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
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ABSTRACT |
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calmodulin kinase II; phospholamban ; calcium ion-adenosinetriphosphatase; sarcoplasmic reticulum
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 (
,
,
, and
) (4). In skeletal muscle, the
,
, and muscle-specific
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 (5759). 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 -,
-, and
-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.
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METHODS |
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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). [-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-
-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
- and
- but not
-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 418% 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 -,
-,
-subunits of CaM kinase II [polyclonal (56), dilution 1:1,000 for
and 1:500 for
/
]. 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 1040 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·min1·nmol1). 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
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The incubation medium used for the assay of Mg2+-dependent Ca2+-ATPase activity was identical to that described for Ca2+ uptake, except that [-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 [
-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 [-32P]ATP (specific activity 200300 counts·min1·pmol1), and SR (25 µg of protein). The phosphorylation reaction was initiated by the addition of [
-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 418% 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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RESULTS |
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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 (3040%) for soleus muscle of the hyperthyroid compared with euthyroid.
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DISCUSSION |
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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., -,
-, and
-isoforms. Previous studies have established that the SR-bound CaM kinase II in skeletal muscle is a heteromultimeric complex composed of
-,
-, and muscle-specific
-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 - and
-subunits; the slow-twitch muscle, on the other hand, has been shown to contain a relatively high level of
-subunit but low levels of holoenzyme as well as
- and
-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.
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ACKNOWLEDGMENTS |
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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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