Coordinate downregulation of CaM kinase II and phospholamban accompanies contractile phenotype transition in the hyperthyroid rabbit soleus

M. Jiang, A. Xu, D.L. Jones, and N. Narayanan

Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada N6A 5C1

Submitted 18 August 2003 ; accepted in final form 19 April 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study investigated the effects of L-thyroxine-induced hyperthyroidism on Ca2+/calmodulin (CaM)-dependent protein kinase (CaM kinase II)-mediated sarcoplasmic reticulum (SR) protein phosphorylation, SR Ca2+ pump (Ca2+-ATPase) activity, and contraction duration in slow-twitch soleus muscle of the rabbit. Phosphorylation of Ca2+-ATPase and phospholamban (PLN) by endogenous CaM kinase II was found to be significantly lower (30–50%) in soleus of the hyperthyroid compared with euthyroid rabbit. Western blotting analysis revealed higher levels of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 (~150%) Ca2+ pump isoform, unaltered levels of SERCA2 Ca2+ pump isoform, and lower levels of PLN (~50%) and {delta}-, {beta}-, and {gamma}-CaM kinase II (40 ~ 70%) in soleus of the hyperthyroid rabbit. SR vesicles from hyperthyroid rabbit soleus displayed approximately twofold higher ATP-energized Ca2+ uptake and Ca2+-stimulated ATPase activities compared with that from euthyroid control. The Vmax of Ca2+ uptake (in nmol Ca2+·mg SR protein–1·min–1: euthyroid, 818 ± 73; hyperthyroid, 1,649 ± 90) but not the apparent affinity of the Ca2+-ATPase for Ca2+ (euthyroid, 0.97 ± 0.02 µM, hyperthyroid, 1.09 ± 0.04 µM) differed significantly between the two groups. CaM kinase II-mediated stimulation of Ca2+ uptake by soleus muscle SR was ~60% lower in the hyperthyroid compared with euthyroid. Isometric twitch force of soleus measured in situ was significantly greater (~36%), and the time to peak force and relaxation time were significantly lower (~30–40%), in the hyperthyroid. These results demonstrate that thyroid hormone-induced transition in contractile properties of the rabbit soleus is associated with coordinate downregulation of the expression and function of PLN and CaM kinase II and selective upregulation of the expression and function of SERCA1, but not SERCA2, isoform of the SR Ca2+ pump.

calmodulin kinase II; phospholamban ; calcium ion-adenosinetriphosphatase; sarcoplasmic reticulum


THYROID HORMONE AND INNERVATION are the two main factors that determine skeletal muscle contractile properties and the slow-twitch vs. fast-twitch muscle fiber phenotype (16, 48). Generally, fast-twitch properties develop in the fast motoneuron, which renders a fiber active <5% of the time (15), or in the complete absence of innervation (17). Development of the slow-twitch phenotype, on the other hand, requires innervation of the fiber with a slow motoneuron, i.e., resulting in virtually continuous low-frequency contractions (40). This plasticity of the phenotype is illustrated by cross-innervation and direct stimulation studies, which have shown that chronic low-frequency activity induces a fast- to slow-twitch transition in adult fast-twitch muscle (5, 51). In contrast, thyroid hormone stimulates the development of fast-twitch fiber characteristics in slow-twitch muscle (30, 39, 51). This is particularly evident in slow-twitch soleus muscle, in which the speed of contraction and relaxation is enhanced in the hyperthyroid state (30, 36), and a decrease in these parameters accompanies the hypothyroid state (30, 45).

Previous studies on the mechanistic basis of thyroid hormone-induced transformation of the slow muscle to fast muscle phenotype have revealed modifications at the level of the myofilament and the sarcoplasmic reticulum (SR). Thus, in the slow-twitch soleus muscle of the rat, thyroid hormone induces the replacement of slow myosin heavy chain isoform (MHC I) with fast MHC (MHC II), which may account for the increase in speed of muscle contraction (6, 19). In the rat soleus, the increased speed of relaxation induced by thyroid hormone is associated with an increased expression of the SR Ca2+-ATPase (10, 45). It has also been reported that the increase in SR Ca2+-ATPase activity in rat soleus muscle is due to thyroid hormone-induced de novo expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 isoform of the Ca2+-ATPase (54). Three genes encode different isoforms (SERCA1, SERCA2, and SERCA3) of the SR Ca2+-ATPase, and SERCA1 is the predominant isoform expressed in fast muscle fibers, whereas SERCA2 is the main isoform normally expressed in slow skeletal muscle and cardiac muscle (18, 28, 29). Thyroid hormone specifically stimulates the production of SERCA1 isoform mRNA and protein, favoring the transformation of slow muscle to fast muscle phenotype (44, 54). The SERCA1 and SERCA2 isoforms, however, have a high degree of sequence homology (~84%), and they share similar transmembrane topologies, tertiary structures, and enzymatic properties (18, 28, 29). The extensive structural homology and similarity in biochemical properties between the Ca2+-ATPase isoforms likely reflect the uniformity in their function, i.e., active Ca2+ transport. On the other hand, the limited structural differences manifested in the Ca2+-ATPase isoforms and their muscle type-specific expression may signify differences in the mechanisms by which their function is regulated physiologically. For example, in cardiac and slow skeletal muscle SR, but not in fast skeletal muscle SR, the Ca2+-ATPase is subject to regulation by phospholamban (PLN), an intrinsic SR protein that is naturally coexpressed with SERCA2 (and not SERCA1) isoform (23, 43, 50). It is widely recognized that dephospho-PLN inhibits Ca2+-ATPase function through physical interaction with the enzyme, and dissociation of the two proteins on phosphorylation of PLN by PKA or Ca2+/calmodulin (CaM)-dependent protein kinase (CaM kinase II) relieves the inhibition (for reviews, see Refs. 23, 43). CaM kinase II exists as a heteromultimeric holoenzyme, encoded by four separate genes ({alpha}, {beta}, {gamma}, and {delta}) (4). In skeletal muscle, the {delta}, {gamma}, and muscle-specific {beta}M subunits of CaM kinase II have been documented to be tightly associated with the SR membrane (3, 9, 42, 52). Recently, Ca2+-dependent processes, other than PLN phosphorylation, have been implicated in the dissociation of Ca2+-ATPase/PLN complex and regulation of SERCA2 function by CaM kinase (2, 35). Furthermore, several studies have demonstrated direct phosphorylation of the SERCA2 isoform of the Ca2+-ATPase by endogenous CaM kinase II (8, 14, 37, 38, 53, 5558); this phosphorylation was shown to result in stimulation of ATP hydrolysis and Ca2+ transport (14, 5558). Although some studies (37, 41) have questioned the physiological significance of SERCA2 phosphorylation, evidence from more recent studies strongly supports the view that SERCA2 phosphorylation is a physiological event that results in stimulation of the Vmax of Ca2+ pumping in native cardiac SR (57–59). The SERCA1 isoform of the Ca2+-ATPase does not undergo phosphorylation by CaM kinase II (14, 53).

