Department of Physiology, The University of Melbourne, Victoria, Australia
Submitted 26 August 2004 ; accepted in final form 6 October 2004
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ABSTRACT |
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aging; calcium; -adrenoceptor; contractility
Neurogenic factors, such as motor unit remodeling and denervation, have been implicated in the age-related changes to skeletal muscle (11). However, myogenic factors are also involved, since the slowing of contraction and relaxation occurs before the onset of muscle wasting (18, 20, 21). Intrinsic changes to skeletal muscle fibers appear to be involved in the deterioration of function with age. Specifically, age-related changes in the proteins regulating Ca2+ handling during excitation-contraction coupling and relaxation have been described (15).
Relaxation of skeletal muscle is thought to involve at least three processes: dissociation of Ca2+ from troponin, detachment of cross bridges, and uptake of Ca2+ by the sarcoplasmic reticulum and myoplasmic Ca2+ buffers (33). The factors regulating intracellular [Ca2+] during skeletal muscle relaxation are fiber type specific. In fast-twitch muscle, it has been shown that parvalbumin contributes at least 50% to the final rate constant of decay of [Ca2+] after a single action potential (2). In slow-twitch muscle (where parvalbumin levels are very low), the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) makes the major contribution to the decay of [Ca2+] (2). Aging has been associated with a decrease in the amount of Ca2+ available for triggering contraction (20) due to dihydropyridine receptor (DHPR) uncoupling from the sarcoplasmic reticulum (SR) Ca2+ release channel (CRC) (5). However, the effects of aging on the kinetic properties of the SERCA are not clear. The reductions in SR Ca2+ resequestration from chemically skinned fast-twitch muscle fibers from aged rats were hypothesized to be caused by an inactivation of SERCA (13). However, predominantly fast-twitch muscles show no age-related alteration in SERCA protein levels, isoform composition, or kinetic properties (4, 7).
To date, the accumulated data have only excluded an effect of aging on the intrinsic properties of SERCA from predominantly fast-twitch muscle. However, SR vesicles isolated from rat slow-twitch muscles (soleus, adductor longus, and vastus intermedius) do show an age-dependent reduction in both SERCA activity and Ca2+ resequestration (32). Furthermore, the age-related inactivation of SERCA activity was associated with a selective nitration of the SERCA2a isoform predominantly at Tyr294-Tyr295 (32), which is close to the Ca2+ translocation sites, suggesting that an age-related decrease in SERCA activity could be related to a decreased binding affinity for Ca2+. It has also been shown that the conformation of the nucleotide-binding domain of SERCA is altered in aged skeletal muscle (3). Therefore, current evidence suggests that an age-related reduction in SERCA activity in slow-twitch muscle may be associated with changes in the environment of nucleotide and/or Ca2+-binding domains.
We have demonstrated previously that 4 wk of daily administration of the 2-adrenoceptor agonist (
2-agonist) fenoterol can attenuate the age-related decrease in rat skeletal muscle mass and strength and decrease the time course of the isometric twitch (22). However, the mechanisms responsible for this change in contractility have not been elucidated. It has been demonstrated that a 6-wk administration of the
2-agonist salbutamol can reduce contraction time in canine skeletal muscle (36). Salbutamol treatment also increased the rate of SR Ca2+ resequestration, which suggested that changes in skeletal muscle contractile parameters were mediated by alterations in SR proteins, namely a
2-agonist-mediated induction of SERCA1 protein levels (36). In addition, salbutamol treatment attenuated the fast twitch-to-slow twitch transformation of myosin heavy-chain (MHC) isoforms, SR protein characteristics, and muscle mechanics that accompany chronic low-frequency stimulation (CLFS) (10). Therefore, it is possible that
2-agonist administration could attenuate an age-related decrease in skeletal muscle relaxation rate by reversing age-related modifications of SR proteins.
We tested the hypothesis that the age-related decrease and the 2-agonist-induced increase in the rate of skeletal muscle relaxation are mediated through alterations in the properties of the SR. We also hypothesized that fenoterol treatment in rats would attenuate age-related changes in the maximal activity (Vmax) of SERCA and determined the underlying mechanisms that could account for changes in SERCA activity, including assessments of SERCA isoform protein levels, SERCA2a nitration levels, and indirect measures of the Ca2+- and ATP-binding affinities of SERCA.
