The major isoform of nitric oxide synthase (NOS) in skeletal muscle is the splice variant of neuronal NOS, termed nNOSμ. Exercise training increases nNOSμ protein levels in rat skeletal muscle, but data in humans are conflicting. We performed two studies to determine 1) whether resting nNOSμ protein expression is greater in skeletal muscle of 10 endurance-trained athletes compared with 11 sedentary individuals (study 1) and 2) whether intense short-term (10 days) exercise training increases resting nNOSμ protein (within whole muscle and also within types I, IIa, and IIx fibers) in eight sedentary individuals (study 2). In study 1, nNOSμ protein was ∼60% higher (P < 0.05) in endurance-trained athletes compared with the sedentary participants. In study 2, nNOSμ protein expression was similar in types I, IIa, and IIx fibers before training. Ten days of intense exercise training significantly (P < 0.05) increased nNOSμ protein levels in types I, IIa, and IIx fibers, a finding that was validated by using whole muscle samples. Endothelial NOS and inducible NOS protein were barely detectable in the skeletal muscle samples. In conclusion, nNOSμ protein expression is greater in endurance-trained individuals when compared with sedentary individuals. Ten days of intense exercise is also sufficient to increase nNOSμ expression in untrained individuals, due to uniform increases of nNOSμ within types I, IIa, and IIx fibers.
- nitric oxide
- nitric oxide synthase
nitric oxide synthases (NOS) represent a family of cytochrome P-450-like flavohemeproteins that catalyze the 5-electron oxidation of a guanidino-nitrogen of l-arginine to form l-citrulline and nitric oxide (NO) (7, 32). In skeletal muscle, NOS appears to play an important role in the regulation of many muscle functions (33), including blood flow (26, 42), contraction (20), and metabolism (33), in particular, skeletal muscle glucose uptake (5, 18, 28, 34). Indeed, we have previously shown in humans that NOS inhibition reduces skeletal muscle glucose uptake during exercise without influencing blood flow (5), an effect that was more pronounced in people with type 2 diabetes compared with healthy controls (18).
There are three distinct NOS isoforms that are expressed in a tissue-specific manner (37). In rodent skeletal muscle, both neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms are expressed within the muscle fibers themselves (21, 26). Human skeletal muscle fibers also contain nNOS, but, in contrast to rodents, eNOS appears to be confined to the endothelium of blood vessels within skeletal muscle (11, 12, 35). It has been shown in both rodents (39) and humans (25, 27) that skeletal muscle expresses an alternatively spliced variant of the conventional nNOS (nNOSα), known as nNOSμ. nNOSμ is larger than nNOSα, due to the insertion of a 102-base-pair sequence between exons 16 and 17, resulting in an extra 34 amino acids (39). It has not been established whether nNOSμ is the only nNOS isoform expressed in human skeletal muscle. There is little expression of inducible NOS (iNOS) in skeletal muscle of healthy humans, although skeletal muscle iNOS is increased in diseases associated with inflammation (1, 14, 41).
Intensive exercise training has consistently been shown to increase skeletal muscle nNOSμ and eNOS protein expression in rats. For instance, 4 to 8 wk of intense treadmill running increases nNOSμ and eNOS protein in rat gastrocnemius and soleus (3, 43). At present, only two studies have examined the effect of exercise training on skeletal muscle nNOS expression in humans, and these studies yielded conflicting results (11, 35). We were intrigued as to why there were conflicting findings regarding the effect of exercise training on nNOSμ protein expression. The disparity in results may, however, have been simply due to differences in the training protocols employed. It appeared likely that training must consist of intense and/or long exercise bouts to cause a consistent increase in skeletal muscle nNOSμ protein expression. This would explain why intense resistance training in humans increases skeletal muscle nNOSμ protein (35), but lower intensity endurance training does not (11). Indeed, the aforementioned studies in rodents showing increased nNOSμ protein expression after training employed exercise bouts that were very intense and/or prolonged in nature (3, 40, 43). Therefore, we examined whether skeletal muscle nNOSμ protein expression was increased at rest in endurance-trained (ET) athletes who had been exposed to a substantial exercise stimulus over many years (study 1). We also examined whether 10 days of very intense endurance-exercise training, which we have previously shown to increase aerobic capacity and alter exercise metabolism (29), increases resting skeletal muscle nNOSμ protein expression in previously sedentary (SED) individuals (study 2). It was hypothesized that skeletal muscle nNOSμ protein would be higher in the ET athletes than SED controls and also that skeletal muscle nNOSμ protein would increase after 10 days of intense exercise training in previously SED individuals.