Despite the impressive evidence documenting muscle type-specific coexpression of SERCA2 and PLN and isoform-specific regulation of SERCA2 by protein phosphorylation, no study has as yet examined the influence of thyroid hormone on these parameters in the context of transformation of slow-twitch muscle to fast-twitch muscle phenotype. It is also noteworthy that all previous studies on the mechanistic basis of thyroid hormone-induced shift in muscle fiber phenotype utilized rat soleus muscle (6, 10, 19, 45); it has been discovered recently that, unlike in other species, rat soleus naturally lacks PLN (8). The aim of the present study was to investigate the influence of thyroid hormone on the expression and function of PLN, SERCA isoforms, and CaM kinase II during transformation of the rabbit soleus muscle from slow-twitch to fast-twitch contractile phenotype following the induction of the hyperthyroid state by administration of L-thyroxine (T4). Contractile properties of the soleus muscle were determined in situ in euthyroid and hyperthyroid rabbits; Western blotting analysis was used to determine changes in the expression of individual proteins; Ca2+-ATPase function was measured by determining Ca2+-activated ATP hydrolysis, as well as ATP-energized Ca2+ transport by SR vesicles; and CaM kinase II function was evaluated from Ca2+/CaM-dependent phosphorylation of SR proteins. The results presented here demonstrate that thyroid hormone-induced transition in contractile properties of the rabbit soleus muscle are associated with coordinate downregulation of the expression and function of PLN and {delta}-, {beta}-, and {gamma}-CaM kinase II and selective upregulation of the expression and function of SERCA1 but not SERCA2 isoform of the SR Ca2+ pump. The findings also imply unique, interactive roles for PLN and CaM kinase II in the SERCA2 isoform-specific physiological regulation of SR Ca2+ pump function.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Twelve-week-old male New Zealand White rabbits were obtained from a local breeder and were maintained on ordinary rabbit chow in the Health Sciences Center animal care facility of this institution. All experiments were approved by the institutional Animal Use Committee and were cared for in accordance with the guidelines of the Canadian Council on Animal Care. Hyperthyroidism was induced by injecting T4 intramuscularly at 200 µg/kg body wt daily for 7 days (1). Age-matched untreated rabbits (euthyroid group) were used as controls.

The rabbits were killed following intravenous administration of pentobarbital sodium (35 mg/kg). Blood samples were collected by cardiac puncture at the same time and centrifuged. The serum samples were treated with polyethylene glycol to precipitate any endogenous antibodies (24), and the hormones were assayed by a fully automated chemiluminescent immunoassay analyzer (Chiron ACS-180, Walpole).

Chemicals. Reagents for electrophoresis were obtained from Bio-Rad Laboratories (Mississauga, ON). 45CaCl2 was purchased from New England Nuclear (Mississauga, ON). [{gamma}-32P]ATP was from Amersham (Oakville, ON). Monoclonal antibody against SERCA1 and SERCA2 isoform of SR Ca2+-ATPase was purchased from Affinity BioReagents (Golden, CO). Anti-PLN monoclonal antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Polyclonal antiphophoserine-16 and antiphosphothreonine-17 PLN antibodies were obtained from Badrilla (Leeds, UK). Anti-{delta}-CaM kinase II polyclonal antibody was a generous gift from Dr. P. Karczewski (Max Delbruck Center for Molecular Medicine). A polyclonal antibody that cross-reacts with {beta}- and {gamma}- but not {delta}-CaM kinase II subunit was obtained from Santa Cruz Biotechnology. All other chemicals were from Sigma Chemical (St. Louis, MO) or BDH Chemicals (Toronto, ON).

Preparation of SR membrane vesicles and muscle homogenate. SR membrane vesicles were prepared as described previously (21). Briefly, the slow-twitch skeletal muscle (soleus) was minced and homogenized in six volumes (based on tissue weight) of ice-cold buffer (10 mM NaHCO3, pH 6.8) by using a Polytron homogenizer (3 bursts of 15-s duration with 30-s intervals, speed setting 5.5; Brinkman Instruments, Westbury, NY). The homogenate was centrifuged at 1,000 g for 10 min at 4°C. The supernatant was decanted and kept in an ice slurry. The pellet was resuspended in four volumes of ice-cold buffer and centrifuged as before. The supernatant was decanted and combined with the first supernatant, and the pellet was discarded. The combined supernatant was centrifuged at 8,000 g for 20 min at 4°C. The supernatant was collected, and the pellet was discarded. Solid KCl (44 mg/ml) was added to the supernatant (final concentration, 0.6 M), swirled to dissolve, left on ice for 25 min, and then centrifuged at 40,000 g for 1 h at 4°C. After isolation, the SR vesicles were suspended in 10 mM Tris-maleate (pH = 6.8) containing 100 mM KCl and stored at –80°C after quick freezing in liquid nitrogen. Protein concentration was determined by the method of Lowry et al. (27) by using bovine serum albumin as standard.

