Rat Diaphragm Research Articles

Contractile properties of the diaphragm in creatine-fed rats.

1. Creatine feeding increases the oxidative capacity of type 1 skeletal muscle fibres and, in soleus muscles, consisting mainly of type 1 fibres, increases fatigue resistance. The diaphragm contains a relatively large content of type 1 fibres and respiratory muscle fatigue is a cause of respiratory failure. The aim of the present study was to determine whether creatine supplements increase fatigue resistance in the diaphragm. 2. Rats were given creatine monohydrate (2.55 g/L) in the drinking water. After 5-6 days, isometric contractile properties were measured in strips of costal diaphragm in Krebs' solution at 30 degrees C. Measurements were also made in soleus muscle strips. Values for strips from creatine-fed rats were compared with those from control rats. 3. Creatine feeding did not increase fatigue resistance and had no effect on twitch or tetanic tension or twitch kinetics in the diaphragm. Creatine increased fatigue resistance in soleus muscles, as reported previously.

An Investigation into the Mechanism of Action of Almitrine on Isolated Rat Diaphragm Muscle Fatigue

Background: Previous studies have shown that almitrine bismesylate, a respiratory stimulant which acts on the mitochondrial electron transport chain, enhances recovery of rat diaphragm muscle from fatigue. Objectives: Our aim is to investigate if the enhanced recovery is due to an anti-oxidant property of almitrine, since the electron transport chain is a major site of intracellular free radical production. Methods: A low-frequency fatigue protocol was used (30 Hz; 250 ms; delivered once every 2 s for 5 min), and the effects of almitrine before and after fatigue onset were compared to those of the anti-oxidant compound N-acetylcysteine (NAC). Results: Almitrine (6 and 10 µg/ml) given before fatigue gave better recovery rates than postfatigue application. In contrast, NAC (100 µM) application before fatigue onset was not as effective as NAC given immediately after the cessation of the fatigue protocol. However, almitrine (6 µg/ml) completely reversed the reduction in baseline twitch tension brought about by a free-radical-producing mixture of FeCl₃ + ADP (1 mM + 2.5 mM, respectively). Conclusion: The results of this study confirm that almitrine enhances recovery from fatigue and, in contrast to NAC prefatigue application, is more effective. Also, almitrine was shown to have an anti-oxidant effect, but it does not act like a typical anti-oxidant.

Dynamics of microvascular oxygen pressure in the rat diaphragm.

The relative amplitudes and rates of increase of muscle blood flow (and O(2) delivery) and O(2) uptake responses determine the O(2) pressure within the muscle microvasculature (Pm(O(2))) across the rest-to-contraction transition. Skeletal muscle function is a primary determinant of pulmonary O(2) uptake kinetics; however, it has never been determined whether the dynamics of muscle Pm(O(2)) are faster in a highly oxidative muscle [e.g., diaphragm (Dia), citrate synthase activity of 39 micromol. min(-1). g(-1)] compared with less oxidative muscles [e.g., spinotrapezius (Spino), citrate synthase activity of 14 micromol. min(-1). g(-1), male Sprague-Dawley rats; Delp MD and Duan C, J Appl Physiol 80: 261-270, 1996]. Phosphorescence quenching techniques (porphyrin dendrimer, R2) were used to determine Pm(O(2)) across the transition to electrically stimulated contractions (1 Hz) within the rat Dia. After a delay of 10.4 +/- 1.3 (SE) s at the beginning of Dia contractions, Pm(O(2)) decreased close to monoexponentially from 42 +/- 2 to 27 +/- 3 Torr (P < 0.05) with an extremely fast time constant of 7.1 +/- 1.1 s. Thus Dia Pm(O(2)) decreased with significantly (P < 0.05) faster kinetics than reported previously for the Spino muscle (delay, 19.2 +/- 2.8 s; time constant Pm(O(2)), 21.7 +/- 2.1 s; Behnke BJ, Kindig CA, Musch TI, Koga S, and Poole DC, Respir Physiol 126: 53-63, 2001). With the use of two specialized muscles with similar fiber-type composition but widely disparate oxidative capacities (Delp MD and Duan C, J Appl Physiol 80: 261-270, 1996), these data demonstrate that Pm(O(2)) kinetics are significantly faster in the highly oxidative Dia compared with the low-oxidative Spino muscle and that this effect is not dependent on muscle fiber-type composition.

