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Expression of phospholemman and its association with Na⁺-K⁺-ATPase in skeletal muscle: effects of aging and exercise training

¹Department of Pharmacology, The Milton S. Hershey Medical Center, College of Medicine, The Pennsylvania State University, Hershey, Pennsylvania; ²Department of Integrative Physiology, University of Colorado, Boulder, Colorado; and ³Program in Molecular and Cellular Cardiology, Department of Medicine, Wayne State University, Detroit, Michigan

Submitted 1 April 2005 ; accepted in final form 10 June 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Phospholemman (PLM) is a recently identified accessory protein of the Na⁺-K⁺-ATPase (NKA), with a high level of expression in skeletal muscle. The objectives of this study are to characterize the PLM in skeletal muscle and to test the hypothesis that, as an accessory protein of NKA, expression of PLM and its association with the -subunits of NKA is regulated during aging and with exercise training. PLM was characterized in skeletal muscle of 6- and 16-mo-old sedentary middle-aged rats (Ms), and the effects of aging and exercise training were studied in Ms, 29-mo-old sedentary senescent, and 29-mo-old treadmill-exercised senescent rats. Expression of PLM was muscle-type dependent, and immunofluorescence study showed that PLM distributed predominantly on the sarcolemmal membrane of the muscle fibers. Anti-PLM antibody reduced activity of NKA, and thus PLM appears to be required for NKA to express its full activity in skeletal muscle. Expression of PLM was not altered with aging but increased after exercise training. Coimmunoprecipitation studies demonstrated that PLM associates with both the ₁- and ₂-subunit isoforms of NKA. Compared with Ms rats, levels of PLM-associated ₁-subunit increased in 29-mo-old sedentary senescent rats, and treadmill exercise has a tendency to partially reverse it. There was no significant change in PLM-associated ₂-subunit with age, and exercise training has a tendency to increase that level. It is concluded that, in skeletal muscle, PLM appears to be a protein integral to the NKA complex and that PLM has the potential to modulate NKA in an isoform-specific and muscle type-dependent manner in aging and after exercise training.

isoform; -subunit; coimmunoprecipitation; FXYD proteins; Fischer 344x Brown Norway rats

Four members of this family (the -subunit, PLM, corticosteroid hormone-induced factor, and FXYD7) have been shown to be associated with Na⁺-K⁺-ATPase (4–6, 12). Of particular interest is PLM, which was originally discovered as a major substrate of protein kinase A and protein kinase C in the plasma membrane of cardiac and skeletal muscle (17, 18, 28, 29, 35). Although several recent publications have begun to characterize PLM, its biochemical and physiological role remains incompletely understood. In neuronal tissues, Feschenko and coworkers (12) demonstrated that PLM is associated with all three -subunit isoforms of the Na⁺-K⁺-ATPase, and the association appears to activate Na⁺-K⁺-ATPase activity without changing the Na⁺ affinity of the enzyme. By contrast, Crambert and coworkers (11) showed that PLM alters both the Na⁺ and K⁺ affinity of Na⁺-K⁺-ATPase when they were coexpressed in Xenopus oocytes. Furthermore, they demonstrated that, in skeletal muscle, PLM associates predominantly with the ₁- and not the ₂-isoform. In myocardium, PLM also appears to be selectively associated with the ₁-isoform, and ischemia increases phosphorylation of PLM and results in increased Na⁺-K⁺-ATPase enzymatic activity (14). In addition, ₁-specific activation by protein kinase A cascade appears to be mediated through phosphorylation of PLM (31). In shark rectal gland, the PLM-like protein has been reported to decrease Na⁺ affinity and enzymatic activity, and phosphorylation of the protein increased Na⁺-K⁺-ATPase activity (21, 22). Together, these data suggest tissue-specific expression and perhaps isoform-specific regulation of Na⁺-K⁺-ATPase by PLM.

In skeletal muscle, Na⁺-K⁺-ATPase plays a critical role in the maintenance of Na⁺-K⁺ homeostasis, which in turn modulates muscle function (7, 9, 23, 26). In previous studies, our laboratory demonstrated that expression of the - and -subunit isoforms is differentially altered during aging and that exercise training was able to reverse some, but not all, of the age-related changes (25, 33). If PLM is indeed an accessory protein of the Na⁺-K⁺-ATPase (12), it may ultimately play a substantial role in modulating skeletal muscle physiology. Therefore, the first objective of the present study was to further characterize the PLM in skeletal muscle in young and middle-aged rats. The second objective was to test, in middle-aged and aged rats, the hypothesis that, as an accessory protein of the Na⁺-K⁺-ATPase, expression of PLM and its association with Na⁺-K⁺-ATPase is altered during aging and after exercise training. Parts of this paper were published previously in abstract form (30).

