Targeted imaging of hypoxia-induced integrin activation in myocardium early after infarction.

The alphavbeta3-integrin is expressed in angiogenic vessels in response to hypoxia and represents a potential novel target for imaging myocardial angiogenesis. This study evaluated the feasibility of noninvasively tracking hypoxia-induced alphavbeta3-integrin activation within the myocardium as a marker of angiogenesis early after myocardial infarction. Acute myocardial infarction was produced by coronary artery occlusion in rodent and canine studies. A novel (111)In-labeled radiotracer targeted at the alphavbeta3-integrin ((111)In-RP748) was used to localize regions of hypoxia-induced angiogenesis early after infarction. In rodent studies, the specificity of (111)In-RP748 for alphavbeta3-integrin was confirmed with a negative control compound ((111)In-RP790), and regional uptake of these compounds correlated with (201)Tl perfusion and a (99m)Tc-labeled nitroimidazole (BRU59-21), which was used as a quantitative marker of myocardial hypoxia. The ex vivo analysis demonstrated that only (111)In-RP748 was selectively retained in infarcted regions with reduced (201)Tl perfusion and correlated with uptake of BRU59-21. In canine studies, myocardial uptake of (111)In-RP748 was assessed using in vivo single-photon-emission computed tomography (SPECT), ex vivo planar imaging, and gamma well counting of myocardial tissue and correlated with (99m)Tc-labeled 2-methoxy-2-methyl-propyl-isonitrile ((99m)Tc-sestamibi) perfusion. Dual-radiotracer in vivo SPECT imaging of (111)In-RP748 and (99m)Tc-sestamibi provided visualization of (111)In-RP748 uptake within the infarct region, which was confirmed by ex vivo planar imaging of excised myocardial slices. Myocardial (111)In-RP748 retention was associated with histological evidence of alphavbeta3-integrin expression/activation in the infarct region. (111)In-RP748 imaging provides a novel noninvasive approach for evaluation of hypoxia-induced alphavbeta3-integrin activation in myocardium early after infarction and may prove useful for directing and evaluating angiogenic therapies in patients with ischemic heart disease.     

Isolated hearts treated with skeletal muscle homogenates exhibit altered function

Skeletal muscle fiber damage and necrosis can result in the release of intracellular molecules into the extracellular environment. These molecules, termed damage-associated molecular patterns (DAMPs), can act as signals capable of initiating immune and/or inflammatory responses through interactions with pattern recognition receptors. To investigate whether skeletal muscle DAMPs interact with the heart and alter cardiac function, isolated rat hearts were perfused for 75 min with buffer containing 1 μg/ml of either soleus (slow), white gastrocnemius (WG, fast), or heat-stressed white gastrocnemius (HSWG) skeletal muscle homogenates. Left ventricular developed pressure (LVDP) and rates of pressure increase/decrease (±dP/dt) were measured using the Langendorff technique. Compared to controls, no changes in LVDP or +dP/dt were observed over the 75-min perfusion when homogenates from the WG muscles were added. In contrast, at 30 min and thereafter, a decreased LVDP and +dP/dt was observed in the hearts treated with soleus muscle homogenates. The hearts treated with HSWG homogenates also showed a decrease in LVDP from 45 min until the end of perfusion. These results suggest that molecules present in slow muscle and heat-stressed muscle are capable of altering cardiac function. Thus, muscle fiber type and/or heat shock protein content of skeletal muscles may be factors that influence cardiac function following skeletal muscle damage.     

No limiting role for glycogenin in determining maximal attainable glycogen levels in rat skeletal muscle

We examined whether the protein level and/or activity of glycogenin, the protein core upon which glycogen is synthesized, is limiting for maximal attainable glycogen levels in rat skeletal muscle. Glycogenin activity was 27.5 +/- 1.4, 34.7 +/- 1.7, and 39.7 +/- 1.3 mU/mg protein in white gastrocnemius, red gastrocnemius, and soleus muscles, respectively. A similar fiber type dependency of glycogenin protein levels was seen. Neither glycogenin protein level nor the activity of glycogenin correlated with previously determined maximal attainable glycogen levels, which were 69.3 +/- 5.8, 137.4 +/- 10.1, and 80.0 +/- 5.4 micromol/g wet wt in white gastrocnemius, red gastrocnemius, and soleus muscles, respectively. In additional experiments, rats were exercise trained by swimming, which resulted in a significant increase in the maximal attainable glycogen levels in soleus muscles ( approximately 25%). This increase in maximal glycogen levels was not accompanied by an increase in glycogenin protein level or activity. Furthermore, even in the presence of very high glycogen levels ( approximately 170 micromol/g wet wt), approximately 30% of the total glycogen pool continued to be present as unsaturated glycogen molecules (proglycogen). Therefore, it is concluded that glycogenin plays no limiting role for maximal attainable glycogen levels in rat skeletal muscle.     

