Chronic hypoxia attenuates resting and exercise-induced VEGF, flt-1, and flk-1 mRNA levels in skeletal muscle.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic mitogen. However, chronic hypoxia is generally not found to increase mammalian skeletal muscle capillarity. We sought to determine the effect of chronic hypoxia (8 wk, inspired O2 fraction = 0.12) on skeletal muscle gene expression of VEGF, its receptors (flt-1 and flk-1), basic fibroblast growth factor, and transforming growth factor-beta1. Wistar rats were exposed to chronic hypoxia (n = 12) or room air (n = 12). After the exposure period, six animals from each group were subjected to a single 1-h treadmill exercise bout (18 m/min on a 10 degrees incline) in room air while the remaining six animals served as rest controls. Morphological analysis revealed that chronic hypoxia did not increase skeletal muscle capillarity. Northern blot analyses showed that chronic hypoxia decreased resting VEGF, flt-1, and flk-1 mRNA by 23, 68, and 42%, respectively (P < 0.05). The VEGF mRNA response to exercise was also decreased (4.1- and 2.7-fold increase in room air and chronic hypoxia, respectively, P < 0.05). In contrast, neither transforming growth factor-beta1 nor basic fibroblast growth factor mRNA was significantly altered by chronic hypoxia. In conclusion, prolonged exposure to hypoxia attenuated gene expression of VEGF and its receptors flt-1 and flk-1 in rat gastrocnemius muscle. These findings may provide an explanation for the lack of mammalian skeletal muscle angiogenesis that is observed after chronic hypoxia.     

Partial activation of thin filaments in resting frog skeletal muscle fibers

X-ray patterns from frog skeletal muscles at rest show a series of relatively weak meridional reflections which may be indexed as the 5, 7, 9, 11 and 13 orders of the repeat period of about 2 x 385 A. According to the model of the thin filaments structure, suggested by V. V. Lednev and G. M. Frank (1977), this period is specific for the activated or "switched on" state of the actin--containing filaments. At the same time, according to the generally accepted model (suggested in 1972), the axial repeat period of the thin filament structure is approximately equal to 385 A and does not depend on the functional state of the muscle. The existence of the repeat period of about 2 x 385 A in the thin filaments of a resting muscle suggests that even at rest the thin filaments of vertebrate skeletal muscle are not completely inhibited. It may be suggested that partial activation of the thin filaments in a resting muscle is the result of formation of long life rigorlike crossbridges, the existence of which was postulated by D. K. Hill in 1968 on the basis of his studies on resting tension in the frog skeletal muscle.     

Neuropeptide Y1 receptor vasoconstriction in exercising canine skeletal muscles.

Existing evidence suggests that neuropeptide Y (NPY) acts as a neurotransmitter in vascular smooth muscle and is coreleased with norepinephrine from sympathetic nerves. We hypothesized that release of NPY stimulates NPY Y(1) receptors in the skeletal muscle vasculature to produce vasoconstriction during dynamic exercise. Eleven mongrel dogs were instrumented chronically with flow probes on the external iliac arteries of both hindlimbs and a catheter in one femoral artery. In resting dogs (n = 4), a 2.5-mg bolus of BIBP-3226 (NPY Y(1) antagonist) infused into the femoral artery increased external iliac conductance by 150 +/- 82% (1.80 +/- 0.44 to 3.50 +/- 0.14 ml.min(-1).mmHg(-1); P < 0.05). A 10-mg bolus of BIBP-3226 infused into the femoral artery in dogs (n = 7) exercising on a treadmill at a moderate intensity (6 miles/h) increased external iliac conductance by 28 +/- 6% (6.00 +/- 0.49 to 7.64 +/- 0.61 ml.min(-1).mmHg(-1); P < 0.05), whereas the solvent vehicle did not (5.74 +/- 0.51 to 5.98 +/- 0.43 ml.min(-1).mmHg(-1); P > 0.05). During exercise, BIBP-3226 abolished the reduction in conductance produced by infusions of the NPY Y(1) agonist [Leu(31),Pro(34)]NPY (-19 +/- 3 vs. 0.5 +/- 1%). Infusions of BIBP-3226 (n = 7) after alpha-adrenergic receptor antagonism with prazosin and rauwolscine also increased external iliac conductance (6.82 +/- 0.43 to 8.22 +/- 0.48 ml.min(-1).mmHg(-1); P < 0.05). These data support the hypothesis that NPY Y(1) receptors produce vasoconstriction in exercising skeletal muscle. Furthermore, the NPY Y(1) receptor-mediated tone appears to be independent of alpha-adrenergic receptor-mediated vasoconstriction.     