In addition to SR membranes, soleus muscle homogenates were used in some experiments. The homogenates were prepared by homogenizing the muscle tissue in six volumes (based on tissue weight) of 10 mM NaHCO3 (pH = 6.8) by using a Polytron homogenizer (three 15-s bursts with 30-s interval between bursts; setting 5.5). The homogenates were filtered through four layers of cheese cloth.

SDS-PAGE and immunoblotting of Ca2+-ATPase, PLN, and CaM kinase II. The protein composition of soleus muscle SR isolated from euthyroid and hyperthyroid rabbits was analyzed by SDS-PAGE, as described previously (20). Western immunoblotting procedure was used to localize and quantify Ca2+-ATPase isoforms, PLN, and CaM kinase II in SR membrane vesicles and soleus muscle homogenates. For this, samples of homogenate or SR vesicles (25 µg protein/lane in each case) were first subjected to SDS-PAGE in 4–18% gradient gels (for Ca2+-ATPase and PLN) or 10% (for CaM kinase II) gels. The fractionated proteins were then electroblotted to nitrocellulose membranes. The membranes were probed with antibodies specific for SERCA1 and SERCA2 Ca2+-ATPase [monoclonal (22), dilution 1:3,000]; PLN [monoclonal (49), 0.5 µg/ml]; and {delta}-, {beta}-, {gamma}-subunits of CaM kinase II [polyclonal (56), dilution 1:1,000 for {delta} and 1:500 for {beta}/{gamma}]. A peroxidase-linked anti-mouse (for Ca2+-ATPase, PLN) or anti-rabbit (for CaM kinase II) IgG at a dilution of 1:5,000 was used as the secondary antibody. Protein bands reactive with antibodies were visualized by using the enhanced chemiluminescence detection system from Amersham. The images of the protein bands were optimized, captured, and analyzed by ImageMaster VDS gel documentation system (Pharmacia Biotech, San Francisco, CA). The Western blotting detection system was determined to be linear with respect to the amount of SR/homogenate protein in the range of 10–40 µg by using this camera-based densitometry method.

Determination of Ca2+ uptake and Ca2+-ATPase activity. ATP-dependent Ca2+ uptake by SR vesicles was measured by using a Millipore filtration technique, as described previously (32). The standard Ca2+ uptake assay medium (total volume, 1 ml) contained 50 mM Tris-maleate (pH 6.8), 5 mM MgCl2, 5 mM ATP, 120 mM KCl, 5 mM potassium oxalate, 5 mM sodium azide, 0.1 mM EGTA, SR membranes (30-µg protein), and varying concentrations of 45CaCl2 (~8,400 counts·min–1·nmol–1). The assays were performed at 37°C. The Ca2+ uptake reaction was initiated by the addition of the membrane fraction after preincubation of the rest of the assay components for 3 min. The initial free Ca2+ concentration in the assay medium was determined by using the computer program of Fabiato (11).

The data on the Ca2+ concentration dependence of Ca2+ uptake were analyzed by nonlinear regression curve fitting by using the SigmaPlot scientific graph program (Systat Software) run on an IBM personal computer (PC). The data were fitted to the equation

where {nu} is the measured Ca2+ uptake activity at a given Ca2+ concentration ([Ca2+]), Vmax is the maximum activity reached, K0.5 is the Ca2+ concentration giving one-half of Vmax, and nH is equivalent to the Hill coefficient.

The incubation medium used for the assay of Mg2+-dependent Ca2+-ATPase activity was identical to that described for Ca2+ uptake, except that [{gamma}-32P]ATP was used instead of nonradioactive ATP, and nonradioactive CaCl2 (free Ca2+, 8.2 µM) was used instead of 45CaCl2. To determine the "basal" ATPase (Mg2+-ATPase) activity, assays were also carried out in the absence of Ca2+ and in the presence of 0.2 mM EGTA. The incubations were carried out at 37°C for 3 min by the addition of SR membranes, and the reaction was stopped by the addition of 1 ml 12% trichloroacetic acid/2 mM KH2PO4. Following this, 0.1 ml each of 25 mM ATP and 0.1% bovine serum albumin was added to the tubes. The tubes were centrifuged (1,000 g, 10 min), and the 32P released from [{gamma}-32P]ATP was extracted and quantitated as described previously (34). The basal ATPase activity was subtracted from the enzyme activity measured in the presence of Ca2+ to obtain the Ca2+-ATPase activity.

Measurement of CaM kinase II-mediated SR protein phosphorylation. Phosphorylation of SR proteins by endogenous CaM kinase II was determined as described previously (55). The phosphorylation assay medium (total volume, 50 µl) contained 50 mM HEPES (pH = 7.4), 10 mM MgCl2, 100 µM CaCl2, 100 µM EGTA, 1 µM CaM, 0.8 mM [{gamma}-32P]ATP (specific activity 200–300 counts·min–1·pmol–1), and SR (25 µg of protein). The phosphorylation reaction was initiated by the addition of [{gamma}-32P]ATP following preincubation of the rest of assay components for 3 min at 37°C. The Ca2+/CaM dependence of phosphorylation was monitored in parallel assays lacking Ca2+ (1 mM EGTA present) and CaM in the assay system. Reactions were terminated after 2 min by the addition of 15 µl of SDS sample buffer, and the samples were subjected to SDS-PAGE in 4–18% gradient gel, stained with Coomassie brilliant blue, dried, and autoradiographed. Quantification of phosphorylation was carried out by liquid scintillation counting after excision of the radioactive bands from the gels, as described previously (20, 56).