Effect of endurance training on the phospholipid content of skeletal muscles in the rat

Only few data are available on the effect of training on phospholipid metabolism in skeletal muscles. The aim of the present study was to examine the effect of 6 weeks of endurance training on the content of particular phospholipid fractions and on the incorporation of blood-borne [14C]-palmitic acid into the phospholipids in different skeletal muscles (white and red sections of the gastrocnemius, the soleus and the diaphragm) of the rat. Lipids were extracted from the muscles and separated using thin-layer chromatography into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, cardiolipin and neutral lipids (this fraction being composed mostly of triacylglycerols). It was found that training did not affect the content of any phospholipid fraction in soleus muscle. It increased the content of sphingomyelin in white gastrocnemius muscle, cardiolipin and phosphatidylethanolamine in red gastrocnemius muscle and phosphatidylinositol in white gastrocnemius muscle and diaphragm. The total phospholipid content in red gastrocnemius muscle of the trained group was higher than in the control group. Training reduced the specific activity of sphingomyelin and cardiolipin in all muscles, phosphatidylcholine in soleus, red, and white gastrocnemius muscles, phosphatidylserine in all muscles, phosphatidylinositol in all except the soleus muscle, and phosphatidylethanolamine in hindleg muscles, but not in the diaphragm compared to the corresponding values in the sedentary group. It was concluded that endurance training affects skeletal muscle phospholipid content and the rate of incorporation of the blood-borne [14C]palmitic acid into the phospholipid moieties.     

Structural and functional remodeling of skeletal muscle microvasculature is induced by simulated microgravity

Hindlimb unloading of rats results in a diminished ability of skeletal muscle arterioles to constrict in vitro and elevate vascular resistance in vivo. The purpose of the present study was to determine whether alterations in the mechanical environment (i.e., reduced fluid pressure and blood flow) of the vasculature in hindlimb skeletal muscles from 2-wk hindlimb-unloaded (HU) rats induces a structural remodeling of arterial microvessels that may account for these observations. Transverse cross sections were used to determine media cross-sectional area (CSA), wall thickness, outer perimeter, number of media nuclei, and vessel luminal diameter of feed arteries and first-order (1A) arterioles from soleus and the superficial portion of gastrocnemius muscles. Endothelium-dependent dilation (ACh) was also determined. Media CSA of resistance arteries was diminished by hindlimb unloading as a result of decreased media thickness (gastrocnemius muscle) or reduced vessel diameter (soleus muscle). ACh-induced dilation was diminished by 2 wk of hindlimb unloading in soleus 1A arterioles, but not in gastrocnemius 1A arterioles. These results indicate that structural remodeling and functional adaptations of the arterial microvasculature occur in skeletal muscles of the HU rat; the data suggest that these alterations may be induced by reductions in transmural pressure (gastrocnemius muscle) and wall shear stress (soleus muscle).     

Effect of plasma free fatty acid concentration on the content and composition of the free fatty acid fraction in rat skeletal muscles.

