Exercise training differentially modifies age-associated alteration in expression of Na+-K+-ATPase subunit isoforms in rat skeletal muscles

The present study tests the hypothesis that endurance exercise training (ETr) reverses age-associated alterations in expression of Na+-K+-ATPase subunit isoforms in rat skeletal muscles. Expression of the isoforms was examined in 16-mo-old sedentary middle-aged, 29-mo-old sedentary senescent, and 29-mo-old treadmill exercise-trained senescent Fischer 344 x Brown Norway rats. Levels of the alpha1-isoform increased with age in red gastrocnemius (GR), white gastrocnemius (GW), and extensor digitorum longus (EDL) muscles, and ETr further increased its levels. Levels of the alpha2-isoform were unchanged in GR, had a strong trend for a decrease in GW, and decreased significantly in EDL. ETr increased expression of the alpha2-isoform in all three muscle groups. There was no increase in expression of the beta1-isoform in GR, GW, or EDL with age, whereas ETr markedly increased its levels in the muscles. There was a marked decrease with age in expression of the beta2-isoform in the muscle groups that was not reversed by ETr. By contrast, beta3-isoform levels increased with age in GR and GW, and ETr was able to reverse this increase. Na+-K+-ATPase enzyme activity was unchanged with age in GR and GW but increased in EDL. ETr increased enzyme activity in GR and GW and did not change in EDL. Myosin heavy chain isoforms in the muscle groups did not change significantly with age; ETr caused a general shift toward more oxidative fibers. Thus ETr differentially modifies age-associated alterations in expression of Na+-K+-ATPase subunit isoforms, and a mechanism(s) other than physical inactivity appears to play significant role in some of the age-associated changes.     

Alpha-catalytic subunits of 5'AMP-activated protein kinase display fiber-specific expression and are upregulated by chronic low-frequency stimulation in rat muscle

5'-AMP-activated protein kinase (AMPK) signaling initiates adaptive changes in skeletal muscle fibers that restore homeostatic energy balance. The purpose of this investigation was to examine, in rats, the fiber-type protein expression patterns of the alpha-catalytic subunit isoforms in various skeletal muscles, and changes in their respective contents within the tibialis anterior (TA) after chronic low-frequency electrical stimulation (CLFS; 10 Hz, 10 h daily), applied for 4 +/- 1.2 or 25 +/- 4.8 days. Immunocytochemical staining of soleus (SOL) and medial gastrocnemius (MG) showed that 86 +/- 4.1 to 97 +/- 1.4% of type IIA fibers stained for both the alpha1- and alpha2-isoforms progressively decreased to 63 +/- 12.2% of type IID/X and 9 +/- 2.4% of IIB fibers. 39 +/- 11.4% of IID/X and 83 +/- 7.9% of IIB fibers expressed only the alpha2 isoform in the MG, much of which was localized within nuclei. alpha1 and alpha2 contents, assessed by immunoblot, were lowest in the white gastrocnemius [WG; 80% myosin heavy chain (MHC) IIb; 20% MHCIId/x]. Compared with the WG, alpha1 content was 1.6 +/- 0.08 (P < 0.001) and 1.8 +/- 0.04 (P < 0.0001)-fold greater in the red gastrocnemius (RG: 13%, MHCIIa) and SOL (21%, MHCIIa), respectively, and increased in proportion to MHCIIa content. Similarly, alpha2 content was 1.4 +/- 0.10 (P < 0.02) and 1.5 +/- 0.07 (P < 0.001)-fold greater in RG and SOL compared with WG. CLFS induced 1.43 +/- 0.13 (P < 0.007) and 1.33 +/- 0.08 (P < 0.009)-fold increases in the alpha1 and alpha2 contents of the TA and coincided with the transition of faster type IIB and IID/X fibers toward IIA fibers. These findings indicate that fiber types differ with regard to their capacity for AMPK signaling and that this potential is increased by CLFS.     

Age-associated differential expression of Na(+)-K(+)-ATPase subunit isoforms in skeletal muscles of F-344/BN rats.