	MATERIALS AND METHODS

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Animals used.   Male Fischer 344x Brown Norway rats (Harlan, Indianapolis, IN) were housed in a 12:12-h light-dark cycle and given standard rat chow and water ad libitum. To specifically identify alterations that are more likely to be the result of aging, without complications from normal maturation, we chose to examine the effects of aging on expression of PLM in 16-mo-old middle-aged rats and 29-mo-old senescent rats in our study. This is because changes that are observed between these age groups are more likely to reflect aging-related alterations rather than changes that may occur as a result of normal development and maturation. However, in experiments where aging is not the main focus, we choose to examine preparations from 6- and 16-mo-old rats to illustrate what typically would be detected in the skeletal muscle of young and middle-aged rats, age groups that investigators are more likely to study. All animal-use protocols were approved by the institutional animal care committees at University of Colorado and The Pennsylvania State University, College of Medicine.

Exercise training of rats.   The protocol for the training of rats was published previously (15, 25). Briefly, the 26-mo-old rats were randomly assigned to either the sedentary or the exercise-trained group. Animals in the trained group underwent 13–14 wk of running on a motorized treadmill. During the first 2 wk, running duration and speed were progressively increased. From the start of week 3 until the death date, the final training protocol consisted of running 5 days/wk up a 10% grade at 14 m/min for 5 min and 17 m/min for 40 min for a total of 45 min. About 24 h (±4 h) after the last bout of exercise training, the rats were anesthetized with intraperitoneal pentobarbital sodium (35 mg/kg) at least 15 min after heparin injection (250 units). Skeletal muscles were dissected after hearts were removed. The red (GR) and white gastrocnemius (GW) muscles were dissected and separated for use in some experiments, whereas in other experiments these muscles were not separated, and the whole gastrocnemius muscle is referred to as mixed gastrocnemius muscle. The three experimental groups were 16-mo-old middle-aged sedentary (Ms); 29-mo-old senescent sedentary (Ss); and 29-mo-old senescent exercise trained (St). Increased citrate synthase activity indicated that the muscles underwent significant adaptation to exercise (25).

Preparation of tissue homogenates.   Total tissue homogenates were prepared as described previously (25). Briefly, skeletal muscles (200–300 mg) were pulverized and homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) at a speed of 6.5 (11.0 full scale) for three 20-s periods at 4°C in a buffer containing 10 mM Tris·HCl (pH 7.5), 1 mM EDTA, and protease inhibitors (in µM: 500 phenylmethylsulfonyl fluoride, 1 leupeptin, 1 pepstatin, and 10 E-64). Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad, Melville, NY).

Preparation of partially purified membrane.   The mixed gastrocnemius muscles from the Ms, Ss, and St rats and from 6-mo-old rats used in the immunofluorescence study were homogenized in ice-cold buffer (200 mg/2 ml) containing (in mM) 250 sucrose, 30 imidazole, and 1 EDTA (pH 7.3) using a Potter-type homogenizer with a motor-driven Teflon pestle. The homogenates were then centrifuged at 3,000 g for 15 min at 4°C, and the supernatants were collected and centrifuged at 140,000 g at 4°C for 1 h. The pellets were then resuspended in 100–200 µl of buffer containing (in mM) 140 NaCl, 25 Imidazole, and 1 EDTA (pH 7.3). Protein concentrations were then determined as described above. These preparations were used in immunoprecipitation experiments.

Enzyme activity assay preparation.   Tissue homogenates were prepared as above and frozen at –80°C and used within a week. The method for Na⁺-K⁺-ATPase enzyme activity assay is similar to that described previously by Feschenko et al. (12) with slight modifications as described previously (25). Briefly, the homogenates were diluted with the homogenizing buffer to 150 µg/40 µl and subjected to rapid "freeze and thaw" 10 times. Total ATPase activity was assayed in a buffer containing (in mM) 100 NaCl, 15 KCl, 5 MgCl₂, 5 NaN₃, 50 Tris·HCl (pH 7.8 at room temperature), and 1 EGTA in 2 mM Tris (pH 7.8, room temperature). After a 10-min preincubation at 37°C, the reaction was started by adding ATP to a final concentration of 5 mM with a trace amount of [-³²P]ATP (ICN, Irvine, CA). Nonspecific ATPase activity was assayed in the presence of 2 mM ouabain. In assays where anti-PLM antibody was added, it was first cleared of contaminants by using a Micron YM-10 centrifugal filter (Millipore, Bedford, MA). Anti-PLM or control IgG (Southern Biotech, Birmingham, AL) (1.5 µg) was incubated with homogenates in assay buffer for 4 h at 4°C before the ATP was added to start the reaction. After a 25-min reaction, 500 µl of the quenching solution (1 NH₂SO₄ and 0.50% ammonium molybdate) were added, and samples were placed on ice. Isobutanol (1 ml) was added, vigorously vortexed, and centrifuged to separate the organic phase from the aqueous phase. The upper organic phase (500 µl) was added to scintillation fluid and counted with a scintillation counter.