Skeletal muscles contain a fraction of free (unesterified) fatty acids. This fraction is very small, but important since it contributes to the creation of the plasma-myocyte free fatty acid concentration gradient. Maintenance of this gradient is necessary for blood-borne fatty acids to be transported into the cell. There are no data on the regulation of the content and composition of the free fatty acid fraction in the cell. The aim of the present study was to examine the effect of an elevation and a reduction in the plasma-borne free fatty acid concentration on the content and composition of the free fatty acid fraction in different skeletal muscle types. The experiments were carried out on male Wistar rats with 280 - 310 g body weight. They were divided into four groups - 1, control; 2, exercised 3 h on a treadmill moving with a speed of 1,200 m/h and set at + 10 degrees incline; 3, treated with heparin; and 4, treated with nicotinic acid. Samples of the soleus as well as the red and white sections of the gastrocnemius muscles were taken. These muscles are composed mostly of slow-twitch oxidative, fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibres, respectively. Lipids were extracted from the muscle samples and from the blood; the free fatty acid fraction was isolated by means of thin-layer chromatography. The individual free fatty acids were identified and quantified using gas-liquid chromatography. The plasma concentration of free fatty acids was as follows: control group, 236.1 +/- 32.9; after exercise, 407.4 +/- 117.5; after heparin, 400.8 +/- 36.8; and after nicotinic acid, 102.5 +/- 26.1 micromol/l (p < 0.01 vs. control values in each case). The total content of the free fatty acid fraction in the control group was as follows: white gastrocnemius, 27.6 +/- 7.3; red gastrocnemius, 52.2 +/- 13.9; soleus, 72.3 +/- 10.2 nmol/g. Elevation in plasma free acid concentration during exercise increased the total content of free fatty acids in the white gastrocnemius (38.7 +/- 13.9) and in the soleus (103.4 +/- 15.9 nmol/g; rest-exercise: p < 0.05 and p < 0.01, respectively), but had no effect in the red gastrocnemius. Neither elevation in the plasma free fatty acid concentration with heparin nor reduction with nicotinic acid affected the total content of the free fatty acid fraction in the muscles examined. The ratio of plasma concentration of individual acid to muscle concentration for the same acid varied greatly, depending on acid, muscle type and experimental group. The ratio was positive (above unity) for each acid almost in all cases with the exception of certain acids in the nicotinic acid-treated group where it was below unity. We conclude that the skeletal myocytes maintain a stable level of free fatty acid fraction in the wide range of plasma free fatty acid concentrations.     

雌、雄草原田鼠外周骨骼肌肌球蛋白重链的表达

已往的研究对于实验室应用的各种啮齿类动物,如大鼠和小鼠骨骼肌蛋白表达的特性已有报道.然而,至今不清楚其它啮齿类动物如野生鼠骨骼肌蛋白的表达或性双态性的特征,而这些野生鼠的行为学、形态学及生理学特点均已有报道.已知骨骼肌的肌球蛋白重链(MHC)成分与其功能特性有关.我们研究了草原田鼠的肱三头肌、胫骨前肌、腓肠肌和比目鱼肌MHC蛋白表达的性别特性.应用SDS聚丙烯酰胺凝胶电泳法测定MHC Ⅰ型、Ⅱa型、Ⅱd/x和Ⅱb型的蛋白表达相对含量.结果表明:与雌鼠相比,雄鼠的比目鱼肌湿重较大,胫骨前肌的MHC Ⅱa蛋白量表达较高.未见骨骼肌重量及MHC蛋白表达含量在雌雄鼠间的性别差异.血中睾酮的浓度差异可能不影响外周骨骼肌蛋白的表达特性.然而,与过去在大鼠、兔和小鼠中的已报道的结果相比,草原田鼠骨骼肌MHC的表达显示了更多异质性.推测这可能与草原田鼠和其它小型哺乳类动物生存的自然环境和功能需要有关.     

Effect of acute exercise on the content of free sphinganine and sphingosine in different skeletal muscle types of the rat.

It has previously been shown that prolonged exercise of moderate intensity reduces the content of ceramide in each type of skeletal muscle. This was accompanied by a reduction in the activity of neutral, Mg++-dependent sphingomyelinase (the major enzyme responsible for ceramide formation from sphingomyelin) in the soleus and red gastrocnemius, but not in the white gastrocnemius (A. Dobrzyń and J. Górski, Am. J. Physiol.: Endorcinol. Metab. 282: E277 - E285, 2002). No other data on regulation of ceramide metabolism in contracting muscles are available. The aim of the present study was to examine the content of sphinganine (a key precursor of ceramide on the de novo synthesis route) and the content of sphingosine (the main product of ceramide catabolism) in different skeletal muscle types after two kinds of acute exercise. The experiments were carried out on 30 male Wistar rats, 250 - 280 g of body weight. The rats were divided equally into three groups: 1 - control, 2 - run until exhaustion (1200 m/h, +10 degree incline), 3 - a group in which the sciatic nerve was stimulated 10 min with tetanic pulses (60 pulses/min). Samples were taken of the soleus and of the red and white section of the gastrocnemius. These muscles are composed mostly of the slow-twitch oxidative, fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibers, respectively. Lipids were extracted with chloroform/methanol. Sphinganine and sphingosine were quantified by high-performance liquid chromatography. At rest, the content of sphinganine in the soleus was higher than in the red gastrocnemius (p < 0.05), and in the latter, it was higher than in the white gastrocnemius (p < 0.01). Prolonged exercise increased the content of sphinganine approximately 6-fold in each muscle. The resting content of sphingosine in the soleus and in the red gastrocnemius was similar--higher than in the white gastrocnemius (p < 0.001 and p < 0.01, respectively). The content of sphingosine increased over 3-fold in the soleus and nearly 2-fold in the red and white sections of the gastrocnemius. Stimulation of the sciatic nerve increased the content of both compounds approximately 2-fold in each muscle. We conclude that acute exercise increases both de novo synthesis and catabolism of ceramide in skeletal muscles. Accumulation of sphingosine in contracting muscles may contribute to the development of fatigue.     