Influence of erythrocyte oxygenation and intravascular ATP on resting and exercising skeletal muscle blood flow in humans with mitochondrial myopathy

Oxygen (O₂) extraction is impaired in exercising skeletal muscle of humans with mutations of mitochondrial DNA (mtDNA), but the muscle hemodynamic response to exercise has never been directly investigated. This study sought to examine the extent to which human skeletal muscle perfusion can increase without reductions in blood oxygenation and to determine whether erythrocyte O₂ off-loading and related ATP vascular mechanisms are impaired in humans with mutations of mtDNA. Leg vascular hemodynamic, oxygenation and ATP were investigated in ten patients with mtDNA mutations and ten matched healthy control subjects: 1) at rest during normoxia, hypoxia, hyperoxia and intra-femoral artery ATP infusion, and 2) during passive and dynamic one-legged knee-extensor exercises. At rest, blood flow (LBF), femoral arterial and venous blood oxygenation and plasma ATP were similar in the two groups. During dynamic exercise, LBF and vascular conductance increased 9–10 fold in the patients despite erythrocyte oxygenation and leg O₂ extraction remained unchanged (p < 0.01). In the patients, workload-adjusted LBF was 28% to 62% higher during submaximal- and maximal exercises and was associated with augmented plasma ATP. The appropriate hemodynamic adjustments during severe hypoxia and ATP infusion suggest that erythrocyte O₂ off-loading and related ATP vascular mechanisms are intact in patients with mtDNA mutations. Furthermore, greater increase in plasma ATP and LBF at a given metabolic demand in the patients, in concert with unchanged oxyhemoglobin, suggest that erythrocyte O₂ off-loading is not obligatory for the exercise-induced increase in blood flow and intravascular ATP concentration.     

Elastic and viscous properties of resting frog skeletal muscle.

The mechanical properties of the resting, whole semitendinosus muscle of the frog have been characterized as functions of both muscle length and temperature. Measurements were made of pseudorandom white noise (PRWN) displacements (less than 10 A/half-sarcomere) applied to the muscle and the force responses to these movements. Signal correlation techniques were then used to obtain the dynamic modulus function for the muscle in the frequency range 2.44-320 Hz. This function was represented by a series combination of a Voigt element and a time delay element for tension propagation along the muscle. A dynamic elastic modulus (E), coefficient of damping (B), and tension transmission velocity (V) were measured for resting muscle on the basis of this model. For each of these parameters, a marked variation with sarcomere length (s) was found. The mean values for E and B at LO (s=2.25 mum) were 1.84+/-0.24 X 10(5) N/m2 and 2.33+/-0.25 X 10(2) Ns/m2, respectively. Further, B demonstrated a negative temperature dependence, Q10=0.78 (P less than 0.05), in the range s=2.6-3.0 mum, while E was not significantly temperature dependent. The length-dependent variations of E and B are interpreted as deriving from both passive muscle elements and attached crossbridges. Velocity was calculated at a single displacing frequency for every experiment; the mean value at LO and all temperatures was v=11.7+/-0.6 m/s. Velocity was also calculated as a function of frequency within several experiments: the results indicate considerable variation of v with frequency.     

Muscle pump does not enhance blood flow in exercising skeletal muscle.

The muscle pump theory holds that contraction aids muscle perfusion by emptying the venous circulation, which lowers venous pressure during relaxation and increases the pressure gradient across the muscle. We reasoned that the influence of a reduction in venous pressure could be determined after maximal pharmacological vasodilation, in which the changes in vascular tone would be minimized. Mongrel dogs (n = 7), instrumented for measurement of hindlimb blood flow, ran on a treadmill during continuous intra-arterial infusion of saline or adenosine (15-35 mg/min). Adenosine infusion was initiated at rest to achieve the highest blood flow possible. Peak hindlimb blood flow during exercise increased from baseline by 438 +/- 34 ml/min under saline conditions but decreased by 27 +/- 18 ml/min during adenosine infusion. The absence of an increase in blood flow in the vasodilated limb indicates that any change in venous pressure elicited by the muscle pump was not adequate to elevate hindlimb blood flow. The implication of this finding is that the hyperemic response to exercise is primarily attributable to vasodilation in the skeletal muscle vasculature.     