Study 1 involved 11 healthy SED males and 10 ET male athletes (V̇o2 peak > 58 ml·kg−1·min−1). Participant characteristics are shown in Table 1. This study was approved by the Monash University Standing Committee on Ethics in Research involving Humans and the Alfred Hospital Ethics Committee and was conducted in accordance with the Declaration of Helsinki of the World Medical Association. ET individuals were typically well-trained cyclists or triathletes, although one runner without any cycling experience also participated in the study. The mean exercise training duration for ET individuals was 6 yr.
Study 2 involved eight healthy SED males. This study was approved by the Human Research Ethics Committee of The University of Melbourne and was conducted in accordance with the Declaration of Helsinki of the World Medical Association. Participant characteristics before the short-term exercise training were (in means ± SE) 23 ± 1 yr, 69.4 ± 3.8 kg, and a V̇o2 peak of 3.1 ± 0.2 l/min, which was equivalent to 44.6 ± 1.3 ml·kg−1·min−1.
In both studies, the SED individuals participated in regular exercise ≤ 1 time per week and had a V̇o2 peak < 47 ml·kg−1·min−1.
Blood Sampling and Analysis
In study 1, blood was obtained by venipuncture in the morning following an overnight fast. Blood was immediately placed on ice, then centrifuged at 1,500 g with the plasma frozen at −80°C for later analysis. Plasma total cholesterol, triglycerides, and glucose concentration were measured using enzymatic, spectrophotometric techniques with a Cobas-BIO centrifugal analyzer (Roche Diagnostic Systems, Basel, Switzerland).
V̇o2 Peak Determination
In both studies V̇o2 peak was determined during continuous incremental cycling to volitional exhaustion on an electronically-braked ergometer (Lode, Groningen, The Netherlands). Expired air was collected into Douglas bags and analyzed for oxygen and carbon dioxide using Exerstress OX21 and CO21 electronic analyzers (Clinical Engineering Solutions, Sydney, Australia) calibrated with gases of known composition. Gas volume was measured by using a dry gas meter (American Meter; Vacumed, Ventura, CA) calibrated against a Tissot spirometer.
At least 7 days following the V̇o2 peak test, the ET and SED participants came to the laboratory for a muscle biopsy. They were asked to refrain from exercise for at least 24 h prior to coming to the laboratory. Approximately 100 mg of skeletal muscle was obtained from the vastus lateralis by using a percutaneous needle biopsy technique (4) under local anesthetic (1% lignocaine; Astra) using suction. The muscle was immediately frozen and stored in liquid nitrogen for later analysis.
The experimental design utilized in this study has previously been described (29). Briefly, 5–7 days after the V̇o2 peak test, participants completed the first experimental trial (pretraining), which involved cycling for 120 min or until exhaustion (whichever occurred first) at ∼65% V̇o2 peak (116 ± 9 Watts). One week after the pretraining trial, participants began a very intense 10-day exercise training program (10 sessions during 2 wk), which involved sessions of cycling for 45–90 min at 75% V̇o2 peak alternated with sessions of interval training at 90–100% V̇o2 peak (6 × 5 min work bouts). Twenty-four to 48 h after the final training session, subjects performed a posttraining exercise trial, which involved cycling at the same absolute workload and duration as before training. In both experimental trials, muscle biopsies were performed at rest, after 30 min of exercise, and immediately after completion of the exercise trial, although only resting data are presented.
