loaded were in the linear range (data not shown). SR Ca2+ uptake and release. Ca2+ uptake and release were measured in 1 ml of incubation buffer containing 92.5 mM KCl, 18.5 mM Tris, 7.5 mM pyrophosphate, 1 mM MgC12, and 2 uM free Ca2+ (pH 7.0). Temperature was maintained at 37°C and the buffer was continually stirred. First, 50 ug of SR protein were added and allowed to equilibrate for 3 min. Uptake was then initiated by the addition of 2 mM Na2ATP and continued until no change in extravesicular free Ca2+ was observed (see Fig 1). After uptake, Ca2+ release was initiated by the addition of 0.05, 0.5, or 5 uM AgNOs. The rates of Ca2+ uptake and release were determined as the steepest negative and positive slope of the free Ca2+ vs. time curve. The total amounts of Ca2+ sequestered and released were computed as the difference between the plateau portions
-3 1.5 -1
Release/Uptake Amount
L;I
1 0.0
Cl589
ATP
i“co 2 g 1.0
0.5 !
MECHANISM
\\’
60s
Fig. 1. Sample recording of free Ca”+ fluxes over time using fura 2 as extravesicular Ca2+ indicator. Sarcoplasmic reticulum (SR) Ca2+ uptake initiated by ATP, and Ca”+ release induced by application of AgN03. Pyrophosphate is SR Ca”+-precipitating ion. Amount of Ca2+ released or taken up by vesicles indicated by vertical arrow (right ).
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of the curve measured before initiation of uptake, at the end of uptake, and at the end of release.All rates and magnitudes of Ca2+ exchange were normalized by SR protein concentration. All samples were run in triplicate with coefficients of variation between 1 and 8%. Extravesicular free Ca2+ was measured with the fluorescent Ca2+ indicator fura 2 (4 pM). Fluorescent changes were monitored with a Jasco CAF-160 fluorometer with excitation light filtered at 340 and 380 nm and emissionlight detected at 500 nm. The ratio (R) of fluorescencedue to excitation and at 340 and 380 nm was used to calculate free Ca2+ in the incubation medium according to the following formula: Ka2+lfree = & x p x [(R - R,,,)/(R - R,,,)l, where the fura 2-Ca2+dissociation constant (&) was assumedto be 200 nM (lo), Rminand h, are the R values measured in the uptake buffer with 10 mM EGTA added and with 1 mM Ca2+added, respectively, l3 is the ratio of fluorescence measured at 380-nm excitation for the EGTA- and Ca2+-supplemented buffers, and [Ca2+lfreeis the free Ca2+concentration. DHP receptor binding. Preliminary experiments were performed to optimize the amount of protein used for binding. DHP binding was measured in filtered homogenates(600 pg) and crude membranes (30 pg) in a buffer containing 5 mM Tris (pH 7.4) and 0.5-50 nM 13H]PN-200-110(New England Nuclear, Boston, MA) to measure bound and free ligand (20); 10 pM unlabeled nisoldipine (BAY K 5552; Dr. F. Seuter, Bayer AG, Leverkusen, Germany) was used to measure nonspecific binding in one of the duplicate set for each radioactive ligand concentration. After a l-h incubation in the dark, total and nonspecific binding were measured by vacuum filtration through Whatman GF/C filters (soaked in 0.33% polyethyleneimine) with a lo-ml ice-cold wash (50 mM Tris and 200 mM choline chloride). The filters were mixed with 3 ml of scintillation cocktail (Beckman, ReadySafe), shaken overnight, and counted. The experiments were repeated twice in duplicate for each of four musclesfrom each group. pH]ryanodine binding. Detailed methods for measuring high-affinity 13Hlryanodinebinding have been described elsewhere (22). Briefly, SR membranes (125 ug/ml) were incubated at 37°C for 3 h in a medium containing 250 mM KCl, 50 pM CaC12,15 mM NaCl, 10 nM [3H]ryanodine, and 20 mM HEPES (pH 7.1). The binding reaction was quenched by rapid filtration through a Whatman GF/B glassfiber filter that was then rinsed with 5 ml of ice-cold buffer. Filters were mixed with 3 ml of scintillation cocktail (Beckman, ReadySafe), shaken overnight, and counted. The experiments were repeated at least twice in duplicate. Nonspecific binding was measured in the presence of a loo-fold excess of unlabeled ryanodine and subtracted before calculation. RESULTS
Effect of increased muscle loading on Ca2+-release proteins. After 5 wk of increased loading, plantaris muscles doubled in size as previously reported (15). Western blot analysis of the oll-DHP receptor and ryanodine receptor showed no changes in the level of expression in SR vesicles from control and hypertrophied plantaris muscles (Fig. 2). The use of ryanodine, a plant-derived alkaloid, has greatly increased our understanding of SR Ca2+ channel function. Ryanodine binds to open SR Ca2+-release channels with nanomolar affinity (22). Ryanodine binding in the presence of Ca2+ provides a quantitative measure of the number of SR channels that open in
A kDa
MECHANISM
Con.