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MATERIALS AND METHODS |
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Contractile properties: maximal rate of twitch and tetanus relaxation.
At the completion of the treatment period, rats were anesthetized with pentobarbital sodium (Nembutal; Rhone Merieux, Pinkenba, QLD, Australia; 60 mg/kg ip). The extensor digitorum longus (EDL) and soleus muscles were surgically excised, and isometric contractile properties were measured in vitro as reported previously (22). In that study, we reported that although absolute one-half relaxation time of the twitch was not different after fenoterol treatment, twitch force was significantly greater. Hence, compared with untreated control rats, the actual rate of relaxation of the twitch response was significantly faster in treated rats (22). Although twitch force of either EDL or soleus muscles was not different between adult and aged rats (22), the ability of fenoterol treatment to increase twitch force warranted closer investigation. Therefore, to further examine the maximal rate of relaxation of the twitch and tetanic responses, we studied the maximum negative derivative of a linear-fit curve calculated over 5 ms for the EDL and 25 ms for soleus muscle by use of custom-written software applications (DR Stom Software Solutions, Ann Arbor, MI) and LabView software (National Instruments, Austin, TX). The maximal rate of relaxation was determined during supramaximal twitches and fused tetani (150 Hz EDL, 120 Hz soleus). Although the EDL and soleus muscles from both hindlimbs were used previously for examination of the effects of fenoterol on contractile properties and -adrenoceptor densities (22), the gastrocnemius muscles were also excised and stored for later biochemical analysis, and these muscles served as the basis for the present study investigating fenoterol's effects on SERCA kinetic properties.
Sample preparation: SR characteristics. While rats were anesthetized, the gastrocnemius muscles were excised from both hindlimbs. The deep red portion of the gastrocnemius muscle (RG) was separated from the predominantly fast-twitch superficial white portion (WG). The rats were killed by the opening of the thoracic cavity and immediate cardiac excision. Crude muscle homogenates were prepared, as described previously (24, 25). SR vesicles were prepared, utilizing a combination of two SR isolation protocols (6, 9), as described previously (24). During the entire homogenization and SR vesicle isolation procedure, samples were immersed in ice. It is critical to keep the samples on ice to avoid temperature-dependent reductions in SERCA activity (27). SR isolation was carried out on the same day as muscle homogenization and was accomplished by sucrose gradient differential centrifugation, using a Beckman ultracentrifuge with a 70.1 Ti fixed-angle rotor. Protein determination of homogenates and SR vesicles was made by the method of Bradford and analyzed in triplicate.
SERCA activity.
Ca2+-induced SERCA activity in enriched SR membranes was analyzed according to the methods of Leberer et al. (14), described previously (24). Ca2+-induced SERCA activity in crude muscle homogenates was analyzed with the use of modifications according to Simonides and van Hardeveld (29), as described previously (25). The reaction buffer for crude muscle homogenates (with SR vesicles in parentheses) contained, in mM, 200 (100) KCl, 20 HEPES, 15 (10) MgCl2, 10 NaN3, 10 phosphoenolpyruvate (PEP), 5 ATP, and 1 EGTA. The pH of the reaction buffer was adjusted to 7.0 at 37°C. Immediately before the reaction was started, 18 U/ml lactate dehydrogenase (LDH), 18 U/ml pyruvate kinase (PK), 0.3 mM NADH, 1 µM Ca2+ ionophore A-23187 (Sigma-Aldrich), and 1530 µl of muscle homogenate or 15 µl of enriched SR membrane were added to 1 ml of reaction buffer. The markers of SERCA enzyme kinetics in muscle homogenates included Vmax, cytosolic free [Ca2+] ([Ca2+]f) required for half-maximal activity (pCa50), and the Hill coefficient (nH), which is a measure of the cooperative Ca2+-binding affinity of the SERCA enzyme. In SR vesicles, only the Vmax was assessed. To assess changes in SR membrane permeability to Ca2+, the assay was also performed without the Ca2+ ionophore A-23187 in a subset of samples for each condition (muscle homogenates only; n = 3). Assays were performed at 37°C and 340 nm (Multiscan Spectrum; Thermo Electron, Waltham, MA). In all cases, the Vmax of SERCA activity was established by progressively raising the [Ca2+]f until a plateau and subsequent decline in SERCA activity occurred. Basal (or Mg2+-ATPase) activity was determined by adding cyclopiazonic acid (CPA), a specific inhibitor of the SERCA enzyme, to a final concentration of 40 µM. The [Ca2+]f for a given day of analysis was measured in triplicate on separate aliquots of reaction buffer with a calcium electrode (model 97-20; Orion, Beverly, MA). SERCA activity was then plotted against the negative logarithm of [Ca2+]f (pCa), as described previously (31). Nonlinear regression analysis was performed with GraphPad Prism v. 4.00 for Windows (GraphPad Software, San Diego, CA) using the following sigmoidal dose-response (variable slope) relationship
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Fluorescence measurements.