Assessment of contractile properties. The contractile properties of soleus muscle were determined in situ, according to the procedure described previously (33). The rabbit was anesthetized with urethane (1 g/kg iv). An incision was made through the skin from the ankle to the knee along the medial side of the right leg. The muscle under investigation was exposed with care so as not to damage the nerve and blood supply. The distal tendon of the muscle was isolated and connected to a hook on the force transducer (FT-10C, Grass Instruments, Quincy, MA) with a piece of braided silk (Ethicon 00). The knee and ankle were fixed on the board. After determination of threshold and maximal voltage, isometric contractions at the muscle length at which twitch force (TF) was maximal (with resting tension being 0) were evoked by electrical field stimulation with supramaximal voltage by use of two platinum plate electrodes positioned on either side of the muscle. The contraction was recorded on a PC Biopac TCI/MP WSW 100 system and sampled at 120 Hz, and the averages of five sequential twitches at 30-s intervals were analyzed by Acqknowledge software for the following parameters: TF, time to peak force (TPF), and time for 25, 50, and 90% relaxation and contraction duration (TPF + time for 50% relaxation). After recording contractions, the muscle was excised and weighed.

Data analysis. Statistical analysis was performed by using SigmaPlot scientific graph program (Systat Software) run on an IBM PC, according to Student's t-test for unpaired data. P < 0.05 was taken as the level of significance. Results were averaged and are expressed as means ± SE of experiments using separate preparations. The n values specified in Fig. 110 legends denote the number of independent determinations by using separate SR/homogenate preparations.



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Fig. 1. Comparison of endogenous calmodulin (CaM) kinase II-mediated phosphorylation of Ca2+-ATPase and phospholamban (PLN) in soleus muscle sarcoplasmic reticulum (SR) from euthyroid and hyperthyroid rabbits. A, left: Coomassie blue-stained SDS-polyacrylamide gel depicting typical protein profiles of soleus muscle SR from euthyroid (EU) and hyperthyroid (Hyper) rabbits, subjected to incubation in the phosphorylation reaction medium in the absence (–) and presence (+) of Ca2+ and CaM, as indicated on top. Right: autoradiogram of the same gel depicting protein phosphorylation due to activation of endogenous CaM kinase II. PLN (H) and PLN (L), high and low molecular weight (MW) forms of PLN, respectively. B: quantitative data on phosphorylation of Ca2+-ATPase and PLN expressed as 32P incorporation per milligram SR protein, as well as the ratio of substrate phosphorylation to the amount of immunoreactive protein. The relative amount of immunoreactive substrate in the euthyroid vs. hyperthyroid state was determined by densitometry of Western immunoblots, as described in METHODS. Values are means ± SE; n = 8. *P < 0.05 vs. euthyroid.

 


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Fig. 10. Effect of activation of endogenous CaM kinase II on Ca2+ uptake activity of soleus muscle SR from euthyroid and hyperthyroid rabbits. A: Ca2+ uptake measured with a subsaturating free Ca2+ concentration of 0.6 µM. B: Ca2+ uptake measured with a saturating free Ca2+ concentration of 8.2 µM. Values are means ± SE; n = 5. *P < 0.05 vs. euthyroid; #P < 0.05 vs. +Ca2+ – CaM.

 

    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Induction of hyperthyroid state and changes in body weight and soleus muscle weight. As shown in Table 1, the blood levels of T4 and triiodothyronine were significantly elevated, whereas thyroid-stimulating hormone levels were significantly decreased in the thyroid hormone-treated (hyperthyroid group) compared with untreated control (euthyroid group) rabbits. A modest (10%), yet significant, decrease in body weight was observed in the hyperthyroid group compared with the euthyroid group. The weight of soleus muscle (pooled from both legs) was significantly lower in hyperthyroid compared with euthyroid rabbits. These data demonstrate that the thyroid hormone treatment protocol used in this study resulted in the development of the hyperthyroid state in the rabbit.


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Table 1. Comparison of body weight, soleus muscle weight, and hormone level in euthyroid and hyperthyroid rabbits

 
Phosphorylation of Ca2+-ATPase and PLN by endogenous CaM kinase II. Ca2+/CaM-dependent protein kinase II, associated with the SR (and present in the cytosol), is known to play an important role in regulating the Ca2+ uptake function of cardiac and slow-twitch skeletal muscle SR through phosphorylation of PLN (23, 43, 50) and Ca2+-ATPase (14, 53, 55). In the present study, endogenous CaM kinase II-mediated protein phosphorylation was determined in SR vesicles isolated from the slow-twitch soleus muscle of euthyroid and hyperthyroid rabbits. In the presence of Ca2+ and CaM, the SR-associated CaM kinase II catalyzed the phosphorylation of Ca2+-ATPase and PLN in soleus muscle SR from euthyroid and hyperthyroid animals (Fig. 1A). Quantitative analysis of 32P incorporation into the peptide bands representing Ca2+-ATPase and PLN, as well as quantification per unit amount of each of the immunoreactive substrate, revealed significantly lower (~30–50%, P < 0.05) phosphorylation of Ca2+-ATPase and PLN in the hyperthyroid compared with euthyroid rabbits (Fig. 1B). The observed decrease in protein phosphorylation may result from a relatively high level of preexisting (basal) phosphorylation of the CaM kinase II substrate. To investigate this possibility, the preexisting phosphorylation level of PLN was determined by Western blotting by using phosphorylation site-specific antibodies (anti-Ser-16 and anti-Thr-17). The results are presented in Fig. 2. It can be seen that the relative amount of Ser-16-phosphorylated PLN (which reflects the preexisting PKA-mediated substrate phosphorylation) did not differ significantly between the euthyroid and hyperthyroid groups. On the other hand, the relative amount of Thr-17-phosphorylated PLN (which reflects the preexisting CaM kinase II-mediated substrate phosphorylation) was significantly lower in the hyperthyroid compared with euthyroid group. Therefore, the decreased CaM kinase II-mediated substrate phosphorylation in the hyperthyroid state observed in this study is not due to relatively high preexisting phosphorylation.