NORMOBARIC HYPOXIA ALTERS METABOLIC PROPERTY IN RAT DIAPHRAGM

It is well established that exposures to hypoxia results in physiological adaptation in both human and animals. Recently, from the viewpoint of erythropoiesis, normobaric hypoxic environment has been applied for conditioning and exercise training in flats. However, no studies have examined the adaptation of respiratory muscle under normobaric hypoxic environment. PURPOSE: To examine effects of normobaric hypoxia on metabolic properties of diaphragm, we measured glycolytic and oxidative enzyme activities of rat diaphragm after 50 days exposure to normobaric hypoxia. Furthermore, we studied their changes after the exposure to hypoxia. METHODS: Seventy male Wistar rats (8 weeks old) were randomly assigned to a normobaric hypoxia (H) group or an aged-matched normoxia (N) group. H group was housed in a normobaric hypoxic room (15.4%O2) for 50days and then it was moved into a normoxic room for 50 days. At the 0, 25th, 50th, 75th, 100th day of the experiment period, 7- 8 rats of each group were anesthetized with pentobarbital sodium. Costal diaphragm was removed, frozen in liquid nitrogen and stored at -80oC until assay. Lactate dehydrogenase (LDH) and citrate synthase (CS) activities were determined spectrophotometrically immediately following homogenization.ü@ RESULTS: LDH activity was increased with aging, on the other hand CS activity was decreased. H group showed significantly (p < 0.05) higher LDH activity (H: 986 ± 56 vs N: 907 ± 44 μmol/mg/min) at the end of the hypoxia period. However the increase was back to the same level of N group at 25 days after the hypoxia period. CS activity did not differ between both groups. As a result, LDH/CS ratio was similar to the change of LDH activity. CONCLUSION: These data indicate that exposure to normobaric hypoxia can enhance glycolytic capacity in diaphragm and that the effect would not be maintained under normoxic condition for so long.

Control of Ser2448 Phosphorylation in the Mammalian Target of Rapamycin by Insulin and Skeletal Muscle Load

We have investigated the effects of insulin, amino acids, and the degree of muscle loading on the phosphorylation of Ser(2448), a site in the mammalian target of rapamycin (mTOR) phosphorylated by protein kinase B (PKB) in vitro. Phosphorylation was assessed by immunoblotting with a phosphospecific antibody (anti-Ser(P)(2448)) and with mTAb1, an activating antibody whose binding is inhibited by phosphorylation in the region of mTOR that contains Ser(2448). Incubating rat diaphragm muscles with insulin increased Ser(2448) phosphorylation but did not change the total amount of mTOR. Insulin, but not amino acids, activated PKB, as evidenced by increased phosphorylation of both Ser(308) and Thr(473) in the kinase. Ser(2448) phosphorylation was also modulated by muscle-loading. Overloading the rat plantaris muscle by synergist muscle ablation, which promotes hypertrophy of the plantaris muscle, increased Ser(2448) phosphorylation. In contrast, unloading the gastrocnemius muscle by hindlimb suspension, which promotes atrophy of the muscle, decreased Ser(2448) phosphorylation, an effect that was fully reversible. Neither overloading nor hindlimb suspension significantly changed the total amount of mTOR. In summary, our results demonstrate that atrophy and hypertrophy of skeletal muscle are associated with decreases and increases in Ser(2448) phosphorylation, suggesting that modulation of this site may have an important role in the control of protein synthesis.

An experimental study of regional chemotherapy using CDDP-loaded microspheres for esophageal cancer.

The purpose of this study was to assess the antitumor effects of cisplatin-loaded microspheres (CDDP-MS) and the efficacy of the administration of CDDP-MS into the mediastinum. To evaluate the antitumor effect, we first performed a paratumoral injection of CDDP-MS to FF6 tumor-bearing DA rats to compare its effect with that of the intraperitoneal injection of a CDDP solution at different doses. In the CDDP-MS groups the tumor growth was effectively delayed in proportion to the dosage of CDDP-MS. All rats treated with the CDDP solution at a dose of 10 mg/kg died within 1 week, while no rats treated with CDDP-MS even at a CDDP dose of 20 mg/kg were lost. In the second experiment, which was designed to determine the delivery of the microspheres-released CDDP to various organs, CDDP-MS was injected directly into the mediastinum via the diaphragm in male Wistar rats. In the CDDP-MS group, the plasma CDDP concentration stayed significantly lower than that in the CDDP solution (intravenous) group while the tissue CDDP concentration in the paratracheal lymph nodes was higher. Moreover, the lymph node-to-kidney platinum ratio was eight times higher in the rats given CDDP-MS intramediastinally than in those given the CDDP solution intravenously. These results demonstrate that a high dose of CDDP can be administered with less systemic side effects by means of encapsulation in the microspheres, and that the administration of CDDP-MS into the mediastinum is more effective for delivering CDDP to the paratracheal lymph nodes. As a regional chemotherapy after esophageal cancer operation, the injection of CDDP-MS into the mediastinum for targeting of the lymph nodes thus promises to be an effective treatment.