The fluorescent probes fluorescein 5-isothiocyanate (FITC; Sigma-Aldrich) and N-cyclohexyl-N'-(dimethylamino--naphthyl)carbodiimide (NCD-4; Molecular Probes, Eugene, OR) were used to indirectly assess the nucleotide- and Ca2+-binding affinities of SERCA, respectively, as previously described (24, 31). An identical amount of enriched SR vesicle protein (500 µg) was labeled with FITC (25 µM, pH 8.8) in the dark for 30 min at 25°C. Fluorescence measurements using the FITC probe were made on a spectrofluorometer (Wallac 1420; Perkin Elmer, Wellesley, MA), based on previous methods (11). Samples were excited at 495 nm, and the maximum emission at 525 nm was recorded. Similarly, samples were labeled with NCD-4 (150 µM, pH 6.2) in the dark for 4 h at 25°C. NCD-4 fluorescence measures were made by exciting samples at 340 nm and recording the maximum emission intensity at 420 nm. The maximum fluorescence intensity for both FITC and NCD-4 in each sample was recorded in triplicate.
SDS-PAGE for MHC determination and Western blotting.
SDS-PAGE was performed to separate and isolate SERCA isoforms according to molecular mass. Immunoblotting was performed using primary monoclonal antibodies specific for rat SERCA1 (110 kDa, IIH11; Affinity Bioreagents, Golden, CO) and SERCA2a (95 kDa, 7E6; Affinity Bioreagents) that were diluted 1:2,500 and 1:1,000 in 10% nonfat milk, respectively. In addition, immunoblotting was performed with a polyclonal antibody specific for nitrotyrosine (A-21285; Molecular Probes) that was diluted 1:1,000 in 10% nonfat milk. Immunoblotting was done in enriched SR vesicles, as described previously (24), with the following minor modifications. After application of the appropriate secondary antibody, each was conjugated with biotinylated streptavidin-alkaline phosphatase. Blots were developed with an alkaline phosphatase conjugate substrate kit with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (BCIP/NBT; 170-6412-1, Bio-Rad, Hercules, CA). Blots were digitized, and relative protein levels were determined by scanning densitometry. Relative protein levels for SERCA1 were quantified using the single band detected at 110 kDa. Immunoblotting revealed several lower-molecular-mass SERCA2a products in addition to the predicted band at 95 kDa. Therefore, for both SERCA2a and nitrotyrosine, bands migrating at an apparent molecular mass of 95 and 70 kDa were summed for quantification. Values (n
5 for each condition) were expressed as a percentage of the muscles from untreated adult rats.
MHC isoform composition was determined in whole muscle homogenates by SDS-PAGE, as reported previously (30), with minor modifications. 2-Mercaptoethanol (0.08%) was added to the running buffer to improve MHC isoform resolution, and proteins were visualized with a commercially available silver stain kit (LC6100; Invitrogen, Carlsbad, CA). Gels were digitized, and relative protein levels were determined by scanning densitometry. Values (n 5 for each condition) were expressed as a percentage of total MHC protein.
