1 Department of Physiological Science, University of California, Los Angeles, Los Angeles, California 90095; and 2 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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The hypothesis that changes in muscle activation and loading regulate the expression and activity of neuronal nitric oxide (NO) synthase (nNOS) was tested using in vitro and in vivo approaches. Removal of weight bearing from rat hindlimb muscles for 10 days resulted in a significant decrease in nNOS protein and mRNA concentration in soleus muscles, which returned to control concentrations after return to weight bearing. Similarly, the concentration of nNOS in cultured myotubes increased by application of cyclic loading for 2 days. NO release from excised soleus muscles was increased significantly by a single passive stretch of 20% or by submaximal activation at 2 Hz, although the increases were not additive when both stimuli were applied simultaneously. Increased NO release resulting from passive stretch or activation was dependent on the presence of extracellular calcium. Cyclic loading of cultured myotubes also resulted in a significant increase in NO release. Together, these findings show that activity of muscle influences NO production in the short term, by regulating NOS activity, and in the long term, by regulating nNOS expression.
rat; calcium; contraction; muscle mechanics
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INTRODUCTION |
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NEURONAL NITRIC OXIDE (NO) synthase (nNOS) has only recently been shown to be expressed by skeletal muscle (14, 25), but the number of physiological processes in which its product, NO, has been implicated has grown rapidly. For example, NO may be involved in the regulation of muscle function by modulating glucose uptake (5, 6, 29), mitochondrial oxygen metabolism (11, 15), blood supply to muscle (11, 34), and contractility (14, 23, 24). In most cases, muscle NO can be viewed as a positive regulator of muscle function, in that NO has been shown experimentally to promote glucose transport and presumably to increase blood supply to muscle through its vasodilatory capabilities. However, the role of NO in regulating force production by muscle is less clear (14, 23, 24).
nNOS is activated by calcium and calmodulin, which provide a primary mechanism for regulating nNOS activity in vivo (7, 12). Increased muscle activation is expected to increase NOS activation that is primarily attributable to increases in cytosolic calcium, presumably of sarcoplasmic reticulum (SR) origin. This expectation has been supported experimentally by measurement of a threefold increase in NO concentration in expired gases during exercise (18) and by high-frequency electrical stimulation of muscle in situ, after which the muscle was excised and shown to release more NO than nonstimulated, excised controls (5). Long-term increases in muscle activation by high-frequency stimulation of motor nerves in vivo have also been shown recently to increase nNOS concentration in muscle (28).
Application of the findings of these previous studies to changes in nNOS activity and expression that may occur in vivo is limited by the experimental perturbations used to produce the changes in nNOS activity and concentration. For example, NO-mediated processes were shown to decrease force production in diaphragm muscle stimulated at 40 Hz, but not at 15 Hz (14). However, mammalian muscle activated at 40 Hz is at its approximate threshold for tetanic fusion frequency, which would not occur normally in diaphragms in vivo. Thus those findings do not substantiate an increase in NO production in muscle that is activated under physiological stimulation frequencies. Other investigations that showed an increase in nNOS activity (5) and concentration (28) employed protocols in which the muscle was stimulated for two 5-min periods at 100 Hz and continuously for 3 wk at 10 Hz, respectively. Although these investigations conclusively demonstrated a positive relationship between muscle activation and nNOS activity and concentration, whether muscle activation at physiological frequencies and durations can also result in an increase in nNOS activation or expression remains unknown.
Investigations of nNOS regulation in muscle have focused on experimental treatments that induced increases in cytosolic calcium by membrane depolarization. However, examination of other cell types has shown that mechanical activity is also a positive regulator of NOS activity and expression. For example, cyclic strains and shear stress applied to endothelial cells in vitro induce an increase in NOS (eNOS) protein and mRNA concentration (4, 33). Similarly, compressive loads applied to bones or cyclic strains applied to osteoblasts or osteocytes stimulate NOS activity (27). Thus it is possible that passive mechanical loading, as well as activation leading to membrane depolarization, may contribute to regulating nNOS activity and expression in muscle cells.