Skeletal muscle expresses multiple isoforms of the Na(+)-K(+)-ATPase. Their expression has been shown to be differentially regulated under pathophysiological conditions. In addition, previous studies suggest possible age-dependent alterations in Na(+)-K(+) pump function. The present study tests the hypothesis that advancing age is associated with altered Na(+)-K(+)-ATPase enzyme activity and isoform-specific changes in expression of the enzyme subunits. Red and white gastrocnemius (Gast) as well as soleus muscles of male Fischer 344/Brown Norway (F-344/BN) rats at 6, 18, and 30 mo of age were examined. Na(+)-K(+)-ATPase activity, measured by K(+)-stimulated 3-O-methylfluorescein phosphatase activity, increased by approximately 50% in a mixed Gast homogenate from 30-mo-old compared with 6- and 18-mo-old rats. Advancing age was associated with markedly increased alpha(1)- and beta(1)-subunit, and decreased alpha(2)- and beta(2)-subunit in red and white Gast. In soleus, there were similar changes in expression of alpha(1)- and alpha(2)-subunits, but levels of beta(1)-subunit were unchanged. Functional Na(+)-K(+)-ATPase units, measured by [(3)H]ouabain binding, undergo muscle-type specific changes. In red Gast, high-affinity ouabain-binding sites, which are a measure of alpha(2)-isozyme, increased in 30-mo-old rats despite decreased levels of alpha(2)-subunit. In white Gast, by contrast, decreased levels of alpha(2)-subunit were accompanied by decreased high-affinity ouabain-binding sites. Finally, patterns of expression of the four myosin heavy chain (MHC) isoforms (type I, IIA, IIX, and IIB) in these muscles were similar in the three age groups examined. We conclude that, in the skeletal muscles of F-344/BN rats, advancing age is associated with muscle type-specific alterations in Na(+)-K(+)-ATPase activity and patterns of expression of alpha- and beta-subunit isoforms. These changes apparently occurred without obvious shift in muscle fiber types, since expression of MHC isoforms remained unchanged. Some of the alterations occurred between middle-age (18 mo) and senescence (30 mo), and, therefore, may be attributed to aging of skeletal muscle.     

Effects of thyroid state on AMP-activated protein kinase and acetyl-CoA carboxylase expression in muscle.

AMP-activated protein kinase (AMPK) consists of three subunits: alpha, beta, and gamma. Two isoforms exist for the alpha-subunit (alpha(1) and alpha(2)), two for the beta-subunit (beta(1) and beta(2)), and three for the gamma-subunit (gamma(1), gamma(2), and gamma(3)). Although the specific roles of the beta- and gamma-subunits are not well understood, the alpha-subunit isoforms contain the catalytic site and also the phosphorylation/activation site for the upstream kinase. This study was designed to determine the role of thyroid hormones in controlling expression levels of these AMPK subunits and of one downstream target, acetyl-CoA carboxylase (ACC), in muscle. AMPK subunit and ACC levels were determined by Western blots in control rats, in rats given 0.01% propylthiouracil (PTU) in drinking water for 3 wk, and in rats given 3 mg of thyroxine and 1 mg of triiodothyronine per kilogram chow for 1 or 3 wk. In gastrocnemius muscle, all isoforms of AMPK subunits were significantly increased in rats given thyroid hormones for 3 wk vs. those treated with PTU. Similar patterns were seen in individual muscle types. Expression of muscle ACC was also significantly increased in response to 3 wk of treatment with excess thyroid hormones. Muscle content of malonyl-CoA was elevated in PTU-treated rats and depressed in thyroid hormone-treated rats. These data provide evidence that skeletal muscle AMPK subunit and ACC expression is partially under the control of thyroid hormones.     

Fiber-type specific expression of α-actinin isoforms in rat skeletal muscle

α-Actinins are actin-binding proteins, and two isoforms (α-actinin-2 and -3) are major structural components of the sarcomeric Z line in mammalian skeletal muscle. Based on human and knockout mice studies, α-actinin-3 is thought to be associated with muscle force output and high contraction velocities. However, fiber-type specific expression of α-actinin isoforms is not well understood and may vary among species. In this study, we investigated the expression of α-actinin isoforms and the difference between fiber types in rat skeletal muscle and compared it with those of humans and mice from previous reports. Soleus and plantaris muscles were analyzed immunohistochemically to identify muscle fiber types and α-actinin protein expression. α-Actinin-2 was stained in all muscle fibers in both the soleus and plantaris muscles; i.e., all α-actinin-3 co-expressed with α-actinin-2 in rat skeletal muscles. The proportions of α-actinin-3 expression, regardless of fiber type, were significantly higher in the plantaris (75.8 ± 0.6%) than the soleus (8.0 ± 1.7%). No α-actinin-3 expression was observed in type I fibers, whereas all type IIx+b fibers expressed α-actinin-3. α-Actinin-3 was also expressed in type IIa fibers; however, approximately 75% of type IIa fibers were not stained by α-actinin-3, and the proportion varied between muscles. The proportion of α-actinin-3 expression in type IIa fibers was significantly higher in the soleus muscle than the plantaris muscle. Our results showed that fiber-type specific expression of α-actinin isoforms in rats is more similar to that in humans compared to that of the mouse, whereas the proportion of α-actinin-3 protein varied between muscles.     