HYP.
-7
219 - a, DHPR
124 111
B
Con.
HYP.
- Ryanodine Receptor
219 Fig. 2. A: Western blot, using monoclonal antibody against ai-subunit dihydropyridine receptor (oi-DHPR; 170 kDa); 20 pg SR protein from control (Con) or hypertrophied (Hyp) plantaris muscles were loaded per lane on a 7% polyacrylamide gel. B: Western blot, using polyclonal antibody against ryanodine receptor (4 polypeptides -565 kDa each); 10 pg of protein were loaded per lane on a 4.5% polyacrylamide gel. Faster-migrating band may be a proteolytic fragment of ryanodine receptor homotetramer or second isoform, as discussed in RESULTS. There is no difference in expression of oi-DHP or ryanodine receptor (both bands of doublet scanned separately and compared1 in hypertrophied muscles. GIBCO BRL prestained protein standards were used as molecular mass markers.
response to activating Ca2’ (pM). In vesicles from control and hypertrophied muscles, the amount of ryanodine bound to the SR Ca2+ channel when stimulated by 50 pM Ca2+ was the same (control = 1.00 ? 0.11 and hypertrophy = 1.02 + 0.10 pmol bound ryanodine/mg SR protein), suggesting no change in the number of Gas+-activated receptors, a result that is in agreement with no difference in receptor expression shown by immunoblotting. In contrast, the rate of AgNOs-induced Ca2+ release in SR vesicles from hypertrophied muscles was decreased (Fig. 3, top right). As expected, increasing the concentrations of AgNOs increased the Ca2+-release rates in vesicles from both control and hypertrophied muscles, but the rate of release was 38-58% lower in SR from hypertrophied muscle. However, the amount of
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iz 2
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control hypertrophied T
i *
:AAliJ K
&
.05pM
SpM
~
Fig. 3. Top left: rate of SR vesicle Ca2+ uptake from control and hypertrophied plantaris muscles, using fura 2 as extravesicular free-Ca2+ indicator. Bottom left: SR Ca2+ uptake amount not different between groups. Top right: rate of AgNOa-induced SR Ca2+ release at various AgNOe concentrations (lAgNO&. Bottom right: SR Ca2+ release amount not different between groups. *Significantly different from control (P < 0.05).
5PM
[AgNOsl 40
s
E
-2 3 s
3o 20
1 d zi
IO
s 0
.05pM
.5pM [AgNOs
Ca2+ released in control and hypertrophied SR was not different (Fig. 3, bottom right). A reduction in SR Ca2+-release rate with no change in the total number of Ca2+ channels (Fig. 2 and ryanodine-binding data) suggests altered SR Ca2+ channel function in hypertrophied muscle.
Figure 3 (top left) shows that the rate of SR Ca2+ uptake decreased by 21% in hypertrophied plantaris muscles, whereas the uptake amount was not different (Fig. 3, bottom left>. This suggests that, although the rate of Ca2+ uptake was slower in hypertrophied muscle, 16
A
l
MAb: IIID5
16
219 kDa
c
.
Unloaded
0 Control
~1, DHPR oo0
10
20
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Control
Soleus
Unloaded
Soleus
)5
0
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25
30
35
4c
Bound (pmol/mg) Control
Unloaded
Fig. 4. A: Western blot using monoclonal antibody (MAb) IIID5 against oi-DHPR (170 kDa). Samples were SR proteins from control and 14-day non-weight-bearing (“unloaded”) soleus muscles; 10 pg protein were loaded per lane on a 7% polyacrylamide gel. B: results of densitometry. GIBCO BRL prestained protein standards were used as molecular mass markers. *Significantly different from control (P < 0.05).
10
Fig. 5. DHP binding of crude membranes from 7-day non-weightbearing (“unloaded”) soleus muscles at several ligand [3H]PN-200110 concentrations (inset). Excess “cold” nisoldipine was used to determine nonspecific binding (515%). Scatchard analysis showed -36% increase in maximum receptor binding capacity and no change in affinity or dissociation constant. DHP binding in whole muscle homogenates from 14-day unloaded soleus muscles showed a similar result with ~30% nonspecific binding.
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the total amount of Ca2+ taken up was not different, and thus the driving force for release could not have contributed to the slower SR Ca2+-release rates.
--II
MECHANISM
Unloaded
..