Evaluation of the cholinomimetic actions of trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana (Gastropoda, Opisthobranchia).

Trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana, negatively modulates vagal response, indicating a probable ability to inhibit cholinergic responses. In the present study, the pharmacological profile of trimethylsulfonium was characterized on muscarinic and nicotinic acetylcholine receptors. In rat jejunum the contractile response induced by trimethylsulfonium (pD2 = 2.46 +/- 0.12 and maximal response = 2.14 +/- 0.32 g) was not antagonized competitively by atropine. The maximal response (Emax) to trimethylsulfonium was diminished in the presence of increasing doses of atropine (P<0.05), suggesting that trimethylsulfonium-induced contraction was not related to muscarinic stimulation, but might be caused by acetylcholine release due to presynaptic stimulation. Trimethylsulfonium displaced [3H]-quinuclidinyl benzilate from rat cortex membranes with a low affinity (Ki = 0.5 mM). Furthermore, it caused contraction of frog rectus abdominis muscles (pD2 = 2.70 +/- 0.06 and Emax = 4.16 +/- 0.9 g), which was competitively antagonized by d-tubocurarine (1, 3 or 10 microM) with a pA2 of 5.79, suggesting a positive interaction with nicotinic receptors. In fact, trimethylsulfonium displaced [3H]-nicotine from rat diaphragm muscle membranes with a Ki of 27.1 microM. These results suggest that trimethylsulfonium acts as an agonist on nicotinic receptors, and thus contracts frog skeletal rectus abdominis muscle and rat jejunum smooth muscle via stimulation of postjunctional and neuronal prejunctional nicotinic cholinoreceptors, respectively.

Effects of hypothyroidism on maximum specific force in rat diaphragm muscle fibers.

We hypothesized that 1) hypothyroidism (Hyp) decreases myosin heavy chain (MHC) content per half-sarcomere in diaphragm muscle (Dia(m)) fibers, 2) Hyp decreases the maximum specific force (F(max)) of Dia(m) fibers because of the reduction in MHC content per half-sarcomere, and 3) Hyp affects MHC content per half-sarcomere and F(max) to a greater extent in fibers expressing MHC type 2X (MHC(2X)) and/or MHC type 2B (MHC(2B)). Studies were performed on single Triton X-permeabilized fibers activated at pCa 4.0. MHC content per half-sarcomere was determined by densitometric analysis of SDS-polyacrylamide gels and comparison with a standard curve of known MHC concentrations. After 3 wk of Hyp, MHC content per half-sarcomere was reduced in fibers expressing MHC(2X) and/or MHC(2B). On the basis of electron-microscopic analysis, this reduction in MHC content was also reflected by a decrease in myofibrillar volume density and thick filament density. Hyp decreased F(max) across all MHC isoforms; however, the greatest decrease occurred in fibers expressing fast MHC isoforms (approximately 40 vs. approximately 20% for fibers expressing slow MHC isoforms). When normalized for MHC content per half-sarcomere, force generated by Hyp fibers expressing MHC(2A) was reduced compared with control fibers, whereas force per half-sarcomere MHC content was higher for fibers expressing MHC(2X) and/or MHC(2B) in the Hyp Dia(m) than for controls. These results indicate that the effect of Hyp is more pronounced on fibers expressing MHC(2X) and/or MHC(2B) and that the reduction of F(max) with Hyp may be at least partially attributed to a decrease in MHC content per half-sarcomere but not to changes in force per cross bridge.

