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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 4, 2004 as doi:10.1096/fj.03-0792fje. |
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* Department of Anatomy, Campus Benjamin Franklin, Charité University Medicine Berlin, Germany;
Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden;
Friedrich-Baur Institute, Department of Neurology, University of Munich, Germany; and
Department of Physiology, CBF,
|| Center for Space Medicine Berlin, Germany
1Correspondence: Charité University Medicine Berlin, Campus Benjamin Franklin, Department of Anatomy and Center for Space Medicine Berlin, Königin-Luise-Strasse 15, D-14195 Berlin, Germany. E-mail: dieter.blottner{at}charite.de
SPECIFIC AIMS
Nitric oxide (NO) generated from NO synthases mediates normal skeletal muscle functions. Biosynthesis of NO apparently is linked to muscle activity, but the distribution and expression of the three major NO synthase 1-3 isoforms under conditions of extended muscle disuse and exercise are still unclear. Our aim was to elucidate whether protein levels and the cellular or subcellular localization patterns of NO synthases underwent significant changes in a mixed fast/slow and slow type skeletal muscle after prolonged disuse in a long-term bed rest study, a useful experimental paradigm of simulated microgravity in ground-based space research. We examined whether resistance exercise performed regularly as a countermeasure to progressive atrophy within 12 wk of strict bed rest would support expression of one or more isoforms of NOS, thereby maintaining normal skeletal muscle functions during immobilization in clinical settings or in human spaceflight.
PRINCIPAL FINDINGS
1. Resistance exercise during experimental bed rest maintained myofiber size and sarcolemmal NOS 1 distribution in a mixed (VL) and a slow type (SOL) muscle
Mechanical deloading of the human body by extended anti-orthostatic minus 6° head-down tilt (HDT) bed rest ultimately leads to exponential development of disuse-induced atrophy of lower limb skeletal muscle groups and deconditioning of major body systems similar to those seen after clinical immobilization or in spaceflight. In a group of male volunteers (n=8, mean age: 32 BMI: 24), short repetitive bouts (4x7 for vastus lateralis and 4x14 for soleus muscle) of resistance training (i.e., eccentric and concentric muscle activity) were performed regularly with maximal working loads every third day throughout 12 wk of HDT bed rest using a gravity-independent spinning fly wheel ergometer (YoYoTM Technology AB, Stockholm, Sweden). A second group of gender- and age-matched volunteers (n=9, mean age: 33; BMI: 23) were without physical training (non-exercise control group) during HDT recumbent position. All subjects were kept in strict HDT position during the course of the study, including the training protocols. Determination of the myofiber cross-sectional areas (CSA) in vastus lateralis (VL) and soleus (SOL) muscle biopsies obtained from each volunteer before and after bed rest (preBR and postBR) revealed substantial atrophy in type I myofibers (–35%) and type II (–20%) compared with subject-matched CSA values before start of bed rest (not shown). In both VL and SOL muscle, resistance exercise significantly increased myofiber CSA at the end of BR, confirming the effectiveness of the fly wheel exercise device to support myofiber morphology and size after extended bed rest (Fig. 1
).
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2. Cellular and subcellular localization of NOS 1-3 isoforms after bed rest and exercise
To further analyze the effects of exercise on the cellular and subcellular distribution of NOS isoforms, monoclonal antibodies against NOS 1 (neuronal), NOS 2 (inducible), and NOS 3 (endothelial) proteins were used for immunostaining protocols on cryosections of biopsy samples before and after HDT and analysis by confocal laser microscopy.
NOS 1: Exercise significantly increased immunostaining patterns of NOS 1 at sarcolemmal membranes preferentially in myofibers II in VL (>45% of relative fluorescence determined by area-based pixel intensity), but not in SOL muscle at the end of the 12 wk of HDT. Apart from increased sarcolemmal NOS 1, confocal microscopy revealed similar dystrophin immunostaining patterns suggesting maintenance of sarcolemmal dystrophin complex distribution, which represents an integral part of the molecular membrane architecture of skeletal myofibers involved in transmembrane signaling processes (Fig. 2
, upper panel).