A1 receptor activation decreases fatigue in mammalian slow-twitch skeletal muscle in vitro.

To test the hypothesis that adenosine improves skeletal muscle cell function, we exposed curarized mouse soleus and extensor digitorum longus (EDL) to a range of concentrations of adenosine (10(-9) M to 10(-5) M). Muscles contracted in Krebs-Henseleit bicarbonate buffer (27 degrees C, 95% O2 and 5% CO2) for 500 ms at 50 Hz once every 90 s. Soleus fatigued significantly less with adenosine present at concentrations of 10(-8) M and higher than with the Krebs-Henseleit vehicle control. Adenosine significantly improved force generation or delayed fatigue of EDL only with the initial adenosine challenge. To investigate the receptor population involved, we exposed soleus to agonists specific for A1 receptors (N6-cyclopentyladenosine, CPA), or A2 receptors (CGS 21680 hydrochloride, CGS), or A3 receptors (N6-benzyl-5'-N-ethylcarboxamidoadenosine, BNECA). CPA (A1) significantly decreased fatigue compared with the Krebs-Henseleit vehicle control at concentrations of 10(-9) M and higher. Muscles exposed to the A2 and A3 agonists did not differ from a Krebs-Henseleit plus methanol control. Phenylephrine (10(-6) M), an alpha-adrenergic agonist that increases the concentration of inositol triphosphate (IP3), significantly improved developed force in soleus. Neither a permeable cAMP analog, 8-bromo-cAMP (10(-5) M), nor a beta, agonist, isoproterenol (10(-6) M), had an effect on force generation in the soleus when compared with a saline control. Thus adenosine slowed fatigue in slow-twitch skeletal muscle through A1 receptors.     

Role of the sympathoadrenal system in the regulation of glycogen metabolism in resting and exercising skeletal muscles.

The purpose of the present study was to characterize the role of catecholamines in the regulation of skeletal muscle glycogen metabolism during exercise. Using the rat hindlimb perfusion technique we have measured skeletal muscle glycogen content, glycogen phosphorylase and synthase activities in sympathectomized and/or demedullated rats under epinephrine treatment (10(-7) M) at rest and during muscle contraction. When epinephrine and/or norepinephrine deficiency was induced, muscle contraction resulted in a decrease in glycogen content (-63%) despite a decrease in glycogen phosphorylase activity ratio (0.25 to 0.11; p less than 0.001) and an increase in glycogen synthase activity ratio (0.13 to 0.27; p less than 0.001). Under these conditions, epinephrine treatment further reduced glycogen content while blunting the changes in the activity ratio of the rate-limiting enzymes. These data indicate that catecholamines do not play a primary role in skeletal muscle glycogen breakdown during acute exercise and suggest that allosteric regulators may be of prime importance.     

VEGF receptor antagonism blocks arteriogenesis, but only partially inhibits angiogenesis, in skeletal muscle of exercise-trained rats