Skeletal muscle fiber type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression in fed and food-deprived rats

Fiber type specificity of pyruvate dehydrogenase (PDH) phosphatase (PDP) was determined in fed (CON) and 48-h food-deprived (FD) rats. PDP activity and isoform protein content were determined in soleus (slow-twitch oxidative), red gastrocnemius (RG; fast-twitch oxidative glycolytic), and white gastrocnemius (WG; fast-twitch glycolytic) muscles. When normalized for mitochondrial volume, there was no difference in PDP activity between muscle types or CON and FD. When expressed per gram wet tissue weight, PDP activity was higher in RG compared with soleus and WG in both CON and FD rats. PDP activities from CON muscles were 1.48 +/- 0.19, 2.68 +/- 0.65, and 1.20 +/- 0.33 nmol x min(-1) x g wet tissue wt(-1) in soleus, RG, and WG, respectively, and decreased in FD muscles (1.22 +/- 0.22, 2.00 +/- 0.57, and 0.84 +/- 0.18 nmol x min(-1) x g wet tissue wt(-1)). This correlated with increased PDP2 protein, however, only in RG, as PDP2 was not detectable in soleus or WG. PDP1 protein was not responsive to food deprivation in all fiber types. In conclusion, PDP activity and protein content were higher in fast-twitch oxidative glycolytic muscles from CON and FD rats, identifying a unique inter- and intramuscular distribution. FD induced a small but significant decrease in PDP activity that was partially due to decreases in PDP2 protein. As a result, coordinate changes to PDP activity opposite to those of the other regulatory enzyme, PDH kinase, during food deprivation would maximize the inactivation of skeletal muscle PDH and enhance carbohydrate conservation during periods of limited carbohydrate supply.     

Assessment of brown adipose tissue activity in rats by 99mTc-sestamibi uptake

Brown adipose tissue (BAT) physiology and imaging have recently attracted considerable attention. BAT is characterized both by enhanced perfusion and increased mitochondrial activity. (99m)Tc-sestamibi is a lipophilic cationic tracer that concentrates in mitochondria. Data on the accumulation of (99m)Tc-sestamibi in BAT are currently lacking. This study investigates the in vivo (99m)Tc-sestamibi uptake in rat BAT. (99m)Tc-sestamibi was administered in male Wistar rats of various age and body size. (99m)Tc-sestamibi uptake was measured in vitro in BAT and white fat (WF) together with cytochrome c oxidase activity. Both (99m)Tc-sestamibi uptake and cytochrome c oxidase activity were higher in BAT than in WF (P<0.05). (99m)Tc-Sestamibi uptake in both BAT and WF was negatively related to body weight (r = -0.96 and -0.89, respectively) as was the BAT/WF uptake ratio (r = -0.85). These data show a higher (99m)Tc-sestamibi uptake in BAT compared to WF, in agreement with the high mitochondrial content and respiratory activity of the former. The strong negative correlation between (99m)Tc-sestamibi uptake in BAT and body weight (negative allometry), is in accordance to increased needs of thermogenesis in smaller animals. Implications of increased (99m)Tc-sestamibi uptake in BAT in radionuclide imaging are also discussed.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Increased DNA fragmentation and altered apoptotic protein levels in skeletal muscle of spontaneously hypertensive rats.