In the present investigation we used three model systems to examine the identity of factors that may regulate nNOS activity and expression in skeletal muscle under stimulation conditions that skeletal muscle normally encounters in vivo. First, we examine whether nNOS expression is modified by changes in muscle loading in vivo by measuring changes in nNOS protein and mRNA after periods of rat muscle hindlimb unloading and subsequent reloading. Second, we test whether brief periods of muscle activation at physiological rates or passive strains of physiological magnitude are capable of producing detectable changes in nNOS activity and whether that activity depends on extracellular calcium. Finally, we test whether cyclic mechanical loading of differentiating myotubes grown in vitro acts as a regulator of nNOS activity and expression.
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MATERIALS AND METHODS |
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Hindlimb unloading-reloading. Female Wistar rats (200-230 g) were subjected to hindlimb unloading for 10 days by a modification of the procedure described by Morey-Holton and Wronski (22) according to protocols approved by the University of California, Los Angeles, Animal Research Committee. Animals were removed from the hindlimb suspension apparatus after 10 days of hindlimb unloading and allowed to recover for 2, 4, or 7 days of normal cage activity, during which hindlimb muscles experienced reloading (8 animals). Rats that did not experience hindlimb unloading were used as ambulatory controls (8 animals). Other rats were subjected to unloading only, without a subsequent period of reloading (8 animals). Animals were killed by injection with pentobarbital sodium (100 mg/kg ip); then the soleus and plantaris muscles were dissected from the hindlimb, weighed, and frozen in liquid nitrogen until analyzed.
Cell culture.
C2C12
muscle cells (American Type Culture Collection) were grown in wells of
a mechanical cell stimulator (MCS; Cell Kinetics, Providence, RI),
which consists of a stainless steel plate containing wells in which the
floor is a transparent Silastic membrane (Dow). Before the cell
cultures were started, the MCS membrane was coated with a 2-mm-thick
layer of 2% gelatin and allowed to dry for 48 h at 37°C. Cells
were then added to each well in 10% fetal bovine serum (FBS) in DMEM
containing penicillin and streptomycin at 37°C and 5%
CO2. Cultures were fed every 48 h
for 6 days, at which time the membranes were covered with confluent
myoblasts. The cultures were transferred to DMEM containing no FBS for
16 h to stimulate fusion, then they were returned to 10% FBS in DMEM. The cultures were maintained for an additional 6-8 days to allow growth of myotubes. At the end of this period the substratum was covered with 15- to 30-µm-diameter myotubes.
Mechanical and electrical stimulation of excised skeletal muscle. Soleus muscles were rapidly dissected from Wistar rats and mounted at rest length by a procedure whereby the tendons of origin and insertion were held in hemostats mounted in a microtensometer. The muscles were rinsed several times in 10 mM HEPES (pH 7.25) containing 135 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2 · 6H2O, and 10 mM glucose and then placed in the same HEPES buffer containing 100 µM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (Cayman Chemical, Ann Arbor, MI). Muscles were then subjected to a variety of stimulation protocols that included 1) 20% passive strain for 2 min, 2) 15 ms of 20-V stimulation applied at 2 Hz for 2 min while at resting length, 3) 2-Hz stimulation while at 20% strain for 2 min, or 4) no strain or stimulation for 2 min. These conditions were chosen because both 20% strain and submaximal activation at 2 Hz are within the natural physiological ranges experienced by vertebrate skeletal muscles. These treatments were performed identically in HEPES buffer or in the same buffer containing no calcium. At the end of each experimental treatment, the buffer was collected and NO release was measured spectrophotometrically according to the technique of Salzman et al. (30) and Amano and Noda (3). Muscle mass was recorded at the end of each experiment.
Cell loading. Myotubes were subjected to cyclic strain by deflecting the center of the Silastic membrane substrata with a piston, causing a 6.7% mean deformation of the membrane and adherent cells. Cells were subjected to five cycles of loading during a 20-s period, then 10 s of no strain. This loading cycle was repeated two more times, followed by no strain for 30 min. Mean strain rate during loading was 3.4%/s. The entire sequence was repeated 95 times over the course of 48 h. Cultures were then inspected visually with an inverted microscope to confirm that the myotubes remained attached to the substratum at the end of experimental loading. Control cultures were grown under identical conditions in the MCS, except they were not subjected to loading.