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NOS 2: The distinct immunostaining patterns of NOS 2 detected before start of HDT in peripheral cells resembling satellite cells as well as in subsarcolemmal focal accumulations of preferentially type 1 myofibers in VL and SOL were no longer detectable after exercise training at the end of HDT (Fig. 2)
. Lack of NOS 2 immunostaining in peripheral cell structures may result from depleted satellite cell pools and/or lack of precursor myonuclei addition to growing and regenerating myofibers by cell fusion, a mechanism that may be no longer functional after extended bed rest with exercise. In addition, NOS 2 immunoreactivity colocalized with the signal-transducing component caveolin 3 at subsarcolemmal domains. Absence of subsarcolemmal NOS 2/caveolin 3 clusters after exercise thus likely reflect redistribution of sarcolemmal microdomains affecting membrane signaling functions yet to be determined (Fig. 2
, middle panel and insets).
NOS 3: Altered patterns of NOS 3 immunoreactivity were found at capillary structures around myofibers of VL and SOL muscle (Fig. 2
, lower panel). After BR and exercise, the capillary-to-fiber ratio (C/F ratio) was significantly increased in VL (preBR: 1.35+0.16; postBR: 2.20±0.34) but not in SOL (preBR: 2.12±0.24; postBR: 2.15±0.25). However, robust NOS 3 immunostaining was detectable in capillary endothelia in both trained VL and SOL. Given the fact that endothelial-derived NO is a potent vasodilator and a possible trigger for neoangiogenesis, these results support the idea of an increased capillarity and elevated levels of NOS 3 in endothelia in VL and, to lesser extent, also in SOL. Thus, exercise training stimulated adaptive enhancement of the local microvascular network expressing NOS 3 as an effective countermeasure against muscle atrophy and its functional deficits after prolonged bed rest. The altered patterns of NOS 1-3 isoform localization in human skeletal muscle cell compartments before and after bed rest and exercise are summarized (cf. Fig. 3
).
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3. Differential NOS 1 protein levels in VL and SOL suggest muscle-specific control of the NOS/NO system in mixed fast and slow muscle types
Determination of the relative proportions of NOS 1-3 protein levels by quantitative immunoblot analysis resulted in complex patterns for VL and SOL after HDT and exercise. Decreased NOS 1 protein levels found in atrophied VL without exercise significantly increased in trained VL, suggesting activity-driven and lasting effects of the countermeasure protocol on the NOS/NO system in fast type VL muscle after prolonged deloading conditions. In SOL, however, major changes in NOS 1 protein levels were not found between untrained control and trained subjects at the end of bed rest. Our results support the idea that alternative signaling mechanisms may exist for one or more isoforms of NOS that may be controlled by 1) differential mechanical loading characteristics, 2) muscle-specific calcium homeostasis, or 3) neuronal triggering inherent to mixed fast and slow muscle type after disuse atrophy or adaptive plasticity by resistance exercise during prolonged bed rest.
CONCLUSIONS
The present investigation provided various evidence for regulatory mechanisms of the NOS/NO system in human skeletal muscle that may be augmented at least partly by resistance exercise training protocol performed during the long-duration bed rest study. The functional significance of altered localization and expression of NOS isoforms in skeletal muscle may be widespread. Exercise clearly affected cellular distribution and subcellular localization patterns of one or more NOS isoforms in mixed fast and slow type human skeletal muscle. Confocal microscopy documented changes in 1) sarcolemmal NOS 1 expression, 2) subsarcolemmal NOS 2/caveolin 3 accumulations, as well as 3) NOS 3 in capillary endothelia, suggesting morphological and functional adaptive processes in the molecular architecture of sarcolemmal microdomains and capillary networks, which remain to be defined by future studies. The unique patterns of NOS 1-3 protein levels in trained skeletal muscle before and after bed rest nevertheless may reflect differential regulatory mechanisms for the muscular NOS/NO system depending on mechanical loading cues inherent to mixed fast-slow and slow human skeletal muscle types, muscle-specific calcium homeostasis, or neuronal control mechanisms that must be considered for adequate countermeasure protocols in order to overcome functional skeletal muscle impairments in clinical settings as well as in extended human spaceflights.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0792fje; doi: 10.1096/fj.03-0792fje
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