Insulin induces translocation of the alpha 2 and beta 1 subunits of the Na+/K(+)-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle.

Unlike glucose transport, where translocation of the insulin-responsive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane is the principal mechanism underlying insulin stimulation, no consensus exists presently for the mechanism by which insulin activates the Na+/K(+)-ATPase. We have investigated (i) the subunit isoforms expressed and (ii) the effect of insulin on the subcellular distribution of the alpha beta isoforms of the Na+/K(+)-ATPase in plasma membranes (PM) and internal membranes (IM) from rat skeletal muscle. Western blot analysis, using isoform-specific antibodies to the various subunits of the Na+/K(+)-ATPase, revealed that skeletal muscle PM contains the alpha 1 and alpha 2 catalytic subunits and the beta 1 and beta 2 subunits of the Na+ pump. Skeletal muscle IM were enriched in alpha 2, beta 1, and beta 2; alpha 1 was barely detectable in this fraction. After insulin treatment, alpha 2 content in the PM increased, with a parallel decrease in its abundance in the IM pool; insulin did not have any effect on alpha 1 isoform amount or subcellular distribution. The beta 1 subunit, but not beta 2, was also elevated in the PM after insulin treatment, but this increase originated from a sucrose gradient fraction different from that of the alpha 2 subunit. Our findings suggest that insulin induces an isoform-specific translocation of Na+ pump subunits from different intracellular sources to the PM and that the hormone-responsive enzyme in rat skeletal muscle is an alpha 2:beta 1 dimer.     

Expression of multiple Na+,K(+)-ATPase genes reveals a gradient of isoforms along the nonpigmented ciliary epithelium: functional implications in aqueous humor secretion.

The Na+,K(+)-ATPase alpha 1, alpha 2, and alpha 3 subunit isoforms have been shown to be differentially expressed in the nonpigmented (NPE) and pigmented (PE) cells of the ocular ciliary epithelium (CE) (Martin-Vasallo et al., J. Cell. Physiol., 141:243-252, 1989; Ghosh et al., J. Biol. Chem., 265:2935-2940, 1990). In this study we analyzed and compared the pattern of expression of the multiple Na+,K(+)-ATPase alpha (alpha 1, alpha 2, alpha 3) subunit genes with the pattern of expression of the Na+,K(+)-ATPase beta (beta 1, beta 2) subunit genes along the bovine CE. We have selected three regions in the CE, referred to as 1) the anterior region of the pars plicata, near the iris; 2) the middle region of the pars plicata; and 3) the posterior region of the pars plana, near the ora serrata. Using isoform-specific cDNA probes and antibodies for the Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunits on Northern and Western blot analysis, we found that mRNA and polypeptides are expressed in all three CE regions with different abundance. The pattern of expression of alpha and beta isoforms detected along the NPE cell layers suggests a gradient of alpha 1, alpha 2, alpha 3, beta 1, and beta 2 mRNAs and polypeptides that correlates with decreasing Na+,K(+)-ATPase activity from the most anterior region at the pars plicata towards the posterior region at the ora serrata. We also found marked differences in the pattern of immunolocalization of Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunit isoforms in different regions of the CE. In the anterior region, NPE cells stained intensely at the basal lateral membrane with specific monoclonal and polyclonal antibodies for each of the alpha (alpha 1, alpha 2, alpha 3) and beta (beta 1, beta 2) Na,K-ATPase isoforms. In the middle and posterior regions of the CE, NPE cells showed lower or absent levels of staining with alpha 1, alpha 2, alpha 3, and beta 1 antibodies, although staining with beta 2 was abundant. In contrast, PE cells throughout the CE were stained at the basal lateral membrane by antibodies to alpha 1 and beta 1, while no staining signals were detected with the rest of the antibodies (i.e. alpha 2, alpha 3, and beta 2). Our results support the conclusion that the three alpha and two beta isoforms of the Na+,K(+)-ATPase are differentially expressed in the two cell layers that make up the CE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression profiling of the gamma-subunit isoforms of AMP-activated protein kinase suggests a major role for gamma3 in white skeletal muscle

Expression patterns of the three isoforms of the regulatory gamma-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The gamma3-isoform appeared highly specific to skeletal muscle, whereas gamma1 and gamma2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of gamma3. In samples of white skeletal muscle, gamma3 clearly appeared to be the most abundant gamma-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of gamma3 mRNA expression, whereas levels of gamma1 and gamma2 remained largely unchanged. However, even in these cultured myotubes, gamma2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a gamma3-specific antibody showed that gamma3 was exclusively associated with the alpha2- and beta2-subunit isoforms. The observation that the AMPKgamma3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant gamma-isoform, strongly suggests that gamma3 has a key role in this tissue.     