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Fig. 2. Comparison of preexisting Ser-16/Thr-17 phosphorylation levels of PLN in soleus muscle SR from euthyroid and hyperthyroid rabbits. Soleus muscle SR from euthyroid and hyperthyroid rabbits was subjected to Western blotting analysis of phosphorylated PLN by using phosphorylation site-specific antibodies (anti-Ser-16 and anti-Thr-17). Bottom: typical immunoblots obtained with 3 separate soleus muscle SR preparations each from euthyroid and hyperthyroid rabbits. Identical amount of SR (25 µg protein) was applied in each lane. Top: bar graphs show the amounts of phosphoserine (A) and phosphothreonine (B) of PLN as determined by computer-assisted scanning densitometry of the immunoblots. Values are means ± SE; n = 3. *P < 0.05 vs. euthyroid.

 
Expression of SERCA1, SERCA2, PLN, and CaM kinase II. To clarify whether the diminished substrate phosphorylation by CaM kinase II in hyperthyroid soleus is associated with changes in the densities of SR Ca2+-ATPase, PLN, or CaM kinase II, the relative levels of each of these proteins in soleus muscle homogenates and SR from euthyroid and hyperthyroid rabbits were determined by quantitative immunoblotting utilizing antibodies specific for SERCA1 and SERCA2 Ca2+-ATPase isoforms, PLN, and {delta}-, {beta}-, and {gamma}-CaM kinase II. The results showed that the relative amounts of {delta}- (40–50%), {beta}- (30–40%), and {gamma}-isoform (45 ~ 70%) subunits of CaM kinase II were markedly diminished (P < 0.05) in hyperthyroid soleus muscle homogenates and SR compared with those of euthyroid (Figs. 3 and 4).



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Fig. 3. Relative amounts of {delta}-CaM kinase II in soleus muscle homogenates and SR from euthyroid and hyperthyroid rabbits, as determined by Western immunoblotting analysis. Bottom: typical immunoblots obtained with 3 separate soleus muscle homogenate (A) and 4 separate soleus muscle SR preparations (B) each from euthyroid and hyperthyroid rabbits. Identical amount of homogenate/SR protein (25 µg) was applied in each lane. Top: bar graphs show the amounts of {delta}-CaM kinase II, as determined by computer-assisted scanning densitometry of the immunoblots. Values are means ± SE; n = 6 (homogenate) or 7 (SR). *P < 0.05 vs. euthyroid.

 


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Fig. 4. Relative amounts of {beta}- and {gamma}-CaM kinase II in soleus muscle homogenates and SR from euthyroid and hyperthyroid rabbits, as determined by Western immunoblotting analysis. The immunoblotting was performed by using a polyclonal antibody, which reacts with {beta}- and {gamma}- but not {delta}-isoform subunits of CaM kinase II (see METHODS). Bottom: typical immunoblots obtained with 3 separate soleus muscle homogenate (A) and 5 separate soleus muscle SR preparations (B) each from euthyroid and hyperthyroid rabbits. Identical amount of homogenate/SR protein (25 µg) was applied in each lane. Top: bar graphs show the amounts of {beta}- and {gamma}-CaM kinase II, as determined by computer-assisted scanning densitometry of the immunoblots. Values are means ± SE; n = 6 (homogenate) or 5 (SR). *P < 0.05 vs. euthyroid.

 
The results presented in Fig. 5A show that the relative amount of SERCA1 was markedly higher (~2.5-fold, P < 0.05) in soleus muscle SR from the hyperthyroid compared with euthyroid rabbits. Similar results were obtained in experiments by using unfractionated soleus muscle homogenates from euthyroid and hyperthyroid rabbits (Fig. 5B). On the other hand, there was no significant difference in the relative amounts of SERCA2 in soleus muscle SR vesicles (Fig. 6A) or homogenates (results not shown) from hyperthyroid and euthyroid rabbits. Interestingly, the relative amount of PLN was significantly decreased (50%, P < 0.05) in SR from hyperthyroid compared with euthyroid rabbits (Fig. 6B). Moreover, a significantly lower PLN-to-SERCA2 ratio was observed in the hyperthyroid rabbit soleus (Fig. 6C).



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Fig. 5. Relative amounts of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 Ca2+-ATPase isoform in soleus muscle homogenates and SR from euthyroid and hyperthyroid rabbits, as determined by Western immunoblotting analysis. Bottom: typical immunoblots obtained with 3 separate soleus muscle SR preparations (A) and 4 separate soleus muscle homogenate preparations (B) each from euthyroid and hyperthyroid rabbits. Identical amount of homogenate/SR (30 µg protein) was applied in each lane. Top: bar graphs show the relative amount of SERCA1, as determined by computer-assisted scanning densitometry of the immunoblots. Values are means ± SE; n = 5. *P < 0.05 vs. euthyroid.

 


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Fig. 6. Relative amounts of SERCA2 Ca2+-ATPase isoform and PLN in soleus muscle SR from euthyroid and hyperthyroid rabbits, as determined by Western immunoblotting analysis. Bottom: typical immunoblots of SERCA2 (A) and PLN (B) obtained by using soleus muscle SR from euthyroid and hyperthyroid rabbits. Identical amount of SR (30 µg protein) was applied in each lane. Top: bar graphs show the relative amounts of SERCA2 (A) and PLN (B), as determined by computer-assisted scanning densitometry of the immunoblots. C: ratio of PLN to SERCA2 immunnoreactive protein. Values are means ± SE; n = 6. *P < 0.05 vs. euthyroid.