Western blot analysis.   PLM and Na⁺-K⁺-ATPase subunits were separated by 4–20% SDS-PAGE and transferred to polyvinylidene difluoride membrane as described previously (25, 33). The antibody to the COOH-terminus of PLM (57–70) was purified on a peptide affinity column and represents antibodies reacting specifically with nonphosphorylated PLM as previously described (19). The antibody to the ₂-isoform (McB2) was kindly provided by K. Sweadner (Harvard University). The antibody to the ₁-isoform (6f) was purchased from Developmental Studies Hybridoma Bank (University of Iowa). Blots probed for PLM were blocked with 5% dry milk in TBS-Tween 20 (Blotto) overnight at 4°C then probed with the anti-PLM antibody (1:1,000) for 2 h. The blots probed for ₁-isoform were blocked in Blotto and probed with anti-₁-antibody (1:250) overnight at 4°C. Bound monoclonal antibodies were detected by horseradish peroxidase-linked goat anti-mouse antibody (Rockland Immunochemicals, Gilbertsville, PA), and bound polyclonal antibodies were detected by horseradish peroxidase-linked goat anti-rabbit IgG (Sigma, St. Louis, MO). The blots probed for ₂-isoform were blocked with TBS-Tween 20 for 1 h, then probed with anti-₂-antibody (1:150) overnight at 4°C. Bound ₂-antibodies were detected by incubating the blots with an unconjugated chicken anti-mouse IgG antibody to amplify the signals (ICN, Costa Mesa, CA) followed by the horseradish peroxidase-linked donkey anti-chicken IgG (ICN, Irvine, CA). Detection was performed with a chemiluminescent method (SuperSignal West Pico or West Femto, Pierce, Rockford, IL), and imaging analysis was performed with a Fujifilm Luminescent Imager (Fuji Photo Film, Japan) using the Image Gauge analysis software. Data are presented in arbitrary chemiluminescent units. All images analyzed were within the linear range of detection.

Immunoprecipitation.   The protocol for immunoprecipitation was basically identical to that described by Feschenko et al. (12). Partially purified membrane preparations (125 µg/50 µl for 6-mo-old rat samples and 100 µg/50 µl for all other samples) were incubated with C₁₂E₈ (6 mg/ml) at room temperature for 20 min with intermittent mixing. The mixtures were diluted 1:1 with buffer B [in mM: 140 NaCl, 25 Imidazole, 1 EDTA (pH 7.3)] and centrifuged at 20,000 g at 4°C for 30 min. The supernatant was precleared of nonspecific binding with 15 µl of Gammabind G-sepharose (Amersham Biosciences, Piscotaway, NJ) for 1 h at room temperature with gentle agitation. The supernatants were then incubated with anti-PLM antibody (0.5 µl/tube) at 4°C overnight. The complexes were immunoprecipitated by incubating with 15 µl of Gammabind G-sepharose for 1 h at room temperature. The samples were centrifuged followed by three washes with buffer B containing 0.05% C₁₂E₈ (buffer C). For the Ms, Ss, and St samples, a final fourth wash in buffer C without NaCl was performed. Samples were incubated with 40 µl of electrophoresis sample buffer at room temperature for 20 min and centrifuged, and the supernatant was saved. Another 30 µl of sample buffer were added to the pellets, and the resuspended mixtures were centrifuged again. Supernatants were combined, then additional -mercaptoethanol (10%) was added to ensure complete dissociation of the IgG chains, and the samples were incubated at 65°C for 10 min and stored frozen at –20°C until used.

Immunofluorescent labeling of skeletal muscle.   Six-month-old rats were anesthetized with intraperitoneal pentobarbital sodium (35 mg/kg) at least 15 min after heparin injection (1,000 IU/kg). Skeletal muscles were dissected and cryoprotected in isopentane, and 8-µm cryostat sections from GR and GW were collected on the same slides to be processed together. The sections were fixed in a 1:1 acetone and methanol solution, incubated in 0.5% NaBH₄ to decrease autofluorescence, and permeabilized in PBS with 0.5% Triton X-100 for 10 min at room temperature. Labeling was performed using monoclonal anti-₂ (McB2; 1:10) and polyclonal anti-PLM (1:50) antibodies. The anti-PLM antibody first was cleared of cross-reacting antibodies by immunoabsorption using a strip that was blotted with the skeletal muscle proteins except PLM. Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 546 goat anti-mouse IgG (1:200) (Molecular Probes, Eugene, OR) were used as secondary antibodies. Microscopy was performed using a Nikon (Melville, NY) fluorescence microscope, and images were processed using Photoshop software (Adobe).