Control
EDL .. . *
Effect of decreased muscle loading on Ca2+-release proteins. Western blot analysis of the oi-DHP receptor
showed a 144% increase in relative expression after 14 days of non-weight bearing (Fig. 4). Consistent with this observation, DHP receptor binding, using the ligand [3H]PN-200-110, showed a 36% increase in maximum binding capacity (B,,,) and no change in affinity (Kd) after 7 days of non-weight bearing in the soleus crude membrane preparation (Fig. 5). Similar results were obtained with DHP binding in whole muscle homogenates of soleus muscles after 14 days of unloading but with -30% nonspecific binding (not shown). Western blot analysis of the ryanodine receptor showed 14 days of non-weight bearing increased ryanodine receptor (SR Ca2+-release channel) expression by 150% (Fig. 6). The increased expression of DHP and ryanodine receptor levels was -80% of that normally seen in control fast-twitch muscle. Doublet on blots of ryanodine receptor. The appearance of the doublet on the ryanodine receptor Western
A
Ab: GP3 C
Ryanodine Receptor
kDa 2191 .
I
Control
1
Soleus
Unloaded
Control
Unloaded
Soleus
Fig. 6. A: Western blot of SR vesicles from control and 14-day unloaded soleus muscles using polyclonal antibody (Ab) GP3 against ryanodine receptor (-565 kDa); 10 pg of protein were loaded per lane on a 4.5% polyacrylamide gel. Both bands of doublet were scanned separately and showed same increase in unloaded vesicles. B: results of densitometry. GIBCO BRL prestained protein standards used as molecular mass markers. Samples are same as those in Fig. 4. *Significantly different from control (P < 0.05).
Ryanodine Receptor
215-
+- ATPase
10570.
*
Calsequestrin?
43281815Fig. 7. Coomassie-stained 3-15% polyacrylamide gel of SR vesicles from control and 14-day unloaded soleus muscles; 5 pg of protein were loaded per lane. EDL, extensor digitorum longus, a fast-twitch muscle used for comparison. A band between 70 and 40 kDa that appears in much greater abundance in unloaded muscles may be a 64-kDa skeletal calsequestrin isoform (6) because a band of similar mobility turned blue with Stains-All staining (data not shown). GIBCO BRL prestained protein standards used as molecular mass markers.
blots (Figs. 2 and 6) as well as that seen on a Coomassiestained gel (Fig. 7) appears in publications by other investigators (5,12, 16,21). The faster-migrating band could be either a proteolytic fragment of the major ryanodine receptor expressed in mammalian skeletal muscle (RyRi) or the expression of a second isoform (RyR,) that was previously found in brain but recently found in low levels in murine skeletal muscle (9). If the faster-migrating immunoreactive band in the present study is RyR,, then it is in much greater abundance relative to RyRi than shown in mouse skeletal muscle (9). On Western blots, the bands were scanned separately, and the differences between the groups were identical when comparing the faster- or the slowermigrating band. Thus, regardless of the identity of the second band, the extent of change in expression was the same as with the 565-kDa band (RyRi 1. Purity and yield of SR vesicles. There was no difference in the purity of SR vesicles from control and hypertrophied plantaris muscles as we have shown previously (15). The yield averaged 0.6 pg SR proteimmg wet muscle mass for plantaris muscles. The purity of SR protein from control and unloaded soleus muscles appeared similar, as shown in the 3-15% polyacrylamide gradient gel (Fig. 7). The yield averaged 0.2 pg SR proteimmg wet muscle mass for soleus muscles. These data suggest that differences in expression are not due to differential recovery of vesicles among groups. Thus the isolated SR is representative of the total muscle SR population. Furthermore, DHP binding in control vs. unloaded soleus crude membrane or whole muscle homogenates had the same outcomes.