 
Ca2+ sequestration function of SR Ca2+-ATPase. The influence of hyperthyroid state on soleus muscle SR Ca2+ sequestration function was studied by determining the ATP-dependent Ca2+ uptake (Ca2+ transport function) and Ca2+-activated ATP hydrolysis (energy transduction function). Figure 7 shows the time course of Ca2+ uptake by soleus muscle SR from euthyroid and hyperthyroid rabbits. The rate of Ca2+ uptake, measured in the presence of 8.2 µM free Ca2+ in the assay medium, was significantly higher (~50% increase, P < 0.05) in the hyperthyroid compared with euthyroid rabbits. Figure 8 shows the effects of varying Ca2+ concentration on ATP-supported Ca2+ uptake by soleus SR from hyperthyroid and euthyroid rabbits. When the free Ca2+ concentration in the assay medium was varied from 0.01 to 8.2 µM, SR from hyperthyroid rabbits showed significantly higher rates of Ca2+ uptake than the membranes from euthyroid rabbits at all Ca2+ concentrations. Analysis of the kinetic parameters revealed that Vmax of Ca2+ uptake was significantly higher in the hyperthyroid compared with euthyroid group; the apparent affinity of Ca2+-ATPase for Ca2+ did not differ significantly between the two groups (Table 2).



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Fig. 7. Time course of ATP-dependent Ca2+ uptake by soleus muscle SR from euthyroid and hyperthyroid rabbits. The Ca2+ uptake rates were measured with a saturating free Ca2+ concentration of 8.2 µM in the assay medium (compare Fig. 8). Values are means ± SE; n = 5. *P < 0.05 vs. euthyroid.

 


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Fig. 8. Effect of varying Ca2+ concentrations on ATP-dependent Ca2+ uptake by soleus muscle SR from euthyroid and hyperthyroid rabbits. Values are means ± SE; n = 5. The kinetic parameters of Ca2+ transport derived from these data are summarized in Table 2.

 

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Table 2. Kinetic parameters of Ca2+ transport by soleus muscle SR from euthyroid and hyperthyroid rabbits

 
Figure 9 compares the Ca2+-ATPase activity of soleus muscle SR from hyperthyroid and euthyroid rabbits, measured at a saturating concentration of free Ca2+ (8.2 µM). The Ca2+-ATPase activity was strikingly higher (~2-fold, P < 0.05) in SR vesicles from hyperthyroid compared with euthyroid rabbits. The efficiency of coupling ATP hydrolysis to Ca2+ transport remained unaltered in the hyperthyroid state (Ca2+ uptake/ATP hydrolysis at saturating free Ca2+: euthyroid, 1.77 ± 0.02; hyperthyroid, 1.72 ± 0.02; n = 5/group).



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Fig. 9. Comparison of Ca2+-activated ATP hydrolysis (Ca2+-ATPase activity) by soleus muscle SR from euthyroid and hyperthyroid rabbits. Values are means ± SE; n = 5. *P < 0.05 vs. euthyroid.

 
Effect of activation of endogenous CaM kinase II on Ca2+ uptake activity of soleus muscle SR. We have shown previously that activation of endogenous CaM kinase II in SR vesicles isolated from rabbit soleus muscle results in stimulation of Ca2+ uptake, apparently due to phosphorylation of Ca2+-ATPase (SERCA2 isoform) and PLN (14). In view of the thyroid hormone-induced alterations in the expression of CaM kinase II, Ca2+-ATPase isoform, and PLN in rabbit soleus, it was of interest to assess potential concomitant changes in the regulatory influence of CaM kinase II activation on Ca2+ uptake activity of soleus muscle SR. The following experiment was performed in this regard. SR vesicles from soleus muscle of euthyroid and hyperthyroid rabbits were preincubated in a phosphorylation reaction medium in the presence of Ca2+ and CaM to permit activation of endogenous CaM kinase II and consequent phosphorylation of Ca2+-ATPase and PLN. SR vesicles subjected to the same preincubation protocol in the absence of Ca2+/CaM in the reaction medium served as the control (unphosphorylated SR) for this experiment. Aliquots of the phosphorylated and unphosphorylated SR vesicles were subsequently transferred to the Ca2+ uptake assay medium to monitor Ca2+ uptake activity. The results showed significant stimulation of Ca2+ uptake activity in the phosphorylated SR compared with unphosphorylated SR from both euthyroid and hyperthyroid rabbit soleus (Fig. 10). This was true at subsaturating (0.6 µM) and saturating (8.2 µM) Ca2+ concentrations. However, the phosphorylation-dependent stimulation of Ca2+ uptake was much lower in the hyperthyroid (~18%) compared with euthyroid (~48%) group. The absolute Ca2+ uptake rates measured in these experiments involving a two-step incubation protocol (at 37°C) were somewhat lower than those observed under standard assay conditions (e.g., Fig. 8). It is noteworthy that the stimulation of Ca2+ uptake observed in these experiments is strictly CaM dependent.

Thyroid hormone-induced alteration of soleus muscle contractile properties. Figure 11 shows typical isometric twitches elicited in situ in the soleus muscle of euthyroid and hyperthyroid rabbits. Twitch characteristics determined from five separate experiments are summarized in Table 3. The TPF and relaxation time were significantly shorter (~30–40%) for soleus muscle of the hyperthyroid compared with euthyroid.



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Fig. 11. Typical isometric twitch of the soleus muscle in a euthyroid rabbit (A) and a hyperthyroid rabbit (B), recorded in situ.

 

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Table 3. Comparison of isometric twitch characteristics in soleus muscle of euthyroid and hyperthyroid rabbits

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The results presented in this study demonstrate that thyroid hormone-induced transition in contractile properties of rabbit soleus muscle is associated with coordinate downregulation of the expression and function of PLN and CaM kinase II ({delta}-, {beta}-, and {gamma}-isoform subunits) and selective upregulation of the expression and function of SERCA1 but not SERCA2 isoform of the SR Ca2+-ATPase. As discussed below, in addition to providing new insights into the mechanisms of thyroid hormone action with respect to the contractile phenotype transition of skeletal muscle, the current observations point to unique, interactive roles for PLN and CaM kinase II in the SERCA2 isoform-specific physiological regulation of SR Ca2+ pump function.