Statistics.   Results are expressed as means ± SD. One-way ANOVA was used to compare group means, and a Duncan test was used in post hoc analysis. One set of data (Fig. 1) was analyzed by repeated-measures ANOVA. Data were examined at P < 0.05 and P < 0.10 to indicate statistical significance and trends, respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Expression of phospholemman (PLM) in red gastrocnemius (GR), white gastrocnemius (GW), and extensor digitorum longus (EDL). Skeletal muscle homogenates (75 µg) from 16-mo-old rats (n = 5) were resolved by SDS-PAGE, and transferred blots were immunoblotted with anti-PLM antibody. Data were normalized to GR values, and repeated-measures ANOVA was used to analyze 2 repeated sets of data. PLM was a cell homogenate from PLM transfected PAC-1 cells, and 17.75 µg were loaded. bSignificantly different from GW (P < 0.05).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

Immunofluorescence labeling of PLM.   To determine the cellular distribution of PLM in the skeletal muscle fibers, immunofluorescent labeling of PLM was performed in GR and GW. As shown in Fig. 2, the ₂-isoform of Na⁺-K⁺-ATPase, a membrane protein, was fairly evenly distributed on the sarcolemmal membrane across fibers in both GR and GW. Some of the fibers showed intracellular labeling; however, it is unclear whether this was a specific signal for ₂. In both skeletal muscles, PLM also can be found prominently labeled on the sarcolemmal membrane.

View larger version (114K): [in this window] [in a new window]   Fig. 2. Immunofluorescent labeling of the Na+-K+-ATPase 2-subunit isoform and PLM in GR and GW. Cryosections from GR and GW were collected on the same slide and processed as described in MATERIALS AND METHODS. A: 2 GR; B: 2 GW; C: PLM GR; D: PLM GW. They were fixed and incubated overnight with 2 and PLM antibodies, and bound antibodies were detected by Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 546 goat anti-mouse IgG (1:200).

View larger version (31K): [in this window] [in a new window]   Fig. 3. Association of the -subunit isoforms with PLM. Coimmunoprecipitation of PLM and the -subunit isoforms was performed using partially purified membrane preparation from mixed GR and GW (125 µg) of 6-mo-old rat. Anti-PLM antibody was used for immunoprecipitation, and the blots were probed with 1, 2, and PLM antibodies. A control with anti-PLM antibody but no membrane preparation (IP Ab) was included to demonstrate a lack of IgG signal at the 100-kDa molecular size range in the coimmunoprecipitation. Another control with membrane preparation but no anti-PLM antibody was also included (No IP Ab). The brain sample because of its abundance in 1, 2, and PLM was used as positive and negative controls. MB, skeletal muscle membrane preparation used for the immunoprecipitation experiments that shows the presence of 1, 2, and PLM in the sample before immunoprecipitation; IP, immunoprecipitation; IB, immunoblotting; SK, skeletal muscle.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Effect of PLM antibody on Na+-K+-ATPase activity. Mixed GR and GW tissue homogenates (n = 3; 150 µg) from a 6-mo-old rat were incubated with 1.5 µg of anti-PLM antibody (anti-PLM), rabbit IgG, or no treatment (control) for 4 h. Enzyme activity was assayed as described in MATERIALS AND METHODS. aSignificantly different from control samples (P < 0.05). bSignificantly different from samples treated with rabbit IgG (P < 0.05).

View larger version (14K): [in this window] [in a new window]   Fig. 5. Relative abundance of PLM in GR (A), GW (B), and EDL (C) muscles of rats with age and exercise training. Tissue homogenates (n = 5) (75 µg) from mature sedentary (Ms), senescent sedentary (Ss), and senescent exercise-trained (St) were resolved by SDS-PAGE. Transferred blots were immunoblotted with PLM antibody. Data were normalized to Ms samples. PLM was a cell homogenate from PLM-transfected PAC-1 cells, and 17.8 µg were loaded. aSignificantly different from Ms samples (P < 0.05). bSignificantly different from Ss samples (P < 0.05).

View larger version (27K): [in this window] [in a new window]   Fig. 6. Levels of 1 and 2 associated with PLM in skeletal muscle of Ms, Ss, and St rats. Partially purified membrane preparations (100 µg; n = 3) from mixed gastrocnemius were immunoprecipitated with anti-PLM antibody, and the blots were probed with 1 (shaded bar), 2 (solid bar), and PLM antibodies. A control with anti-PLM antibody but no membrane preparation (IP Ab) was included to demonstrate a lack of IgG signal in the coimmunoprecipitation. Another control with membrane preparation but no anti-PLM antibody was also included (No IP Ab). Data were normalized to the levels in Ms rats. aSignificantly different from Ms samples (P < 0.05). b'Trend to be different from Ss.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