Triple-labeled immunohistochemistry, using a modified method of Russell et al. (36), was used to examine nNOSμ protein expression in types I, IIa, and IIx fibers. Briefly, skeletal muscle sections (10 μm) were fixed in 4% paraformaldehyde and then incubated with 1% Triton X-100, following which, sections were incubated overnight at 4°C in a solution of 50% preimmune sheep serum (SS) and 3% BSA in PBS. Types I and IIa fibers were detected by incubating sections with monoclonal antibodies raised against human slow (type I; A4.840, mouse IgM) and fast (types IIa; N2.261, mouse IgGγ1) myosin heavy chain, both of which were diluted 1:30 in a solution of 10% SS and 1% BSA in PBS. These antibodies, developed by Dr. H. M. Blau (10, 16), were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development, and maintained by The University of Iowa, Department of Biological Sciences (Iowa City, IA). In conjunction with the A4.840 and N2.261 monoclonal antibodies, nNOSμ was detected by using a mouse IgGγ2a monoclonal antibody (1:200 dilution; BD Biosciences, San Jose, CA). Negative control sections were incubated in 10% SS and 1% BSA in PBS, in the absence of primary antibodies.
After overnight incubation at 4°C, the primary antibodies were visualized by using the following secondary antibodies: goat anti-mouse IgGγ2a conjugated with Alexa Fluor 488 (1:2,000; Molecular Probes, Eugene, OR), goat anti-mouse IgGγ1 conjugated with Alexa Fluor 350 (1:1,000; Molecular Probes), and goat anti-mouse IgM conjugated with Texas Red (1:1,000; Southern Biotechnology Associates, Birmingham, AL). The optimal concentration of each antibody was determined by using serial dilutions. Pre- and posttraining muscle sections for a given time point were mounted onto the same slide for each subject, as well as a separate section that served as the negative control. For each protein measurement, relevant sections from all subjects were treated at the same time. A total of 156 type I fibers, 106 type IIa fibers, and 40 type IIx fibers were counted (total number of fibers counted for all participants combined). Sections were viewed with an Olympus I ×70 microscope, and images were collected using an Optiscan MagnaFire CCD camera and associated software. Fluorescence staining in all images was quantified by semiquantitative densitometric analysis using Imaging Research software (MCID/AIS, Canada). Regions of interest were defined, and the relative fluorescence was expressed in arbitrary units as pixel intensity as a measure of density per unit squared (PSL/mm2).
Western blot analysis.
Thirty to forty milligrams of muscle were homogenized (10 μl/mg of tissue) in buffer A (50 mM Tris·HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 50 mM NaF, 5 mM Na pyrophosphate, 10% glycerol, 1% Triton X-100, 10 μg/ml trypsin inhibitor, 2 μg/ml aprotinin, 1 mM benzamidine, 1 mM PMSF). Protein concentration was determined in triplicate by using a detergent-compatible protein assay (DC protein assay; Bio-Rad Laboratories, Hercules, CA).
In study 1, 80 μl of homogenate were affinity purified using 2′,5′-ADP Sepharose beads (6). All of the 80 μl homogenates contained very close to 50 μg of protein (within 3–5 μg; if there was a greater variation, the results were ignored and the sample was rerun). The fractions were then subjected to SDS-PAGE, and nNOSμ was detected by immunoblotting with a murine monoclonal primary antibody against human nNOS (1:2,000, cat. no. N31020; Transduction Laboratory, Lexington, KY) and a goat anti-mouse secondary antibody (1:2,000; Dako, Glostrup, Denmark). Five microliters of recombinant human nNOS were used as the positive control standard. The blocking buffer contained 5% skim milk powder. Each muscle sample was run in duplicate on two different gels, and then the results were averaged. Based on preliminary findings in a collaborating laboratory (Dr. Zhiping Chen, St. Vincent's Institute of Medical Research, St. Vincent's Hospital, Melbourne, Australia), eNOS was not expected to be detected in human skeletal muscle. As such, eNOS protein expression was probed in only nine skeletal muscle samples (4 SED, 5 ET). A murine monoclonal primary antibody against human eNOS (1:2,000; Transduction Laboratories) and a goat anti-mouse secondary antibody (1:2,000; Dako) was used. The positive control was 5 μl of recombinant human eNOS. Following 60 s of exposure to an enhanced horseradish peroxidase-luminol chemiluminescence reaction kit, the membrane was exposed for 15–60 s to film and was developed with an automated processor. The bands were quantitated (ImageQuant) as the product of band intensity and area divided by background, standardized to the nNOS/eNOS positive control so that different gels could be compared.