Mechanisms of enhanced Ca2+ sequestering activity of hyperthyroid rabbit soleus SR. Evidence from previous immunohistochemical studies (31, 54) indicated that, in the rat soleus, thyroid hormone-induced conversion of muscle fibers from slow-twitch to fast-twitch phenotype is accompanied by an isoform shift in the SR Ca2+-ATPase from SERCA2 to SERCA1 isoform. It was suggested that an increased percentage of fibers expressing SERCA1 Ca2+-ATPase isoform, as well as enhanced SERCA1 protein expression per muscle fiber, contributed to the overall increase in SERCA1 in the hyperthyroid rat soleus (54). In the present study, we observed overexpression of SERCA1 Ca2+-ATPase isoform in hyperthyroid rabbit soleus muscle using Western blotting of muscle homogenates as well as isolated SR vesicles. We found no significant change in the SERCA2 Ca2+-ATPase isoform expression in the hyperthyroid rabbit soleus, whereas a minor increase in SERCA2 expression was reported in hyperthyroid rat soleus (31). The mechanism underlying the apparently selective thyroid hormone stimulation of SERCA1 isoform expression in rabbit soleus is unclear. Thyroid hormone treatment has been shown to result in a strikingly large elevation of the mRNA levels of the SERCA1 isoform (150-fold), relative to the mRNA levels of the SERCA2 isoform (3.5-fold), in rat soleus (44). It is possible that SERCA1 gene expression is repressed in soleus, due to either intrinsic factors or the activity pattern of slow muscle, and the thyroid hormone may act by relieving this repression (44).

Another key finding in the present study is that thyroid hormone causes significantly decreased expression of the Ca2+-ATPase inhibitor protein PLN in rabbit soleus. PLN is generally coexpressed with the SERCA2, but not SERCA1, isoform of the Ca2+-ATPase (23, 43, 50). As noted in the Introduction, all previous studies on the mechanistic basis of thyroid hormone-induced shift in muscle fiber phenotype utilized rat soleus muscle, which naturally lacks PLN. Thus the present study is the first to explore, at the protein level, the influence of thyroid hormone in a slow-twitch skeletal muscle where SERCA2 and PLN coinhabit. The observed downregulation of PLN in the hyperthyroid rabbit soleus occurs in the face of unaltered expression of SERCA2. Consequently, the PLN-to-SERCA2 ratio is decreased, implying that more SERCA2 units can operate free of the inhibitory control by PLN. Despite the intensive studies spanning over the last three decades (for reviews, see Refs. 23, 43), the molecular mechanism of regulation of SERCA2 by PLN still remains unclear. According to the view that has prevailed until recently, a physical interaction of dephospho-PLN with SERCA2 causes inhibition of Ca2+ pump activity, and phosphorylation of PLN (by PKA or CaM kinase II) disrupts this protein-protein interaction, thereby relieving the inhibitory action of PLN. This view has been dispelled by the recent observation that Ca2+ causes dissociation of the PLN-SERCA2 complex in the absence of PLN phosphorylation (2). Because Ca2+ pump function cannot be measured in the absence of Ca2+, this makes it difficult, if not impossible, to determine experimentally the true impact of the physical interaction between PLN and SERCA2 on SERCA2 function. Recent work in our laboratory on PLN regulation of SERCA2 in cardiac SR has suggested that the dissociation of the PLN-Ca2+-ATPase complex is not an autonomous function of Ca2+; rather, it is governed by Ca2+/CaM interaction (35). The potential interplay of CaM-dependent processes other than phosphorylation on the regulation of SERCA2 in soleus muscle remains to be evaluated. As demonstrated in the present study, an abundance of SERCA1 coexists with PLN in the hyperthyroid rabbit soleus. PLN has been shown to be capable of inhibiting SERCA1 when coexpressed in a heterologous system (26, 47). The decreased expression of PLN makes it unlikely that overexpressed SERCA1 is subject to regulation by PLN in the hyperthyroid rabbit soleus.

Analysis of the kinetic properties of the SR Ca2+ transport system revealed markedly enhanced Vmax of Ca2+ sequestration with unaltered apparent affinity of the transport system for Ca2+ in the hyperthyroid soleus. The enhanced Vmax can be attributed mainly to the increase in Ca2+ pump units due to overexpression of SERCA1. Enhanced activity of preexisting SERCA2, due to diminished inhibitory control by PLN, may also contribute to the enhanced SR Ca2+ pump activity of the hyperthyroid soleus. The lack of alteration in Ca2+ affinity reflects the essentially similar intrinsic Ca2+ binding affinities of the SERCA1 and SERCA2 isoforms of the Ca2+-ATPase (28). It is also noteworthy that thyroid hormone did not influence the energetic efficiency of the transport system as the ratio of Ca2+ ion transport to Ca2+-activated ATP hydrolysis remained virtually unaltered in the hyperthyroid soleus SR.