Apoptosis is a highly conserved process that plays an important role in controlling tissue development, homeostasis, and architecture. Dysregulation of apoptosis is a hallmark of numerous human pathologies including hypertension. In the present work we studied the effect of hypertension on apoptosis and the expression of several apoptotic signaling and/or regulatory proteins in four functionally and metabolically distinct muscles. Specifically, we examined these markers in soleus, red gastrocnemius, white gastrocnemius, and left ventricle (LV) of 20-wk-old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Compared with WKY rats SHR had a significantly greater heart weight, LV weight, and mean arterial pressure. In general, SHR skeletal muscle had increased Bax protein, procaspase-3 protein, caspase-3 activity, cleaved poly(ADP-ribose) polymerase protein, and DNA fragmentation as well as decreased Bcl-2 protein and a lower Bcl-2-to-Bax ratio. Subcellular distribution studies demonstrated increased levels of apoptosis-inducing factor protein in cytosolic or nuclear extracts as well as elevated nuclear Bax protein in SHR skeletal muscle. Moreover, heat shock protein 70 in red gastrocnemius and soleus was significantly correlated to several apoptotic factors. With the exception of lower heat shock protein 90 levels in SHR no additional differences in any apoptotic markers were observed in LV between groups. Collectively, this report provides the first evidence that apoptotic signaling is altered in skeletal muscle of hypertensive animals, an effect that may be mediated by both caspase-dependent and -independent mechanisms. This proapoptotic state may provide some understanding for the morphological and functional abnormalities observed in skeletal muscle of hypertensive animals.     

Differential response of rat skeletal muscle glycogen metabolism to testosterone and estradiol.

Although reports on sex steroids have implicated them as promoting protein synthesis and also providing extra strength to the skeletal muscle, it remains unclear whether sex steroids affect glycogen metabolism to provide energy for skeletal muscle functions, since glycogen metabolism is one of the pathways that provides energy for the skeletal muscle contraction and relaxation cycle. The purpose of the current study was to show that testosterone and estradiol act differentially on skeletal muscles from different regions, differentially with reference to glycogen metabolism. To study this hypothesis, healthy mature male Wistar rats (90-120 days of age, weighing about 180-200 g) were castrated (a bilateral orchidectomy was performed to test the significance of skeletal muscle glycogen metabolism in the absence of testosterone). One group of castrated rats was supplemented with testosterone (100 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards). To test whether estradiol has any effect on male skeletal muscle glycogen metabolism 17beta-estradiol (5 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards) was administered to orchidectomized rats. To test whether these sex steroids have any differential effect on skeletal muscles from different regions, skeletal muscles from the temporal region (temporalis), muscle of mastication (masseter), forearm muscle (triceps and biceps), thigh muscle (vastus lateralis and gracilis), and calf muscle (gastrocnemius and soleus) were considered. Castration enhanced blood glucose levels and decreased glycogen stores in skeletal muscle from head, jaw, forearm, thigh, and leg regions. This was accompanied by diminished activity of glycogen synthetase and enhanced activity of muscle phosphorylase. Following testosterone supplementation to castrated rats, a normal pattern of all these parameters was maintained. Estradiol administration to castrated rats did not bring about any significant alteration in any of the parameters. The data obtained suggest a stimulatory effect of testosterone on skeletal muscle glycogenesis and an inhibitory effect on glycogenolysis. Estradiol did not play any significant role in the skeletal muscle glycogen metabolism of male rats.     

Effects of fiber composition and hindlimb unloading on the vasodilator properties of skeletal muscle arterioles.

It has been hypothesized that microgravity-induced orthostatic hypotension may result from an exaggerated vasodilatory responsiveness of arteries. The purpose of this study was to determine whether skeletal muscle arterioles exhibit enhanced vasodilation in rats after 2 wk of hindlimb unloading (HU). First-order arterioles isolated from soleus and white gastrocnemius muscles were tested in vitro for vasodilatory responses to isoproterenol (Iso), adenosine (Ado), and sodium nitroprusside (SNP). HU had no effect on responses induced by Iso but diminished maximal vasodilation to Ado and SNP in both muscles. In addition, vasodilatory responses in arterioles from control rats varied between muscle types. Maximal dilations induced by Iso (soleus: 42 +/- 6%; white gastrocnemius: 60 +/- 7%) and Ado (soleus: 51 +/- 8%; white gastrocnemius: 81 +/- 6%) were greater in arterioles from white gastrocnemius muscles. These data do not support the hypothesis that microgravity-induced orthostatic hypotension results from an enhanced vasodilatory responsiveness of skeletal muscle arterioles. Furthermore, the data support the concept that dilatory responsiveness of arterioles varies in muscle composed of different fiber types.     