In study 2, whole muscle homogenates (40 μg; within the linear range) from six individuals were subjected to SDS-PAGE. nNOSμ and eNOS protein expression were determined by using the primary antibodies described above (both 1:100 dilution); however, no eNOS was detected. iNOS protein expression was assessed by using a rabbit polyclonal antibody (1:100; BD Biosciences, San Jose, CA), and nNOSμ and iNOS were expressed relative to β-Actin (Abcam, Cambridge, UK). Binding of these antibodies was detected by using anti-mouse IRDye 700- and anti-rabbit IRDye 800-labeled secondary antibodies (LICOR Biosciences, Lincoln, NE). Fluorescence was detected and quantified using the Odyssey infrared imaging system (LICOR Biosciences). Each muscle sample was run in duplicate on two different gels, and then the results were averaged.
In nine participants from study 1 (4 SED, 5 ET) total RNA was prepared from frozen muscle biopsy tissue as previously described (2), and the concentration was quantitated by absorbance at 260 nm.
RT-PCR cloning of human nNOS cDNA fragment.
Previous studies have detected the existence of an alternately spliced nNOS mRNA transcript that contains an additional 102 nucleotides inserted between exons 16 and 17 (39) of the conventional nNOSα. Oligonucleotide primers that span exons 16 to 19 were designed based on homologous regions between human and mouse nNOSα allowing for the detection of this putative alternate splicing event in human tissues. Primers corresponding to nucleotides 2492–2516 (5′-TGGAAATGAGGCACCCCAACTCTG-3′) and nucleotides 2930–2953 (5′-GTTCTGGAGCTTCGGCCACAAAGG-3′) of the human nNOSα cDNA sequence (GenBank accession no. D16408) were used to amplify nNOS transcripts from human cDNA samples. As predicted from the known sequence, these primers should amplify a 461-nucleotide fragment of nNOS mRNA in the absence of an insert between the two primers, or a 563-nucleotide fragment of the alternatively spliced nNOSμ transcript. PCR amplification was conducted on 2 μl of human skeletal muscle and human umbilical vein endothelial cell (HUVEC) cDNA with 25 pmol of each primer, 0.2 mM of each 2-deoxynucleotide 5′-triphosphate, 1.5 mM MgCl2, and 2.5 units AmpliTaq-Gold (PerkinElmer). Following an initial polymerase activation step (95°C for 10 min), samples were amplified through 35 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min. Following amplification and agarose gel electrophoresis, cDNA fragments were purified, ligated into pGEM-T (Promega), and sequenced to confirm the identity of the cloned fragments.
RNase protection analysis of differentially spliced nNOS mRNA transcripts.
To distinguish between the two mRNA splice variants (nNOSα and nNOSμ) and to accurately quantitate expression, a solution hybridization, RNase protection assay, using cloned skeletal muscle nNOSμ cDNA was established. This clone contains all of the unique alternatively spliced insert (102 nt) near the 5′ end, as well as 461 nucleotide (exons 16–19) that are common to both nNOS transcripts. The cloned nNOSμ cDNA fragment was used as a template to generate 32P-labeled cRNA probes of 563 nucleotide-spanning exons 16–19 (inclusive of the 102 nucleotide μ-insert) by using SP6 promotor-containing vectors. RNA samples were hybridized overnight, RNase digested, and the protected hybrids were analyzed on nondenaturing 6% polyacrylamide gel and quantitated on a Fuji BAS-1000 phosphoimaging system as described previously (2).