Statistical analyses. Individual variables were compared between groups with a one-way or two-way ANOVA as appropriate. Bonferroni's post hoc multiple comparison procedure was used to detect differences between specific means. Significance was set at P < 0.05. All values are expressed as means ± SE unless specified otherwise.
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RESULTS |
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Fluorescence measurements. The maximum fluorescence intensity of SR vesicles labeled with FITC was 12, 13, and 19% lower in samples from RG of untreated aged rats compared with those from treated aged rats, untreated adult rats, and treated adult rats, respectively (P < 0.05; Fig. 3, A and C). FITC fluorescence was not altered in SR vesicles isolated from WG muscle (Fig. 3A). The maximum emission intensity was unchanged in all conditions for NCD-4 (Fig. 3B).
SDS-PAGE and Western blotting. SERCA1 protein levels in SR vesicles isolated from RG and WG muscle were higher in fenoterol-treated rats. Compared with values for untreated adult rats, RG SERCA1 protein levels were 46 and 42% higher in treated adult and aged rats, respectively (P < 0.05; Fig. 4A). No age-dependent changes in SERCA1 protein levels were apparent in RG or WG. SERCA2a protein levels were not different in WG or RG (Fig. 4B). The SERCA1-to-SERCA2a ratio in RG and WG muscles was greater in fenoterol-treated rats than in untreated rats (P < 0.05, data not shown).
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Determination of the relative MHC composition confirmed that RG muscle contained a different MHC isoform profile compared with WG muscle. Specifically, RG muscle contained 828% of MHC type IIb and 2030% of MHC type I. In contrast, WG muscle contained 4560% MHC type IIb. No MHC type I was detected in WG muscle. In general, aging was associated with a shift toward a higher proportion of slow MHC isoforms (IIB IIa/x
I), whereas fenoterol treatment was associated with a shift toward fast MHC isoforms (I
IIa/x
IIb; Fig. 5).
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DISCUSSION |
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On the basis of the literature to date, the effects of aging on SERCA activity are controversial. It has been argued that aging has no effect on the intrinsic properties of SERCA (15). However, the studies that failed to demonstrate age-related changes in SERCA activity were performed on muscles comprised of fibers with predominantly fast-twitch characteristics (4, 7), with one exception. Narayanan et al. (18) showed that SERCA activity in rat soleus muscle was unchanged despite an age-related depression in Ca2+ resequestration. However, because they assessed SERCA activity at only a single [Ca2+]f (8.21 µM), it cannot be said definitively that the activity measured was the Vmax. Differences in SERCA activity could be masked by possible age-related changes in pCa50 and/or the Hill coefficient.
Our findings are consistent with more recent studies investigating the dependence of muscle fiber type on age-related changes in the kinetic properties of SERCA. Viner et al. (32) showed an age-dependent loss of SERCA activity in SR preparations isolated from muscles with mixed fiber composition but not from preparations containing predominantly fast-twitch muscle fibers.
It has been shown previously that treatment with the 2-agonist salbutamol for 6 wk increased SERCA1 protein levels in canine latissimus dorsi and vastus intermedius muscles (36). Similarly, our findings show that fenoterol treatment increased SERCA1 protein levels in the RG and WG muscle of both adult and aged rats. Furthermore, we (22) have previously shown that fenoterol treatment can reverse age-related muscle wasting and weakness, a finding that is similar in some respects to another study (10), where salbutamol treatment attenuated the muscle atrophy usually associated with CLFS. The results of the present study show that fenoterol treatment reverses the age-related decrease in both the rate of relaxation and the Vmax of SERCA activity, a finding similar to salbutamol treatment attenuating the prolongation of contraction and the depression in SR Ca2+ resequestration concomitant with CLFS (10).