Diminished CaM kinase II regulation of SR Ca2+ pump function in hyperthyroid rabbit soleus. Our results revealed a significant decrease in the endogenous CaM kinase II-mediated phosphorylation of Ca2+-ATPase and PLN in the hyperthyroid rabbit soleus. It is unlikely that differences in the preexisting level of substrate phosphorylation contribute to the thyroid hormone-induced diminished CaM kinase II-medicated SR protein phosphorylation reported here (see Fig. 2). The observed decrease in Ca2+-ATPase and PLN phosphorylation by endogenous CaM kinase II in the hyperthyroid state was accompanied by markedly diminished protein expression levels of all three subunits of CaM kinase II, i.e., {delta}-, {beta}-, and {gamma}-isoforms. Previous studies have established that the SR-bound CaM kinase II in skeletal muscle is a heteromultimeric complex composed of {delta}-, {gamma}-, and muscle-specific {beta}-subunits (3, 9, 42, 52). Therefore, the diminished protein levels of all three subunits in the hyperthyroid state imply diminished levels of functional holoenzyme units. The decreased protein phosphorylation level of PLN is associated, in part, with the downregulated PLN expression. However, the decrease in phosphorylation of both PLN and Ca2+-ATPase was also evident when phosphorylation was quantified per unit amount of each of the immunoreactive substrates, suggesting that thyroid hormone-induced downregulation of CaM kinase II also contributes to the diminished substrate phosphorylation. The phosphorylation of SR Ca2+-ATPase observed in this study confines solely to the SERCA2 isoform, the expression of which remained unaltered in the hyperthyroid soleus. Although the SERCA1 isoform is found to be abundantly expressed in the hyperthyroid soleus, it is not a substrate for CaM kinase II (14). This isoform-specific phosphorylation of the Ca2+-ATPase by CaM kinase II is due to a unique difference in the primary structure of the two isoforms, i.e., SERCA2 but not SERCA1 contains a phosphorylatable serine residue in position 38 of its amino acid sequence (14, 53).

Previous studies have reported quantitative differences in the holoenzyme content and subunit composition of CaM kinase II in slow- vs. fast-twitch rabbit skeletal muscle. Thus the fast-twitch skeletal muscle has been found to contain relatively high levels of CaM kinase II holoenzyme as well as {delta}- and {gamma}-subunits; the slow-twitch muscle, on the other hand, has been shown to contain a relatively high level of {beta}-subunit but low levels of holoenzyme as well as {delta}- and {gamma}-subunits (9, 42). Given such muscle-specific differences, the observed downregulation CaM kinase II protein expression (holoenzyme as well as all three subunits) during slow- to fast-twitch phenotype transition induced by thyroid hormone seems confounding. However, presently, little is known about CaM kinase II substrates in fast muscle SR and the role of CaM kinase II in the regulation of fast muscle SR function. In the case of slow-twitch rabbit soleus, CaM kinase II has been shown to phosphorylate both PLN and SERCA2, resulting in stimulation of SR Ca2+ pump function (14). In this context, the coordinate downregulation of both CaM kinase II and PLN expression can be viewed as a physiological adaptation to prevent overdriving of the SR Ca2+ cycling apparatus. Alternatively, the downregulation of CaM kinase II may reflect the initial stage of transition of slow-twitch type I fibers to the fast-oxidative type IIa fibers following thyroid hormone treatment. In this regard, it is noteworthy that SR isolated from fast-oxidative masseter muscle of the rabbit was found to contain much less CaM kinase II than SR isolated from fast-glycolytic adductor muscle (42). Also, evidence from a recent study has suggested that, in the hyperthyroid state, rat soleus muscle may temporarily acquire unique contractile properties distinct from normal fast and slow fibers (60).

Previous studies have documented that phosphorylation of SERCA2 by CaM kinase II is associated with enhanced Vmax of Ca2+ transport (14, 55, 57, 58), whereas PLN phosphorylation is associated with enhanced Ca2+ binding affinity of the Ca2+-ATPase (23, 43). In the present study, we have observed significant stimulation of Ca2+ sequestration by SR from both euthyroid and hyperthyroid soleus on activation of endogenous CaM kinase II. This stimulatory effect is observed when Ca2+ uptake of SR is measured at subsaturating and saturating concentrations of Ca2+. However, the magnitude of stimulation of Ca2+ uptake is markedly lower in the hyperthyroid compared with euthyroid. Thus the diminished stimulatory effect of CaM kinase activation on Ca2+ uptake by soleus SR in the hyperthyroid state correlates well with the diminished phosphorylation of SERCA2 and PLN.

Relationship between altered SR function and contractile properties in the hyperthyroid rabbit soleus. Thyroid hormone-induced changes in contractile properties of the rabbit soleus muscle reported here include strikingly enhanced isometric TF and decrements in TPF as well as relaxation time. Similar effects of thyroid hormone on twitch characteristics of the rat soleus were reported in previous studies (25, 36). This transition of soleus muscle from the slow-twitch to fast-twitch phenotype involved conversion of the slow MHC (MHC I) to fast MHC (MHC II) isoform at the level of the myofilaments (6, 19, 30, 44), which may account for the enhanced velocity of contraction and decreased TPF. On the other hand, the increase in speed of muscle relaxation can be attributed to the enhanced velocity of cytoplasmic Ca2+ sequestration by the SR, a process facilitated by 1) overexpression of SERCA1 isoform of the Ca2+-ATPase, 2) preservation of the SERCA2 Ca2+ pump units, and 3) attenuation of the inhibitory constraint on Ca2+ pump function through downregulated PLN expression. The increased Vmax of Ca2+ uptake by SR in the hyperthyroid state reflects enhanced Ca2+ storage capacity of this intracellular Ca2+ reservoir. Because the rate of Ca2+ release from the SR is dependent on intraluminal Ca2+ load (12, 46), enhanced Ca2+ storage may favor an increased rate of Ca2+ release during excitation, and this, in turn, may produce the increase in TF of the soleus in the hyperthyroid state.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Canadian Institutes of Health Research Grant MT9553 (to N. Narayanan). M. Jiang is the recipient of a Canadian Institutes of Health Research Graduate Student Scholarship award.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. P. Karczewski, Max Delbruck Center for Molecular Medicine, for the generous gift of {delta}-CaM kinase II polyclonal antibody.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. Narayanan, Dept. of Physiology and Pharmacology, Health Science Center, The Univ. of Western Ontario, London, Ontario, Canada N6A 5C1 (E-mail: njanoor.narayanan{at}fmd.uwo.ca).

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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