The present results show that, in the adult rat, among the three tissues that we examined, EDL expresses higher levels of PLM and GW expresses lower levels, with GR expressing an intermediate level that is not statistically different from EDL or GW. In rat skeletal muscle, GR consists mainly of fast-oxidative glycolytic (60%) and slow-oxidative (30%) fibers, whereas GW consists mainly of fast glycolytic (80%) and fast-oxidative glycolytic (15%). EDL, on the other hand, consists mainly of fast-oxidative glycolytic (40%) and fast glycolytic (50%) (1). Thus our data do not suggest a correlation between muscle fiber type and apparent expression of PLM, but rather expression appears to be dependent on muscle type. The reason underlying this muscle type-dependent difference is not immediately clear. It is well known that postural and locomotive muscles, because of functional differences, may express different levels of proteins. Because both gastrocnemius and EDL are locomotive muscles, it is unlikely that differences in mechanical activation underlie the differences in PLM expression.

Our data show, for the first time using immunofluorescence, a predominance of sarcolemmal distribution of PLM in skeletal muscles. This result is consistent with a large body of evidence, based on membrane fractionation studies, showing that PLM is mainly a sarcolemmal protein (28, 29, 36). However, we cannot exclude the possibility of minor distribution of PLM in other cellular compartments. For example, in rat cardiac myocytes, it has been demonstrated that PLM is distributed in the plasma membrane as well as T tubules (32). Importantly, the even distribution of PLM staining among fibers suggests that there is very little fiber-type-specific expression of PLM in the skeletal muscles that we have examined (fiber-typing data not shown). Because serial sections were not used in the present study, we are unable to discern from the immunofluorescent images whether PLM and the -subunit indeed colocalized on the sarcolemmal membrane. However, in a recent report, Silverman et al. (31) demonstrated, by merging immunofluorescent labeling techniques, the colocalization of PLM and -subunit of Na⁺-K⁺-ATPase in ventricular myocytes.

In our study, incubation of tissue homogenates with anti-PLM antibody decreased Na⁺-K⁺-ATPase enzyme activity. Although we do not know the maximal activity of the Na⁺-K⁺-ATPase in the homogenates, our data suggest that PLM is required for Na⁺-K⁺-ATPase to express its full activity in skeletal muscle. This is similar to the finding by Feschenko et al. (12) who showed that, in neuronal tissue, PLM appears to function as an activator of Na⁺-K⁺-ATPase activity. It is worth mentioning that in the present study exercise training increased PLM by almost 50% in all three skeletal muscle types examined. Yet, in our previous study (25), only GR and GW showed elevated enzyme activity after exercise training. Collectively, the results seem to suggest that increased expression of PLM alone does not necessarily lead to elevated Na⁺-K⁺-ATPase enzyme activity. It is possible that these PLM may be associated with other cellular proteins that PLM is known to be associated with, such as the Na⁺/Ca²⁺ exchanger (24). Presently, it is not understood how the antibody decreases enzymatic activity. Whether the antibody modifies phosphorylation and/or association of PLM with Na⁺-K⁺-ATPase should be the subject of further studies.

Previous results from our laboratory and others have demonstrated that expression of the Na⁺-K⁺-ATPase subunit isoforms in skeletal muscle is dynamically regulated under physiological and pathological conditions. More specifically, we have shown that, with age, the expression of both the - and -subunit isoforms undergoes tissue- and isoform-specific regulation (33). Endurance exercise training was able to reverse some, but not all, of the aging-related deficits (25). Our present data show that the level of expression of PLM was not modified by advancing age but was significantly increased by exercise training in aged animals. To the best of our knowledge, we believe this is the first demonstration that the expression of PLM is modified by a physiological or pathological condition. The increased level of PLM may help to modulate the activity of Na⁺-K⁺-ATPase. In addition, the long-term vs. acute effects of exercise training on the expression of PLM have yet to be established.