All results are expressed as means ± SE and the significance level was set at P < 0.05. In study 1 all parameters were compared using unpaired Student's t-test. In study 2, a paired Student's t-test was used to assess whole muscle protein expression before and after exercise training. A two-factor repeated-measures ANOVA was used to assess nNOSμ protein expression within types I, IIa, and IIx fibers. If the ANOVA was significant (P < 0.05), specific differences were located using the Fisher's least significant difference test.
Study 1: ET Compared with SED
Participant characteristics are presented in Table 1. There were no differences between the two groups for any variables other than exercise parameters. Both absolute (l/min) and relative (ml·kg−1·min−1) V̇o2 peak were significantly (P < 0.05) higher in ET compared with SED.
nNOSμ is the only nNOS expressed in human skeletal muscle.
To clarify previous reports, it was important that we confirm that the only nNOS expressed in the human skeletal muscle samples is nNOSμ. RT-PCR amplification of cDNA derived from cultured HUVEC using the primers corresponding to nucleotides 2492–2516 and nucleotides 2930–2953 of the human nNOSα cDNA generated the predicted 461-nucleotide fragment that corresponded to the previously reported normally spliced nNOSα mRNA (data not shown). In contrast, RT-PCR amplification of human skeletal muscle cDNA revealed a single product that was ∼100 nucleotides longer on agarose gel electrophoresis (data not shown). This skeletal muscle nNOS species was then subcloned into a pGEM-T vector and amplified, and multiple clones were then sequenced to confirm its identity. Sequence analysis revealed that this mRNA species was identical to the reported nNOSα sequence except for the inclusion of a 102-nucleotide insert between exons 16 and 17, as previously reported by other laboratories (25, 27). Alignment of the human nNOSμ sequence with the previously published mouse clone (39) revealed a high degree of sequence homology at both the nucleotide and protein level (data not shown). At the nucleotide level, the human 102-nucleotide, alternatively spliced, nNOSμ insert was 89% (91/102) homologous to the mouse sequence, while at the amino acid level, the human sequence showed 8 amino acid substitutions compared with the mouse (76% amino acid homology; data not shown). The nNOSμ clone was designed so that following RNase protection and analysis on polyacrylamide gels, normally spliced nNOSα transcripts would be represented as 461-nucleotide RNase-resistant bands, whereas nNOSμ transcripts would run as a 563-nucleotide band. We examined muscle samples from nine participants (4 SED, 5 ET), and the nNOSμ mRNA transcript was the only isoform of nNOS detected (Fig. 1). There was no difference in nNOSμ mRNA expression between SED (23 ± 1 arbitrary units, n = 4) and ET (25 ± 2 arbitrary units, n = 5) participants (Fig. 1). Note that because of the small number of participants examined, this result should be considered preliminary and will need to be confirmed in larger studies.
nNOSμ, eNOS, and iNOS protein expression.
Figure 2 demonstrates that there was negligible eNOS protein expression in human skeletal muscle and that there was substantially higher expression of nNOSμ than eNOS. No iNOS could be detected (data not shown). All nNOS protein detected in these human skeletal muscle samples was considered to be nNOSμ protein given the exclusive expression of nNOSμ mRNA in human skeletal muscle as discussed above. The finding that nNOS in human skeletal muscle migrated as a single band on SDS-PAGE supported the assumption that nNOSμ is exclusively expressed in human skeletal muscle (Figs. 2A and 3A). It can also be seen that the nNOS in human skeletal muscle is slightly larger than the recombinant nNOS control, again indicating that it is nNOSμ (Fig. 2A). Skeletal muscle nNOSμ protein content was significantly greater in the ET group (1.63 ± 0.25 relative arbitrary units) compared with the SED group (1.00 ± 0.13 relative arbitrary units; P < 0.05; Fig. 3B).