The metabolic role of branched-chain amino acids

14 C] leucine entering the tissue is decarboxylated in the diaphragm and that a considerable proportion is degraded even further. The percentages of decarboxylation for isoleucine (58%) and valine (31%) also were substantial. Addition of insulin brought about a small but consistent stimulation of leucine oxidation. Moreover, the fraction of isotope taken up by the diaphragm appearing as 14 CO2 did not decline markedly as the concentration of amino acids supplied rose, in contrast with the amount of isotope incorporated into protein, which dropped steadily as the specific activity fell. 3 Further extensive work exploring the possibility that oxidation might be a major metabolic pathway for leucine (and possibly all three BCAAs) in rat diaphragm and in soleus and extensor digitorm longus muscles was published in 1972 by Odessey and Goldberg.4 They determined that the diaphragm posseses a marked ability to degrade leucine 1- 14 Ct o 14 CO2. The metabolism of leucine by the diaphragm depended on the concentration, and leucine uptake and oxidation increased with increasing external concentrations. Further, the amount of leucine degradation relative to its incorporation into protein increased 10-fold as its external concentration was raised. The red soleus and pale extensor degitorum longus muscles exhibited a similar marked capacity for leucine oxidation. The rates of CO2 production, incorporation into protein, and total uptake in these two muscles were lower than those in the diaphragm. The skeletal muscle mass, which constitutes 43% of the body mass, is the major site for leucine oxidation in the body, marking leucine and possibly the other BCAAs as significant energy sources for the muscle. Complete oxidation of leucine in the muscle yields more adenosine triphosphate molecules on a molar basis than complete oxidation of glucose. Further, this inherent ability of muscle to oxidize leucine increases under certain physiologic states such as food deprivation. 5 Later work determined that the rate of oxidation of BCAA in muscle is under metabolic and hormonal regulation. Buse and Reid6 in skeletal muscle and Chua et al.7 in cardiac muscle suggested that leucine also regulates the turnover of protein in muscle cells by inhibiting protein degradation and enhancing protein synthesis. This regulation of muscle protein turnover by leucine influences the transition to negative

Effects of postnatal maturation on energetics and cross-bridge properties in rat diaphragm.

The effects of maturation on cross-bridge (CB) properties were studied in rat diaphragm strips obtained at postnatal days 3, 10, and 17 and in adults (10-12 wk old). Calculations of muscle energetics and characteristics of CBs were determined from standard Huxley equations. Maturation did not change the curvature of the force-velocity relationship or the peak of mechanical efficiency. There was a significant increase in the total number of CBs per cross-sectional area (m) with aging but not in single CB force. The turnover rate of myosin ATPase increased, the duration of the CB cycle decreased, and the velocity of CBs decreased significantly only after the first week postpartum. There was a linear relationship between maximum total force and m (r = 0.969, P < 0.001), and between maximum unloaded shortening velocity and m (r = 0.728, P < 0.001). When this study in the rat and previous study in the hamster are compared, it appears that there are few species differences in the postnatal maturation process of the diaphragm.

Alteration of contractile force and mass in the senescent diaphragm with beta(2)-agonist treatment.

Aging is associated with a decrease in diaphragmatic maximal tetanic force production (P(o)) in senescent rats. Treatment with the beta(2)-agonist clenbuterol (CB) has been shown to increase skeletal muscle mass and P(o) in weak locomotor skeletal muscles from dystrophic rodents. It is unknown whether CB can increase diaphragmatic mass and P(o) in senescent rats. Therefore, we tested the hypothesis that CB treatment will increase specific P(o) (i.e., force per cross-sectional area) and mass in the diaphragm of old rats. Young (5 mo) and old (23 mo) male Fischer 344 rats were randomly assigned to one of the following groups (n = 10/group): 1) young CB treated; 2) young control; 3) old CB treated; and 4) old control. Animals were injected daily with either CB (2 mg/kg) or saline for 28 days. CB increased (P < 0.05) the mass of the costal diaphragm in both young and old animals. CB treatment increased diaphragmatic-specific P(o) in old animals (approximately 15%; P < 0.05) but did not alter (P > 0.05) diaphragmatic-specific P(o) in young animals. Biochemical analysis indicated that the improved maximal specific P(o) in the diaphragm of CB-treated old animals was not due to increased myofibrillar protein concentration. Analysis of the myosin heavy chain (MHC) content of the costal diaphragm revealed a CB-induced increase (P < 0.05) in type IIb MHC and a decrease in type I, IIa, and IIx MHC in both young and old animals. These data support the hypothesis that CB treatment can restore the age-associated decline in both diaphragmatic-specific P(o) and muscle mass.