Training-induced changes in skeletal muscle Na+-K+ pump number and isoform expression in rats with chronic heart failure.

The mechanisms responsible for the decrements in exercise performance in chronic heart failure (CHF) remain poorly understood, but it has been suggested that sarcolemmal alterations could contribute to the early onset of muscular fatigue. Previously, our laboratory demonstrated that the maximal number of ouabain binding sites (B(max)) is reduced in the skeletal muscle of rats with CHF (Musch TI, Wolfram S, Hageman KS, and Pickar JG. J Appl Physiol 92: 2326-2334, 2002). These reductions may coincide with changes in the Na(+)-K(+)-ATPase isoform (alpha and beta) expression. In the present study, we tested the hypothesis that reductions in B(max) would coincide with alterations in the alpha- and beta-subunit expression of the sarcolemmal Na(+)-K(+)-ATPase of rats with CHF. Moreover, we tested the hypothesis that exercise training would increase B(max) along with producing significant changes in alpha- and beta-subunit expression. Rats underwent a sham operation (sham; n = 10) or a surgically induced myocardial infarction followed by random assignment to either a control (MI; n = 16) or exercise training group (MI-T; n = 16). The MI-T rats performed exercise training (ET) for 6-8 wk. Hemodynamic indexes demonstrated that MI and MI-T rats suffered from severe left ventricular dysfunction and congestive CHF. Maximal oxygen uptake (Vo(2 max)) and endurance capacity (run time to fatigue) were reduced in MI rats compared with sham. B(max) in the soleus and plantaris muscles and the expression of the alpha(2)-isoform of the Na(+)-K(+)-ATPase in the red portion of the gastrocnemius (gastrocnemius(red)) muscle were reduced in MI rats. After ET, Vo(2 max) and run time to fatigue were increased in the MI-T group of rats. This coincided with increases in soleus and plantaris B(max) and the expression of the alpha(2)-isoform in the gastrocnemius(red) muscle. In addition, the expression of the beta(2)-isoform of the gastrocnemius(red) muscle was increased in the MI-T rats compared with their sedentary counterparts. This study demonstrates that CHF-induced alterations in skeletal muscle Na(+)-K(+)-ATPase, including B(max) and isoform expression, can be partially reversed by ET.     

Nonuniform changes in arteriolar myogenic tone within skeletal muscle following hindlimb unweighting.

Hindlimb unweighting (HLU) has been shown to alter myogenic tone distinctly in arterioles isolated from skeletal muscles composed predominantly of fast-twitch (white gastrocnemius) compared with slow-twitch (soleus) fibers. Based on these findings, we hypothesized that HLU would alter myogenic tone differently in arterioles isolated from distinct fiber-type regions within a single skeletal muscle. We further hypothesized that alterations in myogenic tone would be associated with alterations in voltage-gated Ca(2+) channel current (VGCC) density of arteriolar smooth muscle. After 14 days of HLU or weight bearing (control), first-order arterioles were isolated from both fast-twitch and mixed fiber-type regions of the gastrocnemius muscle, cannulated, and pressurized at 90 cmH(2)O. Mixed gastrocnemius arterioles of HLU rats demonstrated increased spontaneous tone [43 +/- 5% (HLU) vs. 27 +/- 4% (control) of possible constriction] and an approximately twofold enhanced myogenic response when exposed to step changes in intraluminal pressure (10-130 cmH(2)O) compared with control rats. In contrast, fast-twitch gastrocnemius arterioles of HLU rats demonstrated similar levels of spontaneous tone [6 +/- 2% (HLU) vs. 6 +/- 2% (control)] and myogenic reactivity to control rats. Neither KCl-induced contractile responses (10-50 mM KCl) nor VGCC density was significantly different between mixed gastrocnemius arterioles of HLU and control rats. These results suggest that HLU produces diverse adaptations in myogenic reactivity of arterioles isolated from different fiber-type regions of a single skeletal muscle. Furthermore, alterations in myogenic responses were not attributable to altered VGCC density.     