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The large relative difference in expression of the Ca2+ATPase in Fig. 7 is dealt with in detail elsewhere (15). DISCUSSION
The major finding of the present study is that chronic changes in weight-bearing condition can modulate the SR Ca2+-release mechanism. Non-weight bearing for 14 days leads to atrophy of the soleus muscle, significantly increased (-150%) expression of both the DHP and ryanodine receptors, and increased B,,, of DHP binding. The fact that both proteins increased expression to the same extent suggests that their stoichiometry was unaltered. These changes in protein expression are likely pretranslationally regulated, at least in the case of the (x1-DHP receptor, because we previously showed that 14 days of non-weight bearing induced a 200% increase in (x1-DHP receptor mRNA expression in rat soleus muscles (14). Unfortunately, the very slow rate of Ca2+ uptake of soleus vesicles and the limiting amounts of starting material from small rat soleus muscles precluded measurement of SR Ca2+ release. Five weeks of increased weight bearing (via surgical ablation of synergistic muscles) lead to muscle hypertrophy in the plantaris and no difference in the relative levels of expressed cxl-DHP or ryanodine receptor. Furthermore, the number of open Ca2+ channels, activated by micromolar concentrations of Ca2+ and measured by [“Hlryanodine binding, was not different between groups. With similar numbers of Ca2+-release channels, however, the rate of AgNOs-induced Ca2+ release was 3858% slower in vesicles from hypertrophied muscles. Ag + has been shown to induce Ca2+ release through the Ca2+ -release channel and not via nonspecific diffusion (28). These data therefore suggest that the Ca2+-release function appears to be modulated posttranslationally in hypertrophied muscle. Possible explanations for the decreased Ca2+-release rate are 1) a decrease in the density of channels, 2) a decrease in the driving force for Ca2+ release, 3) an alteration in Ca2+-release channel function/regulation, which could alter gating characteristics such as open probability, channel conductance, or open-time constants. Data presented herein obviate the first two possibilities. There was no difference in the relative density of ryanodine receptors ( Ca2 + -activated ryanodine binding and immunoblotting). There was also no difference in the total amount of Ca 2+ taken up by the vesicles during active loading, and thus the driving force for Ca2+ release was similar between groups. The third explanation is more plausible given the existing data. Our results suggest that there is a decreased responsiveness of the Ca2+ -release mechanism to Ag+ stimulation. Reduced sulfhydryl reactivity of Ca2+ channels resulting in a lower number of channels in the open state was suggested by Favero et al. (8) as a possible mechanism of reduced Ag+-induced Ca2+ release in SR vesicles from muscles fatigued by an acute exercise bout. It is possible that the Ca2+ -release channel is regulated by the redox state of sulfhydryl groups (l), and sulfhydryl modification by an allosteric interaction may regulate Ca2+-release channel function as postulated in muscle
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MECHANISM
after acute exercise (8). Alterations in muscle redox equilibria have been suggested to contribute to a decline in muscle function (7, 24). In vivo, alterations in the phosphorylation state (for recent reference, see Ref. 11) or regulation by calmodulin binding (see Ref. 27 for review) of the ryanodine receptor may differ in the hypertrophied state. Phenotypic changes in the SR Ca2+-release mechanism would involve alterations in the number of DHP or ryanodine receptors rather than isoform transitions, since the same isoform is expressed in both slow- and fast-twitch muscles, with fast-twitch muscles having significantly greater expression levels (17, 18, 21). Ca2+-release channel components in differentiated muscle such as the (xl-DHP receptor and the ryanodine receptor are significantly reduced in chronic stimulated canine latissimus dorsi muscle (21). The lack of similar changes in hypertrophied muscle suggests that the total amount of muscle activation may not have been great enough to alter expression levels of DHP and ryanodine receptors as seen with chronic electrical stimulation. However, posttranslational modification of SR Ca2+-release channel function may represent an intermediate step that ultimately leads to alterations in the level of protein expression. Collectively, these data in addition to the increased channel expression in non-weight-bearing muscle in the present study suggest that components of the SR Ca2+-release mechanism can be controlled by and are responsive to chronic alterations in contractile activity. Alterations in the level of expression of Ca2+ release proteins appear to regulate greater muscle activity than that required to modify Ca 2+-ATPase expression. We thank Dr. K. Campbell (University of Iowa) for providing the al-subunit DHP receptor and ryanodine receptor antibodies and Dr. F. Seuter from Bayer AG for the gift of the nisoldipine. The skillful assistance of Shih-Yao Chen is appreciated. The crude membrane DHP-binding experiments were done in the laboratory of Dr. Javier Navarro at Boston University. This work was supported by the National Institute ofArthritis and Muscoloskeletal and Skin Diseases Grants AR-41705 and AR-41727. S. C. Kandarian is an Established Investigator of the American Heart Association. Address for reprint requests: S. Kandarian, Boston Univ., 635 Commonwealth Ave., Boston, MA 02215. Received
10 August
1995;
accepted
in final
form
1 December
1995.
REFERENCES 1. Abramson, J. J., and G. Salama. Critical sulfhydryls regulate calcium release from sarcoplasmic reticulum. J. Bioenerg. Biomembr. 21: 283-294,1989. 2. Booth, F. W., and B. S. Tseng. Olympic goal: molecular and cellular approaches to understanding muscle adaptation. News PhysioZ. Sci. 8: 165-169, 1993. 3. Briggs, F. N., K. F. Lee, J. J. Feher, A. S. Wechsler, K. Ohlendieck, and K. Campbell. Ca-ATPase isozyme expression in sarcoplasmic reticulum is altered by chronic stimulation of skeletal muscle. FEBS Lett. 259: 269-272, 1990. 4. Briggs, F. N., K. F. Lee, A. W. Wechsler, and L. R. Jones. Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca 2+-ATPase. J. Biol. Chem. 267: 2605626061,1992. 5. Chen, S. R. W, D. M. Vaughan, J.A.Airey, R. Coronado, and D. H. MacLennan. Functional expression of cDNA encoding the
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7.
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11.
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15.
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18.
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