Because multiple isoforms of Na⁺-K⁺-ATPase are typically expressed in a particular tissue, the question of whether PLM associates only with certain isoforms and, therefore, regulates Na⁺-K⁺-ATPase in an isoform-specific manner is an intriguing one. In the central nervous system, PLM appears to associate equally with the ₁-, ₂-, and ₃-subunit isoforms (12), whereas in skeletal and cardiac muscle PLM reportedly was associated exclusively with the ₁-isoform (11, 14, 31). By contrast, our coimmunoprecipitation data show the association of PLM with both the ₁- and ₂-isoforms. The reason underlying the apparent discrepancy between our results and the previously published data is not immediately clear. Because the report by Crambert et al. (11) did not specify the type of muscle used in their study, it cannot be determined whether muscle type could explain the discrepancy. However, association of PLM with an -subunit isoform may be dictated by the relative distribution of the isoform in the sarcolemma and T tubule (13, 31). For example, in guinea pig ventricular myocytes, it has been shown that ₂ interacts very little with PLM because ₂ is predominantly localized to T tubules and PLM is predominantly localized to peripheral sarcolemma (31). Thus our data appear to suggest that, in skeletal muscle, PLM and the ₁- and ₂-isoforms are all localized in similar subcellular compartments. Our findings may be significant in that, in skeletal muscle, the ₂-isoform is the major Na⁺-K⁺-ATPase isoform being expressed (20, 27). If PLM had associated minimally to this isoform, as reported by others, it would have suggested that PLM could only modulate a relatively small fraction of the Na⁺-K⁺-ATPase in skeletal muscle. The importance of PLM in regulating skeletal muscle Na⁺-K⁺-ATPase, if it indeed occurs, could thus be questioned. On the contrary, our result demonstrates that PLM has the potential to modulate a majority of the Na⁺-K⁺-ATPase in skeletal muscle, suggesting perhaps a more significant role of PLM in skeletal muscle physiology than previously envisioned.

It is worth noting that our coimmunoprecipitation data showed that, in skeletal muscle and brain tissues, the same amount of PLM appears to be associated with very different amounts of the ₁- and ₂-subunits (Fig. 3). This suggests that there may be tissue-dependent differential association of PLM with the -subunits due to either the different amounts of -subunits being expressed in these tissues and/or different affinity of association. The data further suggest that not all PLM is associated with the -subunits, which is perhaps expected since PLM may serve other cellular functions (32, 35, 38).

Although expression of PLM was unaltered by age, the levels of PLM-associated ₁-isoform increased with age and had a tendency to be partially reversed by exercise training. On the other hand, PLM-associated ₂-isoform did not change with age but had a tendency to increase after exercise training. This increase in PLM-associated ₁ compared with PLM-associated ₂ with age suggests that PLM may play a more important role in regulating ₁ during aging. The changes in PLM-associated -subunit isoforms appear to follow similar trends with the changes in overall expression of the -subunit isoforms with age and after exercise training, as we have reported previously (25). Specifically, we showed that ₁ expression increases with age and that exercise training has marginal effect on its expression. There was a trend for decreased ₂ levels with age, and exercise training significantly increased the levels.

A limitation in the design of the experiments is that the sample size in two of the experiments (Figs. 4 and 6) is small (n = 3). The reason for the small sample size in those experiments is due to the limited amount of antibody that we had and the large amount of antibody that is required for performing the enzyme activity assay and immunoprecipitation. Because of the small sample size, our analysis may be prone to type II error. The fact that statistical significance was still found between some of the treatment groups would seem to suggest that, despite the potential pitfall, the analysis was able to discern at least the more substantial changes.

Functional significance of the modulation of Na⁺-K⁺-ATPase activity by PLM in skeletal muscle remains to be fully elucidated. However, a recent study by Jia et al. (16) demonstrated that, in the myocardium of PLM-deficient mice, there was a reduction in Na⁺-K⁺-ATPase activity, changes in Na⁺-K⁺-ATPase isoform expression, an increase in ejection fraction, and an increase in cardiac mass. Thus this appears to be the clearest demonstration yet of the important physiological role of PLM with respect to Na⁺-K⁺-ATPase at the cellular and functional levels.

In conclusion, PLM appears to be an integral part of Na⁺-K⁺-ATPase in skeletal muscle and has the potential to modulate the Na⁺-K⁺-ATPase in an isoform-specific and muscle type-dependent manner during aging and after exercise training.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by National Institutes of Health Grants AG-16822 (to Y.-C. Ng) and HL-40306 (to R. L. Moore).

	ACKNOWLEDGMENTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We thank Dr. K. Sweadner for generously providing the anti-₂-subunit antibody. The monoclonal 6f antibody developed by Dr. D. M. Fambrough was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA.

	FOOTNOTES

 

Address for reprint requests and other correspondence: Y.-C. Ng, Dept. of Pharmacology, College of Medicine, The Pennsylvania State Univ., Milton S. Hershey Medical Center, 500 Univ. Dr., Hershey, PA 17033 (e-mail: ycn1{at}psu.edu)

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.