Western blotting. Immediately after the last cycle of myotube loading, the culture media were removed from all loaded and control cultures, and the cultures were rinsed briefly with DMEM. Myotubes were collected by adding reducing sample buffer to each culture (80 mM Tris, pH 6.8, containing 0.1 M dithiothreitol and 70 mM SDS) (16) and scraping the myotubes from the membranes with a rubber policeman. The myotube extract was then heated to 100°C for 1 min, and the protein concentration was determined by the method of Minamide and Bamburg (21).
Whole soleus muscle samples that were obtained after hindlimb unloading and reloading treatments were homogenized in a Dounce homogenizer in reducing sample buffer, then they were boiled for 1 min and centrifuged to remove particulates, and the protein concentration of the supernatant fraction was measured. Samples of 35 or 50 µg each were loaded on 8% polyacrylamide gels that were prepared according to Laemmli (16). Gels were then electrophoretically transferred to nitrocellulose (8). Protein blots were incubated with goat anti-rat brain nNOS (19) diluted 1:200 in 50 mM Tris, pH 7.6, containing 150 mM NaCl, 0.1% NaN3, 0.05% Tween 20, and 3% BSA. After extensive buffer washing of the blots, they were incubated with an alkaline phosphatase-conjugated second antibody and then incubated in alkaline phosphatase substrate.Northern blots. Whole soleus muscle RNA was isolated according to the technique of Chomczynski and Sacchi (10). The final RNA pellet was dissolved in 25 µl of 10 mM Tris, pH 8.0, containing 1 mM EDTA, and the concentration was determined by absorbance at 260 nm. RNA was loaded onto 1.2% formaldehyde-agarose gels and electrophoresed overnight at 25 V. The samples were electrophoretically transferred to uncharged nylon membranes, ultraviolet cross-linked with 150 mJ, and stained with methylene blue to verify uniformity of loading.
Nylon membranes were prehybridized in 1× Denhardt's solution, 4× SSC (0.03 M citric acid trisodium-0.3 M sodium chloride), 1% SDS, and 100 µg/ml salmon sperm DNA for 2-4 h at 65°C. Hybridization was carried out in 1× Denhardt's solution, 4× SSC, 1% SDS, 100 µg/ml salmon sperm DNA, and 10% sodium dextran sulfate with 2 × 106 cpm/ml ofDensitometry. Relative quantities of nNOS protein and mRNA in Western and Northern blots were determined by scanning densitometry (Alpha Innotec, San Leandro, CA) and expressed in arbitrary units. Relative quantities of myosin heavy chain in Coomassie blue-stained gels were similarly determined by densitometry. The significance of differences between the relative quantities of any given protein or mRNA in experimental and control samples was tested by the Mann-Whitney test, with confidence limit set at P < 0.05.
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RESULTS |
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nNOS expression is positively regulated by muscle activity in vivo. Modifications in muscle loading by hindlimb suspension or by suspension followed by muscle reloading caused changes in the concentration of nNOS and nNOS mRNA in rat soleus and plantaris muscles. Hindlimb suspension caused a reduction in the relative quantity of nNOS by 43% after 10 days of unloading compared with ambulatory controls (Figs. 1 and 2). After 2 days of reloading, nNOS concentration had returned to levels that did not differ significantly from ambulatory controls. In contrast, the relative quantity of myosin heavy chain did not differ significantly from ambulatory controls after 10 days of unloading or after 2 days of reloading (Fig. 2).
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Mechanical loading and electrical stimulation each increase NOS
activity in skeletal muscle.
Basal level of NO release from single dissected soleus muscles
varied substantially between animals, with a range of 5-25 pmol
NO · mg1 · min
1
[13.8 ± 1.58 (SE)
pmol · mg
1 · min
1,
n = 47]. We tested whether the
difference in NO release corresponded to differences in nNOS
concentration in muscles of different masses and found, by using
immunoblot comparisons of nNOS concentration in small and large
muscles, that large soleus muscles contained a higher NOS concentration
than small soleus muscles (Fig. 4). However, regression analysis showed no significant relationship between
basal level of NO release per muscle and muscle mass (Spearman coefficient of correlation =
0.127,
P = 0.45; Fig.