Isoforms of Na+, K+-ATPase in human prostate; specificity of expression and apical membrane polarization

The cellular distribution of Na+, K+-ATPase subunit isoforms was mapped in the secretory epithelium of the human prostate gland by immunostaining with antibodies to the alpha and beta subunit isoforms of the enzyme. Immunolabeling of the alpha1, beta1 and beta2 isoforms was observed in the apical and lateral plasma membrane domains of prostatic epithelial cells in contrast to human kidney where the alpha1 and beta1 isoforms of Na+, K+-ATPase were localized in the basolateral membrane of both proximal and distal convoluted tubules. Using immunohistochemistry and PCR we found no evidence of Na+, K+-ATPase alpha2 and alpha3 isoform expression suggesting that prostatic Na+, K+-ATPase consists of alpha1/beta1 and alpha1/beta2 isozymes. Our immunohistochemical findings are consistent with previously proposed models placing prostatic Na+, K+-ATPase in the apical plasma membrane domain. Abundant expression of Na+, K+-ATPase in epithelial cells lining tubulo-alveoli in the human prostate gland confirms previous conclusions drawn from biochemical, pharmacological and physiological data and provides further evidence for the critical role of this enzyme in prostatic cell physiology and ion homeostasis. Na+, K+-ATPase most likely maintains an inwardly directed Na+ gradient essential for nutrient uptake and active citrate secretion by prostatic epithelial cells. Na+, K+-ATPase may also regulate lumenal Na+ and K+, major counter-ions for citrate.     

Expression of Na+,K(+)-ATPase alpha and beta subunit isoforms in the motor neurons of the rat spinal cord

The data concerning the distribution of Na+,K(+)-ATPase alpha and beta subunit isoforms in the spinal cord and partly in the motor neurons of the ventral horns are limited. The lumbo-sacral portion of the spinal cord of adult rats was immunotested with polyclonal antibodies (UBI, NY) specific for alpha 1, alpha 2, alpha 3 and beta 1, beta 2 isoforms. After paraformaldehyde perfusion and postfixation, free-floating 50 microns thick vibratome sections were immunostained with Vectastatin Elite ABC. Sites of bound primary antibodies were visualized by incubation in DAB-H2O2 substrate medium. The histochemical technique revealed immunostaining for all five isoforms of Na+,K(+)-ATPase in the motor neurons. The findings show a principal similarity in the distribution pattern of the immuno-like reactivity for alpha 1 and alpha 2 isoforms, the staining of the pericarya being more or less continuous with that of the microenvironment. The immunostaining for beta 2 (in comparison with alpha 1 and alpha 2) outlines the pericarya of the motor neurons slightly better, whereas the staining for beta 1 outlines them extremely sharply. The immunostaining pattern for the alpha 3 isoform differs considerably from that for the other isoforms. The immuno-like reactivity for this isoform is concentrated at the surface of the pericarya and processes of the motor neurons. Accumulation of alpha 3 immunoreactivity on the surface of the motor neurons might reflect the intensive traffic of the alpha 3 isoform from the pericaryon to the plasma membrane and the processes of the neurons. The findings from the investigations performed here support the opinion, that, in addition to the conventional catalytic role in Na+,K(+)-ATPase activity, Na+,K(+)-ATPase isozymes play a part in different specific phenomena in the nervous system.     

Vascular smooth muscle expresses a truncated Na+, K(+)-ATPase alpha-1 subunit isoform.

By regulating transmembrane Na+ and K+ concentrations and membrane potential, the Na+,K(+)-ATPase plays an important role in regulating cardiac, skeletal, and smooth muscle function. A high degree of amino acid sequence and structural identity characterizes the three Mr 100,000 Na+,K(+)-ATPase alpha subunit isoforms expressed in cardiac and skeletal muscle. Strikingly, vascular smooth muscle utilizes alternative RNA processing of the alpha-1 gene to express a structurally distinct Mr approximately 65,000 isoform, alpha 1-T (truncated). Analysis of both its mRNA and protein structure reveals that alpha-1-T represents a major, evolutionarily conserved, truncated Na+,K(+)-ATPase isoform expressed in vascular smooth muscle. This demonstrates an unexpected complexity in the regulation of vascular smooth muscle Na+,K(+)-ATPase gene expression and suggests that a structurally novel, truncated alpha subunit may play a role in vascular smooth muscle active ion transport.     