	REFERENCES

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1. Armstrong RB and Phelps RO. Muscle fiber type composition of the rat hindlimb. Am J Anat 171: 259–272, 1984.[CrossRef][Web of Science][Medline]
2. Azuma KA, Hensley CB, Tang M, and McDonough AA. Thyroid hormone specifically regulates skeletal muscle Na⁺-K⁺-ATPase alpha 2- and beta 2-isoforms. Am J Physiol Cell Physiol 265: C680–C687, 1993.[Abstract/Free Full Text]
3. Azuma KK, Hensley CB, Putnam DS, and McDonough AA. Hypokalemia decreases Na⁺-K⁺-ATPase alpha 2- but not alpha 1-isoform abundance in heart, muscle, and brain. Am J Physiol Cell Physiol 260: C958–C964, 1991.[Abstract/Free Full Text]
4. Beguin P, Crambert G, Guennoun S, Garty H, Horisberger JD, and Geering K. CHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the gamma-subunit. EMBO J 20: 3993–4002, 2001.[CrossRef][Web of Science][Medline]
5. Beguin P, Crambert G, Monnet-Tschudi F, Uldry M, Horisberger JD, Garty H, and Geering K. FXYD7 is a brain-specific regulator of Na,K-ATPase alpha 1-beta isozymes. EMBO J 21: 3264–3273, 2002.[CrossRef][Web of Science][Medline]
6. Beguin P, Wang XY, Firsov D, Puoti A, Claeys D, Horisberger JD, and Geering K. The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function. EMBO J 16: 4250–4260, 1997.[CrossRef][Web of Science][Medline]
7. Bundgaard H, Schmidt TA, Larsen JS, and Kjeldsen K. K⁺ supplementation increases muscle [Na⁺-K⁺-ATPase] and improves extrarenal K⁺ homeostasis in rats. J Appl Physiol 82: 1136–1144, 1997.[Abstract/Free Full Text]
8. Chen LS, Lo CF, Numann R, and Cuddy M. Characterization of the human and rat phospholemman (PLM) cDNAs and localization of the human PLM gene to chromosome 19q13.1. Genomics 41: 435–443, 1997.[CrossRef][Web of Science][Medline]
9. Clausen T and Everts ME. Regulation of the Na,K-pump in skeletal muscle. Kidney Int 35: 1–13, 1989.[Web of Science][Medline]
10. Crambert G, Beguin P, Uldry M, Monnet-Tschudi F, Horisberger JD, Garty H, and Geering K. FXYD7, the first brain- and isoform-specific regulator of Na,K-ATPase: biosynthesis and function of its posttranslational modifications. Ann NY Acad Sci 986: 444–448, 2003.[Web of Science][Medline]
11. Crambert G, Fuzesi M, Garty H, Karlish S, and Geering K. Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties. PNAS 99: 11476–11481, 2002.[Abstract/Free Full Text]
12. Feschenko MS, Donnet C, Wetzel RK, Asinovski NK, Jones LR, and Sweadner KJ. Phospholemman, a single-span membrane protein, is an accessory protein of Na,K-ATPase in cerebellum and choroid plexus. J Neurosci 23: 2161–2169, 2003.[Abstract/Free Full Text]
13. Fransen P. Phospholemman, a chaperone of Na⁺,K⁺-ATPase? Cardiovasc Res 65: 13–15, 2005.[Free Full Text]
14. Fuller W, Eaton P, Bell JR, and Shattock MJ. Ischemia-induced phosphorylation of phospholemman directly activates rat cardiac Na/K-ATPase. FASEB J 18: 197–199, 2004.[Abstract/Free Full Text]
15. Jew KN and Moore RL. Exercise training alters an anoxia-induced, glibenclamide-sensitive current in rat ventricular cardiocytes. J Appl Physiol 92: 1473–1479, 2002.[Abstract/Free Full Text]
16. Jia LG, Donnet C, Bogaev RC, Blatt RJ, McKinney CE, Day KH, Berr SS, Jones LR, Moorman JR, Sweadner KJ, and Tucker AL. Hypertrophy, increased ejection fraction, and reduced Na,K-ATPase activity in phospholemman-deficient mice. Am J Physiol Heart Circ Physiol 288: H1982–H1988, 2005.[Abstract/Free Full Text]
17. Jones LR, Besch HR Jr, Fleming JW, McConnaughey MM, and Watanabe AM. Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities. J Biol Chem 254: 530–539, 1979.[Abstract/Free Full Text]
18. Jones LR, Maddock SW, and Hathaway DR. Membrane localization of myocardial type II cyclic AMP-dependent protein kinase activity. Biochim Biophys Acta 641: 242–253, 1981.[Medline]
19. Kelly C, Ram M, Francis S, and Cala S. Identification of a cytoskeleton-bound form of phospholemman with unique C-terminal immunoreactivity. J Membr Biol 201: 1–9, 2004.[CrossRef][Web of Science][Medline]
20. Lavoie L, Levenson R, Martin-Vasallo P, and Klip A. The molar ratios of alpha and beta subunits of the Na-K-ATPase differ in distinct subcellular membranes from rat skeletal muscle. Biochemistry 36: 7726–7732, 1997.[CrossRef][Medline]