Effect of prolonged undernutrition on rat diaphragm mitochondrial respiration.

Previous studies have shown that undernutrition induces an impairment of the respiratory muscle function in patients with chronic lung disease. To explain this, we hypothesized that undernutrition could decrease oxidative metabolism in the diaphragm. We therefore examined the effect of prolonged undernutrition on diaphragm mitochondrial oxygen uptake with pyruvate and palmitate as substrates in adult rats. Ten rats served as controls (CTL). Ten nutritionally deprived rats (ND) received 40% of their estimated daily nutrition. Five weeks of undernutrition induced a 33% decrease in state 3 respiration with pyruvate plus malate as substrate (993 +/- 171 versus 1488 +/- 167 nmol atomic O/mg/min, P < 0.01) and a 39% decrease with palmitate plus malate (516 +/- 89 versus 850 +/- 165 nmol atomic O/mg/min, P < 0.05). With succinate plus rotenone, there was no significant difference in the respiratory rate between groups. In the ND group, we found a significant decrease in citrate synthase activity (P < 0.01), and also in reduced nicotinamine adenine dinucleotide (NADH) dehydrogenase activity (P < 0.05), which cannot alone induce such a state 3 respiratory decrease. This showed that undernutrition in rat diaphragm does not induce an alteration in protein complexes I, II, III, and IV, or the F complex containing the mitochondrial ATPase of the electron transport chain. In conclusion, the main result of this study was that prolonged undernutrition induced a decrease in mitochondrial respiration secondary to a significant reduction in NADH generation by the Krebs cycle, which may affect respiratory muscle function with implications for patient care.

Mechanical Ventilation Protects against Diaphragm Injury in Sepsis

Overproduction of nitric oxide (NO) with attendant oxidative and nitrosative stress has been implicated in sepsis-induced diaphragm dysfunction. Here we determined the impact of controlled mechanical ventilation (MV) on rat diaphragm sarcolemmal injury, inducible NO synthase (iNOS) expression, and oxidative stress during endotoxemia. At 4 h after injection of endotoxin, impaired sarcolemmal integrity and decreased force production by the diaphragm were observed in spontaneously breathing rats. The use of MV during endotoxemia largely eliminated sarcolemmal damage and significantly improved diaphragm force production. These benefits were not associated with alterations in either iNOS expression or protein carbonyls (marker of oxidation), which remained abnormally elevated in septic diaphragms despite MV. Therefore, we hypothesized that the protection afforded by MV was due to its ability to decrease the level of mechanical stress placed on the sarcolemma, because the latter could be hyperfragile in the setting of increased oxidative stress. Using an in vitro system to independently modulate oxidative and mechanical stresses, we confirmed that these two factors act together in a synergistic fashion to favor sarcolemmal injury. Accordingly, our data suggest that MV protects the diaphragm during sepsis by abrogating an injurious interaction between oxidative and biomechanical stresses imposed on the sarcolemma.

Microvascular remodelling after endurance training with Co2+ treatment in the rat diaphragm and hind-leg muscles.

This study was designed to examine the changes in capillary geometry, especially the distribution of arteriolar and venular capillaries, in the skeletal muscles of female Wistar rats after endurance training with and without chronic CoCl(2) administration. Four groups of rats were used: non-treated sedentary, non-treated training, Co(2+)-treated sedentary, and Co(2+)-treated training. Exercise training by running lasted for 5 weeks at 25 m/min on a 20% gradient, 10-60 min/d, 5 d/week. The Co(2+)-treated rats drank water containing 0.01% CoCl(2) for 5 weeks. Morphological findings were obtained from the soleus (SOL), deep (PLd) and superficial (PLs) portions of plantaris, and diaphragm (DIA) muscles. Co(2+) administration significantly increased the blood hemoglobin concentration by approximately 25% with and without training. Only in DIA, the Co(2+) treatment alone significantly increased total capillary density and the capillary-to-fiber ratio (C : F) (p<0.05). Both training groups with and without Co(2+) administration showed a significant increase in the C : F in SOL and PLd (p<0.05). In PLd, the increase was significantly greater in the Co(2+)-treated training group than in the non-Co(2+)-treated training group (p<0.05). Training significantly increased the proportion of arteriolar capillaries while it decreased that of venular capillaries in both SOL and PLd (p<0.05). These changes were also observed in PLd after training with Co(2+). The densities of VEGF-positive and TGF-beta1-positive capillaries remained unchanged in all muscle portions examined after either Co(2+) administration or exercise training. These results suggest that chronic Co(2+) administration causes adaptive changes in the oxygen transport system in respiratory muscle and facilitates exercise-induced angiogenesis in hind-leg muscles.