Both collateral vessel enlargement (arteriogenesis) and capillary growth (angiogenesis) in skeletal muscle occur in response to exercise training. Vascular endothelial growth factor (VEGF) is implicated in both processes. Thus we examined the effect of a VEGF receptor (VEGF-R) inhibitor (ZD4190, AstraZeneca) on collateral-dependent blood flow in vivo and collateral artery size ex vivo (indicators of arteriogenesis) and capillary contacts per fiber (CCF; an index of angiogenesis) in skeletal muscle of both sedentary and exercise-trained rats 14 days after bilateral occlusion of the femoral arteries. Total daily treadmill run time increased appreciably from approximately 70 to approximately 100 min (at 15-20 m/min, twice per day) and produced a large (approximately 75%, P < 0.01) increase in calf muscle blood flow and a greater size of the collateral artery (wall cross-sectional area). ZD4190, which previously has been shown to inhibit the activity of VEGF-R2 and -R1 tyrosine kinase in vitro (IC50 = 30 and 700 nM, respectively), completely blocked the increase in collateral-dependent blood flow and inhibited collateral vessel enlargement. Thus exercise-stimulated collateral arteriogenesis appears to be completely dependent on VEGF-R signaling. Interestingly, enhanced mRNA expression of the VEGF family ligand placental growth factor (2- to 3.5-fold), VEGF-R1 (approximately 2-fold), and endothelial nitric oxide synthase (2- to 3.5-fold) in an isolated collateral artery implicates these factors as important in arteriogenesis. Training of ischemic muscle also induced angiogenesis, as shown by an increase (approximately 25%, P < 0.01) in CCF in white gastrocnemius muscle. VEGF-R inhibition only partially blocked (P < 0.01) but did not eliminate the increase (P < 0.01) in capillarity. Our findings indicate that VEGF-R tyrosine kinase activity is essential for collateral arteriogenesis and important for the angiogenesis induced in ischemic muscle by exercise training; however, other angiogenic stimuli are also important for angiogenesis in flow-limited active muscle.     

Ouabain stimulates unidirectional and net potassium efflux in resting mammalian skeletal muscle.

The present study compared ouabain-sensitive unidirectional K+ flux into (JinK) and out of (JoutK) perfused rat hindlimb skeletal muscle in situ and mouse flexor digitorum brevis (FDB) in vitro. In situ, 5 mM ouabain inhibited 54 +/- 4% of the total JinK in 28 +/- 1 min, and increased the net and unidirectional efflux of K+ within 4 min. In contrast, 1.8 mM ouabain inhibited 40 +/- 8% of the total JinK in 38 +/- 2 min, but did not significantly affect JoutK. In vitro, 1.8 and 0.2 mM ouabain decreased JinK to a greater extent (83 +/- 5%) than in situ, but did not significantly affect 42K loss rate compared with controls. The increase in unidirectional K+ efflux (JoutK) with 5 mM ouabain in situ was attributed to increased K+ efflux through cation channels, since addition of barium (1 mM) to ouabain-perfused muscles returned JoutK to baseline values within 12 min. Perfusion with 5 mM ouabain plus 2 mM tetracaine for 30 min decreased JinK 46 +/- 9% (0.30 +/- 0.03 to 0.16 +/- 0.02 micromol x min(-1) x g(-1)), however tetracaine was unable to abolish the ouabain-induced increase in unidirectional K+ efflux. In both rat hindlimb and mouse FDB, tetracaine had no effect on JoutK. Perfusion of hindlimb muscle with 0.1 mM tetrodotoxin (TTX, a Na+ channel blocker) decreased JinK by 15 +/- 1%, but had no effect on JoutK; subsequent addition of ouabain (5 mM) decreased JinK a further 32 +/- 2%. The ouabain-induced increase in unidirectional K+ efflux did not occur when TTX was perfused prior to and during perfusion with 5 mM ouabain. We conclude that 5 mM ouabain increases the unidirectional efflux of K+ from skeletal muscle through a barium and TTX-sensitive pathway, suggestive of voltage sensitive Na+ channels, in addition to inhibiting Na+/K+-ATPase activity.     

Sensitivity of the soleus muscle to insulin in resting and exercising rats with experimental hypo- and hyper-thyroidism.

1. The effects of hypothyroidism (caused by surgical thyroidectomy followed by treatment for 1 month with propylthiouracil) and of hyperthyroidism [induced by subcutaneous administration of L-tri-iodothyronine (T3)] on glucose tolerance and skeletal-muscle sensitivity to insulin were examined in rats. Glucose tolerance was estimated during 2 h after subcutaneous glucose injection (1 g/kg body wt.). The sensitivity of the soleus muscle to insulin was studied in vitro in sedentary and acutely exercised animals. 2. Glucose tolerance was impaired in both hypothyroid and hyperthyroid rats in comparison with euthyroid controls. 3. In the soleus muscle, responsiveness of the rate of lactate formation to insulin was abolished in hypothyroid rats, whereas the sensitivity of the rate of glycogen synthesis to insulin was unchanged. In hyperthyroid animals, opposite changes were found, i.e. responsiveness of the rate of glycogen synthesis was inhibited and the sensitivity of the rate of lactate production did not differ from that in control sedentary rats. 4. A single bout of exercise for 30 min potentiated the stimulatory effect of insulin on lactate formation in hyperthyroid rats and on glycogen synthesis in hypothyroid animals. 5. The data suggest that thyroid hormones exert an interactive effect with insulin in skeletal muscle. This is likely to be at the post-receptor level, inhibiting the effect of insulin on glycogen synthesis and stimulating oxidative glucose utilization.     