Diminished overload-induced hypertrophy in aged fast-twitch skeletal muscle is associated with AMPK hyperphosphorylation.

Skeletal muscle mass declines with age, as does the potential for overload-induced fast-twitch skeletal muscle hypertrophy. Because 5'-AMP-activated protein kinase (AMPK) activity is thought to inhibit skeletal muscle protein synthesis and may therefore modulate muscle mass and hypertrophy, the purpose of this investigation was to examine AMPK phosphorylation status (a marker of AMPK activity) and its potential association with the attenuated overload-induced hypertrophy observed in aged skeletal muscle. One-week overload of fast-twitch plantaris and slow-twitch soleus muscles was achieved in young adult (8 mo; n = 7) and old (30 mo; n = 7) Fischer344 x Brown Norway male rats via unilateral gastrocnemius ablation. Significant (P < or = 0.05) age-related atrophy (as measured by total protein content) was noted in plantaris and soleus control (sham-operated) muscles. In fast-twitch plantaris muscles, percent hypertrophy with overload was significantly attenuated with age, whereas AMPK phosphorylation status as determined by Western blotting [phospho-AMPK (Thr172)/total AMPK] was significantly elevated with age (regardless of loading status). There was also a main effect of loading on AMPK phosphorylation status in plantaris muscles (overload > control). Moreover, a strong and significant negative correlation (r = -0.82) was observed between AMPK phosphorylation status and percent hypertrophy in the overloaded plantaris muscles of all animals. In contrast to the plantaris, overload-induced hypertrophy of the slow-twitch soleus muscle was similar between ages, and AMPK phosphorylation in this muscle was also unaffected by age or overload. These data support the possibility that an age-related elevation in AMPK phosphorylation may partly contribute to the attenuated hypertrophic response observed with age in overloaded fast-twitch plantaris muscle.     

Phosphate uptake in rat skeletal muscle is reduced during isometric contractions.

During contractions, there is a net efflux of phosphate from skeletal muscle, likely because of an elevated intracellular inorganic phosphate (P(i)) concentration. Over time, contracting muscle could incur a substantial phosphate deficit unless P(i) uptake rates were increased during contractions. We used the perfused rat hindquarter preparation to assess [(32)P]P(i) uptake rates in muscles at rest or over a range of energy expenditures during contractions at 0.5, 3, or 5 Hz for 30 min. P(i) uptake rates were reduced during contractions in a pattern that was dependent on contraction frequency and fiber type. In soleus and red gastrocnemius, [(32)P]P(i) uptake rates declined by approximately 25% at 0.5 Hz and 50-60% at 3 and 5 Hz. Uptake rates in white gastrocnemius decreased by 65-75% at all three stimulation frequencies. These reductions in P(i) uptake are not likely confounded by changes in precursor [(32)P]P(i) specific activity in the interstitium. In soleus and red gastrocnemius, declines in P(i) uptake rates were related to energy expenditure over the contraction duration. These data imply that P(i) uptake in skeletal muscle is acutely modulated during contractions and that decreases in P(i) uptake rates, in combination with expected increases in P(i) efflux, exacerbate the net loss of phosphate from the cell. Enhanced uptake of P(i) must subsequently occur because skeletal muscle typically maintains a relatively constant total phosphate pool.     

Angiotensin-converting enzyme inhibition attenuates myonuclear addition in overloaded slow-twitch skeletal muscle