Study 2: Short-Term Exercise Training
In study 2, iNOS was barely detectable in human skeletal muscle (Fig. 4A), and eNOS expression could not be detected at all (data not shown). Ten days of exercise training increased whole muscle (Western blot analysis) nNOSμ protein significantly (29 ± 10% vs. pretraining, P < 0.05; Fig. 4A). There was no effect of exercise training on whole muscle iNOS protein expression (Fig. 4A). Before exercise training, nNOSμ protein expression did not differ between types I, IIa, and IIx fibers as assessed by immunohistochemistry (Fig. 4B and Fig. 5). The increase in whole muscle nNOSμ expression following exercise training (Fig. 4A) was due to a uniform increase in nNOSμ protein within types I, IIa, and IIx fibers (average of ∼53% increase vs. pretraining, P < 0.05; Fig. 4B). Accordingly, nNOSμ protein expression remained similar between types I, IIa, or IIx fibers after exercise training.
The main findings of this study were that 1) skeletal muscle nNOSμ protein is higher in ET compared with SED individuals, and 2) that 10 days of intense endurance exercise training increases skeletal muscle nNOSμ protein in previously SED individuals. A second important finding was that nNOSμ protein was expressed uniformly within types I, IIa, and IIx fibers of SED men and that exercise training increases nNOSμ protein to a similar extent in all skeletal muscle fiber types.
Our results indicate that if endurance exercise training is prolonged (e.g., 6 yr in study 1) or intense (e.g., 10 days of very intense endurance exercise training in study 2), increases in skeletal muscle nNOSμ protein consistently occur. This phenomenon likely explains why 90 days of bed rest combined with intense resistance exercise increases skeletal muscle nNOSμ protein content (35), but 28 days of mild endurance exercise does not increase skeletal muscle nNOSμ protein content in people with type 2 diabetes and matched controls (Bradley SJ, Kingwell BA, Canny BJ, McConell GK, unpublished observations). However, it is not clear why a relatively intense 6 wk of knee-extensor training or 6 wk of running training has been shown to have no effect on skeletal muscle nNOS protein content (11). Studies in animals have shown that a variety of short-term training protocols (all involved intense and/or prolonged exercise sessions) result in an increase in skeletal muscle nNOS protein expression (3, 40, 43).
We found that there was a similar distribution of nNOSμ protein expression within types I, IIa, and IIx fibers in human skeletal muscle, regardless of training status (Fig. 4B). These findings are consistent with those of Grozdanovic et al. (13), but differ with those of Frandsen et al. (12) who reported higher nNOS expression in type I than in type II muscle fibers of humans. It should be noted though that no quantification was presented in the latter study (12). Importantly, our findings of an increase in whole muscle nNOSμ protein (Fig. 4A) with short-term exercise training in study 2 support the immunohistochemistry results from the same study, which showed a uniform increase in nNOSμ within types I, IIa, and IIx fibers after training (Fig. 4B). Although nNOSμ protein was most concentrated around the sarcolemma, there was evidence of diffuse expression throughout the myocyte (Fig. 5). Such findings are in line with others who have found a similar pattern of nNOSμ protein expression using immunohistochemistry of human skeletal muscle (35).
The results of this study support the contention that nNOSμ is the exclusive variant of nNOS expressed in human skeletal muscle (27). This was the case in both ET and SED humans. Our results differ with Larsson and Phillips (25) who reported both nNOSα and nNOSμ mRNA in human skeletal muscle. We found only one cDNA, one mRNA, and one protein for nNOS, and all were verified as being nNOSμ rather than conventional nNOSα. The nucleotide and deduced amino acid sequence for nNOSμ were identical to that reported previously (25, 27). The functional significance of this variant remains unknown, although some aspects of the potential effect of the mu insert have been identified. Rodent nNOSμ exhibits similar catalytic activity and calcium dependence to nNOSα (39), however, nNOSμ consumes NADPH and reduces cytochrome c at approximately half the rate of nNOSα (24). Further, the in vitro half-lives of rodent nNOSα and nNOSμ are 12 and 30 min, respectively (24). We have previously shown that skeletal muscle nNOSμ is phosphorylated by AMP-activated protein kinase during exercise in humans (9), but at this time, the physiological significance of the nNOSμ protein in human skeletal muscle remains to be determined.