5).
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nNOS expression and activity are positively regulated by muscle
loading in vitro.
C2C12
myotubes ~20 µm in diameter that were grown on Silastic membranes
and then subjected to cyclic loading for 48 h showed a significant 27%
increase (P < 0.05) in the relative
quantity of nNOS compared with nonloaded controls. Unstrained, control C2C12
myotubes released 0.60 ± 0.03 (SE) pmol
NO · mg1 · min
1
(n = 3) over the course of a 2-h
experiment. Myotubes subjected to cyclic stretching for 2 h showed a
42% increase in NO production (0.85 ± 0.03 pmol
NO · mg
1 · min
1,
n = 3).
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DISCUSSION |
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The results of the present investigation show that mechanical loading is a positive regulator of NOS expression and activity in developing myotubes and in fully differentiated skeletal muscle. For example, the present data show that NO production by muscle increases by ~20% after a single stretch or after 2 min of submaximal stimulation at 2 Hz and that weight-bearing muscles of ambulatory controls contain 75% more nNOS per unit muscle mass than muscles of animals experiencing periods of muscle unloading. Thus mechanical activity of muscle can influence NO production by muscle in the short term, by regulating NOS activity, and in the long term, by regulating nNOS expression. Because muscle is the primary source of NO in mammals, the influence of muscle loading on nNOS expression and activation can be expected to have systemic influences during periods of muscle loading and inactivity.
The magnitude of NO released by unstimulated myotubes in the present
investigation is similar to that measured in muscle and other cell
types in previous investigations. For example, unstimulated muscle
cells, endothelial cells, and bone cells in vitro show a basal level of
NO release of ~0.5-2 pmol
NO · mg1 · min
1.
Lee et al. (17) found by measuring the conversion of
[14C]arginine to
[14C]citrulline in
muscle cell extracts that 0.1 pmol
NO · mg
1 · min
1
was generated by extracts of cells collected before fusion, which increased to ~2.4 pmol
NO · mg
1 · min
1
generated by extracts of muscle cells collected during fusion, then
returned to ~0.1 pmol
NO · mg
1 · min
1
after fusion. Although the measurement of conversion of arginine to
citrulline in extracts is a relative measure of NOS concentration and
not a measurement of activity in the intact cells, the values obtained
were similar in magnitude to the measurements of NO release from intact
muscle cells determined in the present investigation (0.6 pmol
NO · mg
1 · min
1).
The release of NO from fully differentiated muscle in the present
investigation is several times greater than that determined in a
previous investigation (~1 pmol
NO · mg1 · min
1)
(5). However, much of this discrepancy may be attributable to
reductions in NO release that occur over the course of muscle incubation in vitro. In the previous study the average NO release into
the media was measured over a 1-h incubation after dissection, whereas
in the present investigation the rate of NO release was averaged for a
2-min period after dissection. Similarly, the values for NO release
after 2 min of submaximal stimulation at 2 Hz measured in the present
investigation (~17 pmol
NO · mg
1 · min
1
released into the medium during stimulation) are much greater than the
NO release measured previously after two 5-min periods of tetanic
stimulation at 100 Hz (~2.3 pmol
NO · mg
1 · min
1
average release into the medium during 1-h incubation after completion of the stimulation procedure) (5). This discrepancy can also be
attributed to the decrease in basal level of NO release over the course
of the 1-h incubation in the previous study but is also attributable to
the rapid rate at which muscle returns to basal level of NO release
after stimulation. We found that NO release by muscle returns to basal
levels within 2 min after the completion of stimulation.