The effects of glutamate on spontaneous acetylcholine secretion processes in the rat neuromuscular synapse.

Experiments on rat diaphragm muscles showed that glutamate (10 microM-1 mM) had no effect on the mean frequency, interspike intervals, and amplitude-time characteristics of miniature endplate potentials, but had a suppressive action on non-quantum secretion (the intensity of which was assessed in terms of the H effect). The effect of glutamate was markedly concentration-dependent and was completely overcome by blockade of NMDA receptors, inhibition of NO synthase, and by binding of NO molecules in the extracellular space by hemoglobin. It is suggested that glutamate can modulate the non-quantum release of acetylcholine, initiating the synthesis of NO molecules in muscle fibers via activation of NMDA receptors followed by the retrograde action of NO on nerve terminals.

Sarcomeres Are Added in Series to Emphysematous Rat Diaphragm After Lung Volume Reduction Surgery

The diaphragm adapts to its shortened state in experimental emphysema primarily by losing sarcomeres in series, thus reducing its optimal operating length. One would expect improved diaphragmatic function after lung volume reduction surgery (LVRS) only if the muscle can readapt to its elevated, lengthened postoperative position by either adding back sarcomeres or lengthening sarcomeres. We used a model of elastase-induced emphysema in rats to test the hypothesis that sarcomere addition occurs following LVRS. A cohort of emphysematous rats was created by the intratracheal instillation of elastase. Five months after the instillation, one group of rats underwent measurement of in situ costal diaphragm length via laparotomy, the determination of optimal muscle fiber operating length (Lo) on stimulated diaphragm strips in vitro, and the measurement of sarcomere length by electron microscopy on strips fixed at Lo. Another group of rats underwent LVRS or sham sternotomy 5 months after the instillation, and 5 months following the operation these animals underwent the same series of diaphragmatic studies. Lo was significantly greater in rats that underwent LVRS than those that underwent sternotomy (mean [+/- SE] Lo after LVRS, 2.50 +/- 0.08 cm; mean Lo after sternotomy, 2.27 +/- 0.06 cm; p = 0.013). There was no significant difference in sarcomere lengths between the two groups (2.95 +/- 0.04 vs 3.04 +/- 0.04 microm, respectively; p = 0.10). Using Lo as the length basis, the mean sarcomere number was calculated to be 8,712 +/- 192 in animals that had undergone LVRS and 7,144 +/- 249 in animals that had undergone sternotomy (p < 0.001). Sarcomere length is not significantly altered but sarcomeres are added in series following LVRS in this experimental model of emphysema/LVRS. It is likely that this sarcomere addition is a prerequisite to the improvement in inspiratory muscle function that has been observed following LVRS in humans.

Free radicals in hypoxic rat diaphragm contractility: no role for xanthine oxidase.

Recent evidence indicates that hypoxia enhances the generation of oxidants. Little is known about the role of free radicals in contractility of the rat diaphragm during hypoxia. We hypothesized that antioxidants improve contractility of the hypoxic rat diaphragm and that xanthine oxidase (XO) is an important source of free radicals in the hypoxic diaphragm. The effects of N-acetylcysteine (NAC; 18 mM), Tiron (10 mM), and the XO inhibitor allopurinol (250 microM) were studied on isometric and isotonic force generation during hypoxia (PO(2) approximately 7 kPa). NAC and Tiron decreased maximal force generation, slowed the shortening velocity, and decreased the power output. Fatigue rate was decreased in the presence of either NAC or Tiron. Allopurinol did not alter the contractility or fatigability of the diaphragm. During hyperoxia (PO(2) approximately 85 kPa), neither NAC nor allopurinol affected the contractility or fatigability of the diaphragm. Thus free radicals play a significant role in diaphragm contractility during hypoxia. Whether antioxidants exert a beneficial or harmful effect on muscle performance depends on the contraction pattern of the muscle. Free radicals generated by XO do not play a role in diaphragm contractility during either hypoxia or hyperoxia.