Na+,K+-ATPase trafficking in skeletal muscle: insulin stimulates translocation of both alpha 1- and alpha 2-subunit isoforms

We determined insulin-stimulated Na(+),K(+)-ATPase isoform-specific translocation to the skeletal muscle plasma membrane. When rat muscle plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of alpha(2)- but not alpha(1)-subunits was detected. However, using cell surface biotinylation techniques, an insulin-induced membrane translocation of both alpha(1) and alpha(2)-subunits in rat epitrochlearis muscle and cultured human skeletal muscle cells was noted. Na(+),K(+)-ATPase alpha-subunit translocation was abolished by the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, as well as by the protein kinase C inhibitor GF109203X. Thus, insulin mediates Na(+),K(+)-ATPase alpha(1)- and alpha(2)-subunit translocation to the skeletal muscle plasma membrane via a PI 3-kinase-dependent mechanism.     

Examination of the calcium-modulated protein S100 alpha and its target proteins in adult and developing skeletal muscle.

In this study radioimmunoassay, immunohistochemistry, Northern blot analysis, and a gel overlay technique have been used to examine the level, subcellular distribution, and potential target proteins of the S100 family of calcium-modulated proteins in adult and developing rat skeletal muscles. Adult rat muscles contained high levels of S100 proteins but the particular form present was dependent on the muscle type: cardiac muscle contained exclusively S100 alpha, slow-twitch skeletal muscle fibers contained predominantly S100 alpha, vascular smooth muscle contained both S100 alpha and S100 beta, and fast-twitch skeletal muscle fibers contained low but detectable levels of S100 alpha and S100 beta. While the distribution of S100 mRNAs paralled the protein distribution in all muscles there was no direct correlation between the mRNA and protein levels in different muscle types, suggesting that S100 protein expression is differentially regulated in different muscle types. Immunohistochemical analysis of the cellular distribution of S100 proteins in adult skeletal muscles revealed that S100 alpha staining was associated with muscle cells, while S100 beta staining was associated with nonmuscle cells. Radioimmunoassays of developing rat skeletal muscles demonstrated that all developing muscles contained low levels of S100 alpha at postnatal day 1 and that as development proceeded the S100 alpha levels increased. In contrast to adult muscle S100 alpha expression was confined to fast-twitch fibers in developing skeletal muscle until postnatal day 21. At postnatal day 1, developing contractile elements were S100 alpha positive, but no staining periodicity was detectable. At postnatal day 21, S100 alpha exhibited the same subcellular localization as seen in the adult: colocalization with the A-band and/or longitudinal sarcoplasmic reticulum. Comparison of the S100 alpha-binding protein profiles in fast- and slow-twitch fibers of various species revealed few, if any, species- or fiber type-specific S100 binding proteins. Isolated sarcoplasmic reticulum fractions and myofibrils contained multiple S100 alpha-binding proteins. The colocalization of S100 alpha and S100 alpha-binding proteins with the contractile apparatus and sarcoplasmic reticulum suggest that S100 alpha may regulate excitation and/or contraction in slow-twitch fibers.     

Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass

The present study tested the hypothesis that exercise with a large compared with a small active muscle mass results in a higher contraction-induced increase in Na(+)-K(+)-ATPase mRNA expression due to greater hormonal responses. Furthermore, the relative abundance of Na(+)-K(+)-ATPase subunit alpha(1), alpha(2), alpha(3), alpha(4), beta(1), beta(2), and beta(3) mRNA in human skeletal muscle was investigated. On two occasions, eight subjects performed one-legged knee extension exercise (L) or combined one-legged knee extension and bilateral arm cranking (AL) for 5.00, 4.25, 3.50, 2.75, and 2.00 min separated by 3 min of rest. Leg exercise power output was the same in AL and L, but heart rate at the end of each exercise interval was higher in AL compared with L. One minute after exercise, arm venous blood lactate was higher in AL than in L. A higher level of blood epinephrine and norepinephrine was evident 3 min after exercise in AL compared with L. Nevertheless, none of the exercise-induced increases in alpha(1), alpha(2), beta(1), and beta(3) mRNA expression levels were higher in AL compared with L. The most abundant Na(+)-K(+)-ATPase subunit at the mRNA level was beta(1), which was expressed 3.4 times than alpha(2). Expression of alpha(1), beta(2), and beta(3) was less than 5% of the alpha(2) expression, and no reliable detection of alpha(3) and alpha(4) was possible. In conclusion, activation of additional muscle mass does not result in a higher exercise-induced increase in Na(+)-K(+)-ATPase subunit-specific mRNA.