Effects of muscle activation on fatigue and metabolism in human skeletal muscle.

Increasing stimulation frequency has been shown to increase fatigue but not when the changes in force associated with changes in frequency have been controlled. An effect of frequency, independent of force, may be associated with the metabolic cost resulting from the additional activations. Here, two separate experiments were performed on human medial gastrocnemius muscles. The first experiment (n = 8) was designed to test the effect of the number of pulses on fatigue. The declines in force during two repetitive, 150-train stimulation protocols that produced equal initial forces, one using 80-Hz trains and the other using 100-Hz trains, were compared. Despite a difference of 600 pulses (23.5%), the protocols produced similar rates and amounts of fatigue. In the second experiment, designed to test the effect of the number of pulses on the metabolic cost of contraction, 31P-NMR spectra were collected (n = 6) during two ischemic, eight-train stimulation protocols (80- and 100-Hz) that produced comparable forces despite a difference of 320 pulses (24.8%). No differences were found in the changes in P(i) concentration, phosphocreatine concentration, and intracellular pH or in the ATP turnover produced by the two trains. These results suggest that the effect of stimulation frequency on fatigue is related to the force produced, rather than to the number of activations. In addition, within the range of frequencies tested, increasing total activations did not increase metabolic cost.     

Lower skeletal muscle capillarization and VEGF expression in aged vs. young men.

Recently, we observed that muscle capillarization, vascular endothelial growth factor (VEGF) protein, and the VEGF mRNA response to acute exercise were lower in aged compared with young women (Croley AN, Zwetsloot KA, Westerkamp LM, Ryan NA, Pendergast aged men, Hickner RC, Pofahl WE, and Gavin TP. J Appl Physiol 99: 1875-1882, 2005). We hypothesized that similar age-related differences in muscle capillarization and VEGF expression would exist between young and aged men. Skeletal muscle biopsies were obtained from the vastus lateralis before and at 4 h after a submaximal exercise bout for the measurement of morphometry, capillarization, VEGF, KDR, and Flt-1 in seven aged (mean age 65 yr) and eight young (mean age 21 yr) sedentary men. In aged compared with young men, muscle capillary contacts and capillary-to-fiber perimeter exchange index were lower regardless of fiber type. Muscle VEGF mRNA and protein were lower in aged men both at rest and 4 h postexercise. Exercise increased muscle VEGF mRNA and protein and KDR mRNA independent of age group. There were no effects of exercise or age on muscle Flt-1 mRNA or protein or KDR protein. These results confirm that skeletal muscle capillarization and VEGF expression are lower in aged compared with young men.     

Glucocorticoid-receptor activation in hypertrophied skeletal muscle

This investigation was undertaken 1) to determine whether the increased glucocorticoid-receptor binding activities, observed in hypertrophied plantaris muscles, are associated with a reduced ability to undergo receptor activation and 2) to examine whether glucocorticoid-receptor complexes in hypertrophied muscles undergo a shift in the relative distribution of the two thermally activated receptor forms (termed binder II and corticosteroid binder IB) to a distribution that is found in slow-twitch or heart muscle types. Plantaris muscles of female adrenalectomized rats, enlarged by surgical removal of synergists, were 60% heavier and had higher glucocorticoid cytosol binding (125 +/- 14 vs. 79 +/- 8 fmol/mg protein) than these muscles of controls. Activation, which was quantitated by the ability of the steroid-receptor complex to bind to DNA, was similar in overloaded and control muscles (57 +/- 2 vs. 62 +/- 4%). Diethylaminoethyl-cellulose chromatography of activated receptors showed approximately 16% of the radioactivity appearing as binder II and 38% as binder IB in both hypertrophied and control muscles. These results show that although enlarged plantaris muscles are undergoing certain fast- to slow-twitch biochemical transformations, the activated glucocorticoid-receptor distribution does not shift to that observed in slow fibers.