Because optimal overload-induced skeletal muscle hypertrophy requires ANG II, we aimed to determine the effects of blocking ANG II production [via angiotensin-converting enzyme (ACE) inhibition] on potential mediators of hypertrophy in overloaded skeletal muscle, namely, myonuclear addition and fibroblast content. In a 2 x 2 design, adult (200-225 g) female Sprague-Dawley rats were placed into one of four groups (n = 8/group): 7-day skeletal muscle overload, sham operation, 7-day skeletal muscle overload with ACE inhibition, or sham operation with ACE inhibition. Functional overloads of the plantaris and soleus muscles were produced via bilateral surgical ablation of the synergistic gastrocnemius muscle, and ACE inhibition was accomplished by the addition of the ACE inhibitor enalapril maleate to the animals' daily drinking water (0.3 mg/ml). Myonuclear addition and extrasarcolemmal nuclear proliferation, as measured by in vivo 5-bromo-2'-deoxyuridine labeling, were significantly (P < or = 0.05) increased by overload in both the slow-twitch soleus and fast-twitch plantaris muscles. Furthermore, ACE inhibition attenuated these overload-induced increases in the soleus muscle but not in the plantaris muscle. However, the effect of ACE inhibition on soleus extrasarcolemmal nuclei was not likely due to differences in fibroblast content because overload elicited significant increases in vimentin-positive areas in soleus and plantaris muscles, and these areas were unaffected by ACE inhibition in either muscle. There was no effect of ACE inhibition on any measure in sham-operated muscles. Collectively, these data indicate that ANG II may mediate the satellite cell response to overload in slow-twitch soleus but not in fast-twitch plantaris muscles and that this effect may occur independently of changes in fibroblast content.     

Apoptotic responses to hindlimb suspension in gastrocnemius muscles from young adult and aged rats

Although apoptosis has been demonstrated in soleus during hindlimb suspension (HS), it is not known whether apoptosis is also involved in the loss of muscles dominated by mixed fibers. Therefore, we examined the apoptotic responses in gastrocnemius muscles of young adult and aged Fischer 344 x Brown Norway rats after 14 days of HS. The medial gastrocnemius muscle wet weight significantly decreased by 30 and 32%, and muscle wet weight normalized to the animal body weight decreased by 11 and 15% in young adult and aged animals, respectively, after HS. The extent of apoptotic DNA fragmentation increased by 119 and 61% in suspended muscles from young and aged rats, respectively. Bax mRNA increased by 73% in young muscles after HS. Bax and Bcl-2 protein levels were greater in suspended muscles relative to control muscles in both age groups. The level of cytosolic mitochondria-housed apoptotic factor cytochrome c was significantly increased in the mitochondria-free cytosol of suspended muscles from young and aged rats. In contrast, the release/accumulation of AIF, a caspase-independent apoptogenic factor, was exclusively expressed in the suspended muscles from aged rats. Our data also show that aging favors the proapoptotic signaling in skeletal muscle by altering the contents of Bax, Bcl-2, Apaf-1, AIF, caspases, XIAP, Smac/DIABLO, and cytochrome c. Furthermore, these results indicate that apoptosis occurs not only in slow-twitch soleus muscle but also in the mixed-fiber (predominately fast fibered) gastrocnemius muscle. Our data are consistent with the hypothesis that apoptotic signaling differs in young adult and aged gastrocnemius muscles during HS.     

An investigation of arterial insufficiency in the rat hindlimb. Correlation of skeletal muscle bloodflow and glucose utilization in vivo.

Muscle bloodflow and the rate of glucose uptake and phosphorylation were measured in vivo in rats 7 days after unilateral femoral artery ligation and section. Bloodflow was determined by using radiolabelled microspheres. At rest, bloodflow to the gastrocnemius, plantaris and soleus muscles of the ligated limb was similar to their respective mean contralateral control values; however, bilateral sciatic nerve stimulation at 1 Hz caused a less pronounced hyperaemic response in the muscles of the ligated limb, being 59, 63 and 49% of their mean control values in the gastrocnemius, plantaris and soleus muscles respectively. The rate of glucose utilization was determined by using the 2-deoxy[3H]glucose method [Ferré, Leturque, Burnol, Penicaud & Girard (1985) Biochem. J. 228, 103-110]. At rest, the rate of glucose uptake and phosphorylation was statistically significantly increased in the gastrocnemius and soleus muscles of the ligated limb, being 126 and 140% of the mean control values respectively. Bilateral sciatic nerve stimulation at 1 Hz caused a 3-5-fold increase in the rate of glucose utilization by the ligated and contralateral control limbs; furthermore, the rate of glucose utilization was significantly increased in the muscles of the ligated limb, being 140, 129 and 207% of their mean control values respectively. For the range of bloodflow to normally perfused skeletal muscle at rest or during isometric contraction determined in the present study, a linear correlation between the rate of glucose utilization and bloodflow can be demonstrated. Applying similar methods of regression analysis to glucose utilization and bloodflow to muscles of the ligated limb reveals a similar linear correlation. However, the rate of glucose utilization at a given bloodflow is increased in muscles of the ligated limb, indicating an adaptation of skeletal muscle to hypoperfusion.