The effect of hypoxia on shortening contractions in rat diaphragm muscle.

Hypoxia is known to reduce isometric contractile properties of isolated rat diaphragm bundles. Its effect on isotonic contractile properties (i.e. force-velocity relationship and power output) has not been studied. We hypothesized that hypoxia reduces velocity of shortening and consequently power output of the unfatigued muscle, and shortens endurance time during isotonic contractions. Force-velocity relationship, power output, and fatigue resistance of rat diaphragm muscle bundles were measured during hypoxia (PO2: 6.6 +/- 0.2 kPa) and compared with hyperoxia (PO2: 91.8 +/- 0.7 kPa). Force was clamped from 1 to 100% of maximal tetanic force (Po). Fatigue during isotonic contractions was induced by repeated stimulation every 2 s at a clamp level of 33% of Po. Hypoxia did not affect isometric force generation compared with hyperoxia, nor contraction or relaxation time. In contrast, maximum shortening velocity decreased significantly (hypoxia: 4.2 +/- 0.3, hyperoxia: 6.0 +/- 0.2 Lo/s, P < 0.05). The force-velocity curve shifted downwards (P < 0.05). Hypoxia lowered power output at each load compared with hyperoxia (P < 0.05). The isotonic endurance time was shorter during hypoxia compared with hyperoxia (80 +/- 2 vs. 130 +/- 3 s, P < 0.05). These data show that hypoxia depresses isotonic contractile properties and power output, and reduces endurance time during repeated isotonic contractions.

Differential expression of Tie-2 receptors and angiopoietins in response to in vivo hypoxia in rats.

In this study, we assessed the effects of in vivo hypoxia on the expression of Tie-2 receptors and angiopoietins in various organs of conscious rats and correlated these effects with the expression of hypoxia-inducible factor-1 (HIF-1). RT-PCR and Southern blotting were used to amplify mRNA expression of angiopoietin-1, -2, and -3, Tie-2, and HIF-1 alpha in tissues of normoxic and hypoxic (fraction of inspired oxygen of 9--10% for either 12 or 48 h) rats. Hypoxia provoked a decline in angiopoietin-1 mRNA and Tie-2 mRNA, protein, and phosphorylation levels in the lung, liver, cerebellum, and heart but not in the kidney and diaphragm. In comparison, hypoxia raised the levels of angiopoietin-2 mRNA in the cerebellum and angiopoietin-3 mRNA in the lung, kidney, and diaphragm. HIF-1 alpha mRNA was abundant in most organs of normoxic rats but was significantly induced in the kidney and diaphragm of hypoxic rats. We conclude that in vivo hypoxia exerts inhibitory effects on the activity of the angiopoietin-1/Tie-2 receptor pathway through reduction of angiopoietin-1 and upregulation of angiopoietin-2 and -3. Induction of angiopoietin-3 in the kidney and diaphragm of hypoxic rats could be mediated through the HIF-1 transcription factor.

Modulation by dietary restriction in gene expression related to insulin-like growth factor-1 in rat muscle.

Physiological adaptations induced by dietary restriction might include the modulation of the insulin-like growth factor 1 (IGF-1) axis. We investigated the effects of dietary restriction on aging-dependent changes in plasma level of IGF-1 and gene expression levels of type-1 IGF receptor (IGFR), insulin receptor substrate-1 (IRS-1), and IGF-1 in the diaphragm and quadriceps femoris muscle (QFM) of male F344 rats. The animals were fed ad libitum throughout life (AL), or provided with 70% of diet of AL rats from 6 weeks of age (DR). The plasma IGF-1 and steady-state levels of the genes were quantified by radioimmunoassay and the reverse transcription-polymerase chain reaction method, respectively. Immunohistochemical analysis in tissue sections was also performed for IGFR. Our results showed that dietary restriction: 1) decreased the plasma level of IGF-1; 2) increased the steady-state level of IGFR-mRNA at 6 and 16 months of age. and the peptide level at 6 months; 3) maintained IGF-1- and IRS-1-mRNA at a level similar to that in AL rats; and 4) delayed or inhibited an aging-dependent increase in IGFR-mRNA in the muscles. The present results suggest that dietary restriction could modulate IGF-1 signaling by augmenting local tissue response to IGF-1 and by maintaining the local production of the peptide, even though plasma IGF-1 is reduced.