The dependence of muscle loading- or activation-induced increases in NOS activity on the presence of extracellular calcium was unexpected. We anticipated that muscle activation in calcium-free buffer would have little detectable effect on activation-induced NO release, because the SR is the source of most calcium released into the cytosol during muscle activation. This finding has bearing on previous speculations concerning a negative-feedback loop between nNOS activity and SR calcium release channel activity on the basis of the observations that addition of NO donors to SR vesicle preparations reduced the rate of calcium release from SR membrane vesicles and reduced open probability of SR calcium release channels in bilayer preparations (20). In this feedback loop, increased SR calcium release would increase nNOS activity, which would then inhibit SR calcium release and, subsequently, reduce nNOS activity. The results of the present investigation indicate that NO is not functioning as a negative-feedback system, because the increase in nNOS activity during muscle activation appears to be attributable to influx of extracellular calcium rather than SR calcium release. Thus the level of activation-induced increases in nNOS activity is expected to be independent of the possible inhibition of calcium release channels by NO.
The positive relationship between mechanical loading and NOS expression also occurs for eNOS, which suggests that there may be similar mechanical signal transduction mechanisms that influence the expression of nNOS in muscle and eNOS in endothelial cells. Cyclic loading of aortic endothelial cells in vitro resulted in a significant increase in eNOS protein and mRNA (4), and exercise increased eNOS mRNA concentration in dog vessels in vivo (32). Thus, just as in skeletal muscle, endothelial cells exhibit mechanisms for increasing NO release in the short term, by regulating NOS activity, and in the long term, by regulating NOS expression.
Although the functional significance of increased eNOS activity and expression in endothelial cells subjected to mechanical loading has been clearly established as a mechanism inducing vascular smooth muscle relaxation (26), the functional significance of increased NO release in skeletal muscle that is subjected to increased mechanical loading or activation appears to be more complex. The increases in muscle NOS activity (5; present investigation), nNOS protein concentration (28; present investigation), and nNOS mRNA concentration (present investigation) that result from increased activation have most clear functional significance in increasing glucose transport and contributing to vasodilation of vessels that supply the muscle. Strong evidence exists to show that NO plays an important role in regulation of glucose transport in muscle (6, 29), although no direct evidence has been published to show that muscle-derived NO contributes significantly to increase vascular smooth muscle relaxation.
Previous investigations have examined the possibility that increased NO release during muscle activation may modulate force production in skeletal muscle by a mechanism similar to that which has been well established for smooth muscle. From a teleological point of view, this would seem a poor design, because the force-generating ability of muscle would be inhibited by NO-mediated mechanisms when muscle is being activated to generate force. Thus far, investigations have shown that stimulation of skeletal muscle at approximate tetanic fusion frequencies can decrease force production through a pathway mediated by NO and cGMP (14). However, stimulation at 15 Hz, which would not produce tetanus, did not produce a significant NO-mediated effect on force production (14). A more recent study has shown that the unloaded shortening velocity and maximum isometric force of muscle stimulated at 200 Hz, which is well above the threshold frequency for tetanic fusion, were not affected by NOS inhibition, although NOS inhibition resulted in a decrease in loaded shortening velocity and power production (23). Thus there are data to support a role for muscle-derived NO in decreasing force production and for increasing power, but neither effect has been demonstrated at physiological stimulation frequencies. Functional interpretation of these findings is further complicated by the observation that NO donors added to excised muscle preparations can significantly increase tetanic force production (23, 24).
An unexplored possibility for the functional significance of increased NOS activation and expression that result from increased muscle loading is that it may provide a mechanism for regulating inflammation caused by increased muscle use. Previous studies have shown that NO inhibits leukocyte adhesion to vascular endothelium in muscle tissue (1) and that NO can induce apoptosis of inflammatory cells (2, 31). Data from the present study show that muscle unloading results in a downregulation of muscle NOS, so that when the muscle is reloaded, its ability to generate NO on loading is diminished. We hypothesize that the reduction of NO-generating capacity after periods of muscle unloading is linked to the subsequent inflammation of muscle subjected to reloading and are currently testing this hypothesis.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. Bettie Sue Masters and Timothy McCabe for providing NOS antibodies.
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FOOTNOTES |
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This investigation was supported by National Institutes of Health Grants AR-40343, HL-06296, GM-52419, and GM-31296 and Robert A. Welch Foundation Grant AQ-1192.
Address for reprint requests: J. G. Tidball, Dept. of Physiological Science, University of California, Los Angeles, Los Angeles, CA 90095-1527.
Received 3 October 1997; accepted in final form 6 April 1998.
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