21. Mahmmoud YA, Cramb G, Maunsbach AB, Cutler CP, Meischke L, and Cornelius F. Regulation of Na,K-ATPase by PLMS, the phospholemman-like protein from shark: molecular cloning, sequence, expression, cellular distribution, and functional effects of PLMS. J Biol Chem 278: 37427–37438, 2003.[Abstract/Free Full Text]
22. Mahmmoud YA, Vorum H, and Cornelius F. Identification of a phospholemman-like protein from shark rectal glands. Evidence for indirect regulation of Na,K-ATPase by protein kinase c via a novel member of the FXYDY family. J Biol Chem 275: 35969–35977, 2000.[Abstract/Free Full Text]
23. McDonough AA, Thompson CB, and Youn JH. Skeletal muscle regulates extracellular potassium. Am J Physiol Renal Physiol 282: F967–F974, 2002.[Abstract/Free Full Text]
24. Mirza MA, Zhang XQ, Ahlers BA, Qureshi A, Carl LL, Song J, Tucker AL, Mounsey JP, Moorman JR, Rothblum LI, Zhang TS, and Cheung JY. Effects of phospholemman downregulation on contractility and [Ca²⁺]_i transients in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol 286: H1322–H1330, 2004.[Abstract/Free Full Text]
25. Ng YC, Nagarajan M, Jew KN, Mace LC, and Moore RL. Exercise training differentially modifies age-associated alteration in expression of Na⁺-K⁺-ATPase subunit isoforms in rat skeletal muscles. Am J Physiol Regul Integr Comp Physiol 285: R733–R740, 2003.[Abstract/Free Full Text]
26. Nielsen OB and Clausen T. The Na⁺/K⁺-pump protects muscle excitability and contractility during exercise. Exerc Sport Sci Rev 28: 159–164, 2000.[Medline]
27. Orlowski J and Lingrel JB. Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic alpha isoform and beta subunit mRNAs. J Biol Chem 263: 10436–10442, 1988.[Abstract/Free Full Text]
28. Palmer CJ, Scott BT, and Jones LR. Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. J Biol Chem 266: 11126–11130, 1991.[Abstract/Free Full Text]
29. Presti CF, Jones LR, and Lindemann JP. Isoproterenol-induced phosphorylation of a 15-kilodalton sarcolemmal protein in intact myocardium. J Biol Chem 260: 3860–3867, 1985.[Abstract/Free Full Text]
30. Reis JJ, Cala SE, Jew KN, Mace LC, Moore RL, and Ng YC. Expression of phospholemman in aged skeletal muscle: effect of exercise training (Abstract). FASEB J 18: 346, 2004.
31. Silverman DBZ, Fuller W, Eaton P, Deng J, Moorman JR, Cheung JY, James AF, and Shattock MJ. Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase. Cardiovasc Res 65: 93–103, 2005.[Abstract/Free Full Text]
32. Song J, Zhang XQ, Ahlers BA, Carl LL, Wang J, Rothblum LI, Stahl RC, Mounsey JP, Tucker AL, Moorman JR, and Cheung JY. Serine 68 phospholemman is critical in modulation of contractility, [Ca²⁺]_i transients and Na⁺/Ca²⁺ exchange in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol 288: H2342–H2354, 2005.[Abstract/Free Full Text]
33. Sun X, Nagarajan M, Beesley PW, and Ng YC. Age-associated differential expression of Na⁺-K⁺-ATPase subunit isoforms in skeletal muscles of F-344/BN rats. J Appl Physiol 87: 1132–1140, 1999.[Abstract/Free Full Text]
34. Sweadner KJ and Rael E. The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression. Genomics 68: 41–56, 2000.[CrossRef][Web of Science][Medline]
35. Walaas SI, Czernik AJ, Olstad OK, Sletten K, and Walaas O. Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate phospholemman, an insulin and adrenaline-regulated membrane phosphoprotein, at specific sites in the carboxy terminal domain. Biochem J 304: 635–640, 1994.[Web of Science][Medline]
36. Walaas SI, Horn RS, Albert KA, Adler A, and Walaas O. Phosphorylation of multiple sites in a 15,000 dalton proteolipid from rat skeletal muscle sarcolemma, catalyzed by adenosine 3',5'-monophosphate-dependent and calcium/phospholipid-dependent protein kinases. Biochim Biophys Acta 968: 127–137, 1988.[Medline]
37. Walaas SI, Horn RS, Nairn AC, Walaas O, and Adler A. Skeletal muscle sarcolemma proteins as targets for adenosine 3':5'-monophosphate-dependent and calcium-dependent protein kinases. Arch Biochem Biophys 262: 245–258, 1988.[CrossRef][Web of Science][Medline]
38. Zhang XQ, Qureshi A, Song J, Carl LL, Tian Q, Stahl RC, Carey DJ, Rothblum LI, and Cheung JY. Phospholemman modulates Na⁺/Ca²⁺ exchange in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol 284: H225–H233, 2003.[Abstract/Free Full Text]

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