In addition to the analysis of nNOS, this study investigated the expression of eNOS and iNOS in human skeletal muscle. Previous studies investigating eNOS expression in human skeletal muscle have provided conflicting results. Brenman et al. (8) and Hickner et al. (15) reported eNOS protein in human skeletal muscle, but Nakane et al. (30) found none. Frandsen et al. (12) investigated the location of eNOS in human skeletal muscle by immunohistochemistry. Unlike rat skeletal muscle (21), no eNOS protein was detected within skeletal muscle myocytes themselves, with eNOS immunoreactivity confined to the microvascular endothelium and the endothelium of larger vessels (12). By using immunoblotting, the present study detected little eNOS protein in skeletal muscle biopsies from either the ET or SED subjects in study 1 (Fig. 2B) and was unable to detect any eNOS protein in study 2. Similarly, a recent study using immunoblotting in humans found barely detectable levels of eNOS protein in skeletal muscle (35). Therefore, taken together, the results of the present and previous studies indicate that there is much lower expression of eNOS in human skeletal muscle than nNOSμ. iNOS was barely detectable in immunoblots of these healthy human participants (Fig. 4A). It will be important to determine whether exercise training decreases the elevated iNOS expression that is observed in people with type 2 diabetes (41).
Skeletal muscle nNOSμ protein was 60% higher in the ET compared with the SED participants of study 1, but nNOSμ mRNA abundance was similar in the two groups. This suggests that translational factors contribute to the higher nNOSμ protein in the trained participants. However, it is important to note that exercise may increase skeletal muscle nNOSμ mRNA content acutely after exercise but then return to basal levels. All participants in study 1 were requested to refrain from exercise for 24 h prior to their biopsy and so any acute changes in nNOSμ mRNA could not be examined. It has been shown that the mRNA abundance of some other key metabolic regulators in skeletal muscle, such as the glucose transporter GLUT-4, increase transiently in the hours following an exercise bout but return to basal levels at 24 h, while protein level remains elevated (22, 23, 45). It is therefore likely that skeletal muscle nNOSμ exhibits a similar pattern.
The higher skeletal muscle nNOSμ protein expression after exercise training is likely to be associated with greater production of NO by skeletal muscle (38, 40, 43). NO has been implicated in a wide range of diverse processes that have implications for both exercise performance and risk for vascular and metabolic diseases. NO production from skeletal muscle may influence exercise capacity via effects on glucose uptake, oxidative phosphorylation, blood delivery (vasodilation), contractility, and excitation-contraction coupling (for a review see Ref. 19). There is also evidence that NO plays a role in skeletal muscle mitochondrial biogenesis (31, 44). We have preliminary evidence that skeletal muscle nNOSμ protein is reduced in people with type 2 diabetes (Bradley SJ, Kingwell BA, Canny BJ, McConell GK, unpublished observations), who are known to have reduced mitochondrial biogenesis (17). Therefore, the higher skeletal muscle nNOSμ expression after exercise training may, in part, contribute to the well-known protective effects of exercise with regard to cardiovascular and metabolic diseases.
In conclusion, ET males have higher skeletal muscle nNOSμ protein content than SED males, and intense short-term exercise training in previously SED males increases skeletal muscle nNOSμ protein content. Skeletal muscle nNOSμ protein content is similar in types I, IIa, and IIx skeletal muscle fibers and the increase in whole muscle nNOSμ protein content following short-term exercise training is due to similar increases in types I, IIa, and IIx muscle fibers.
This work was supported by grants from Diabetes Australia and the National Health and Medical Research Council of Australia.
Present address: R. Lee-Young. Dept. of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN (email:).
The authors would like to thank the participants for taking part in these experiments and also Dr. Dominic Autelitano for assistance with mRNA measurements.
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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