SERCA has a relatively long half-life (14 days) that is extended by
27% in aged muscle (8). Therefore, SERCA has a high potential for posttranslational modification, particularly in aged muscle. The ATP-binding domain of SERCA appears to be particularly susceptible to posttranslational modification, which may result in altered enzymatic activity. For example, hydroxyl radicals have been shown to inhibit SERCA function in cardiac and skeletal muscle by altering the ATP-binding site (35). Aged skeletal muscle shows an altered ATP-binding site conformation (3). CLFS has been shown to decrease FITC binding, which corresponded to an increase in the number of inactive SERCA enzymes (16). It should be noted that FITC labels Lys515 in the nucleotide-binding domain of SERCA and, as such, is sensitive to changes in the environment around that residue. Our results demonstrate an age-related reduction in FITC-binding affinity, indicating that a similar mechanism may mediate this effect during both aging and CLFS. We have also shown that fenoterol treatment can attenuate the age-related decreases in FITC-binding affinity. This measure could reflect SERCA E2-to-SERCA E1 transitions and/or alterations in the environment of the nucleotide-binding domain (19).
Our findings are consistent with previous reports demonstrating a selective age-related nitration of the SERCA2a isoform (32). It appears that nitration of SERCA2a is, in part, due to a peroxynitrite-mediated process (28). However, the mechanisms linking nitration of SERCA2a to reductions in enzyme activity have not been elucidated. To our knowledge, this is the first report to demonstrate that treatment with a 2-agonist reverses the age-related accumulation of nitrotyrosine residues on the SERCA2a isoform. The mechanism underlying this effect is not known; however, we have shown that fenoterol increases SERCA1 protein levels, and it is possible that the synthesis of SERCA1 protein and/or the consequent increase in the ratio of SERCA1 to SERCA2a could reduce the amount of SERCA2a available for nitration.
Age-related alterations in the CRC, such as DHPR-CRC uncoupling (4), may be associated with skeletal muscle twitch prolongation (4). We have demonstrated that the underlying mechanisms of age-related alterations in the rate of skeletal muscle relaxation are clearly fiber-type specific. Our results demonstrate no age-related changes in SERCA activity in fast-twitch muscle; hence, alterations in excitation-contraction coupling remain a likely explanation for age-related changes in contractile properties in fast muscle (20). However, our results demonstrate that, in slow-twitch muscle, uncoupling of the DHPR-CRC complex is not the only age-related alteration to the proteins involved in excitation contraction and relaxation. Although SERCA activity is not the only factor controlling the rate of skeletal muscle relaxation, it is reasonable to associate decreased SERCA activity with the reduced rate of relaxation in muscles of aged rats. In addition, given the coordination between SR Ca2+ resequestration and Ca2+ release (23), it is possible that alterations in SERCA activity in slow-twitch muscle may manifest as changes in SR release kinetics and ultimately as changes in contractile parameters, such as force production (20). We have provided evidence that an age-related reduction in SERCA activity may be mediated through SERCA2a nitration and/or alterations in the environment of the nucleotide-binding domain of SERCA. Several other mechanisms may also be important. Phosphorylation of SR-bound calmodulin kinase II (CaMKII) increases the Vmax of SERCA2a (34). The protein levels of - and
-CaMKII subunits are conserved through aging of rat muscle (15). However, the enzymatic activity of CaMKII has not been assessed during aging, and this assessment could prove useful. It is unlikely that the actions of fenoterol are mediated through CaMKII phosphorylation, since this pathway is only activated by
1-adrenoceptor stimulation in rat cardiac muscle (1).
In summary, we demonstrated that aging was associated with a decrease in the rate relaxation of both slow- and fast-twitch muscles. Aging was also associated with depression in the Vmax of rat SERCA in muscle with a mixed fiber type but not in predominately fast-twitch muscle. The mechanism underlying the depression in the Vmax of SERCA may be alterations in the environment of the nucleotide-binding domain and/or an age-related selective nitration of SERCA2a. We also demonstrated that treatment with the 2-agonist fenoterol reversed the depression in the Vmax of SERCA. We provided evidence that the changes in SERCA activity are related to alterations in SERCA1 protein levels, alterations in the environment of the nucleotide-binding domain, and the accumulation of nitrotyrosine residues on SERCA2a. Taken together, these findings show that the underlying mechanisms responsible for age-related changes in skeletal muscle contractile properties are fiber type specific, whereas the effects of fenoterol are independent of age and fiber type.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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