Chronic intermittent hypoxia impairs endothelium-dependent dilation in rat cerebral and skeletal muscle resistance arteries

The goal of the present study was to evaluate the effects of relatively short-term chronic intermittent hypoxia (CIH) on endothelial function of resistance vessels in the skeletal muscle and cerebral circulations. Sprague-Dawley rats were exposed to 14 days of CIH (10% fraction of inspired oxygen for 1 min at 4-min intervals, 12 h/day, n = 6). Control rats (n = 6) were housed under normoxic conditions. After 14 days, resistance arteries of the gracilis muscle (GA) and middle cerebral arteries (MCA) were isolated and cannulated with micropipettes, perfused and superfused with physiological salt solution, and equilibrated with 21% O2-5% CO2 in a heated chamber. The arteries were pressurized to 90 mmHg, and vessel diameters were measured via a video micrometer before and after exposure to ACh (10-7-10-4 M), sodium nitroprusside (10-6 M), and acute reduction of Po2 in the perfusate/superfusate (from 140 to 40 mmHg). ACh-induced dilations of GA and MCA from animals exposed to CIH were greatly attenuated, whereas responses to nitroprusside were similar to controls. Dilations of both GA and MCA in response to acute reductions in Po2 were virtually abolished in animals exposed to CIH compared with controls. These findings suggest that exposure to CIH reduces the bioavailability of nitric oxide in the cerebral and skeletal muscle circulations and severely blunts vasodilator responsiveness to acute hypoxia.     

Elevated salt intake impairs dilation of rat skeletal muscle resistance arteries via ANG II suppression

Vasodilator responses were assessed in resistance arteries (100-200 microm) isolated from the gracilis muscle of normotensive rats after changes in dietary salt intake. Sprague-Dawley rats were maintained on either a high-salt (HS) diet (4.0% NaCl) or a low-salt (LS) diet (0.4% NaCl) for 4-8 wk (chronic) or 3 days (short-term) with water ad libitum. One group of short-term HS rats received a continuous intravenous infusion of a low dose (5 ng x kg(-1) x min(-1)) of ANG II to prevent the ANG II suppression that occurs with HS diet. Short-term and chronic HS diet eliminated arterial dilation in response to ACh and reduced PO(2) (30-40 mmHg) and the stable prostacyclin analog iloprost. ANG II infusion preserved the response to these vasodilator stimuli in short-term HS animals. Dilator responses to sodium nitroprusside and forskolin were unaffected by HS diet. These findings suggest that ANG II suppression during HS diet impairs vascular relaxation mechanisms upstream from the cAMP and cGMP second messenger systems.     

Aging induces muscle-specific impairment of endothelium-dependent dilation in skeletal muscle feed arteries.

We tested the hypothesis that aging decreases endothelium-dependent vasodilation in feed arteries perfusing rat skeletal muscle. In addition, we tested the hypothesis that attenuated vasodilator responses are associated with decreased endothelial nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) expression. Soleus feed arteries (SFA) and gastrocnemius feed arteries (GFA) were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats. Feed arteries from the right hindlimb were cannulated with two glass micropipettes for examination of endothelium-dependent [acetylcholine (ACh)] and endothelium-independent [adenosine (Ado) or sodium nitroprusside (SNP)] vasodilator function. Feed arteries from the left hindlimb were frozen and used to assess eNOS and SOD-1 protein and mRNA expression. In SFA, endothelium-dependent dilation to ACh was reduced in old rats (0.9 +/- 0.04 vs. 0.8 +/- 0.03), whereas dilator responses to Ado and SNP were similar in SFA of young and old rats. In GFA, vasodilator responses to ACh, Ado, and SNP were not altered by age. eNOS and SOD-1 protein expression declined with age in SFA (-71 and -54%, respectively) but not in GFA. eNOS and SOD-1 mRNA expression were not altered by age in SFA or GFA. Collectively, these data indicate aging induces muscle-specific impairment of endothelium-dependent vascular function in SFA.     

Interaction of myogenic mechanisms and hypoxic dilation in rat middle cerebral arteries

The goal of this study was to determine how myogenic responses and vascular responses to reduced Po(2) interact to determine vascular smooth muscle (VSM) transmembrane potential and active tone in isolated middle cerebral arteries from Sprague-Dawley rats. Stepwise elevation of transmural pressure led to depolarization of the VSM cells and myogenic constriction, and reduction of the O(2) concentration of the perfusion and superfusion reservoirs from 21% O(2) to 0% O(2) caused vasodilation and VSM hyperpolarization. Myogenic constriction and VSM depolarization in response to transmural pressure elevation still occurred at reduced Po(2). Arterial dilation in response to reduced Po(2) was not impaired by pressure elevation but was significantly reduced at the lowest transmural pressure (60 mmHg). However, the magnitude of VSM hyperpolarization was unaffected by transmural pressure elevation. This study demonstrates that myogenic activation in response to transmural pressure elevation does not override hypoxic relaxation of middle cerebral arteries and that myogenic responses and hypoxic relaxation can independently regulate vessel diameter despite substantial changes in the other variable.     

IGF-I stimulates protein synthesis in skeletal muscle through multiple signaling pathways during sepsis

Chronic septic abscess formation causes an inhibition of protein synthesis in gastrocnemius not observed in rats with a sterile abscess. Inhibition is associated with an impaired mRNA translation initiation that can be ameliorated by elevating IGF-I but not insulin. The present study investigated the ability of IGF-I signaling to stimulate protein synthesis in gastrocnemius by accelerating mRNA translation initiation. Experiments were performed in perfused hindlimb preparations from rats 5 days after induction of a septic abscess. Protein synthesis in gastrocnemius from septic rats was accelerated twofold by the addition of IGF-I (10 nM) to perfusate. IGF-I increased the phosphorylation of translation repressor 4E-binding protein-1 (4E-BP1). Hyperphosphorylation of 4E-BP1 in response to IGF-I resulted in its dissociation from the inactive eukaryotic initiation factor (eIF) 4E.4E-BP1 complex. Assembly of the active eIF4F complex (as assessed by the association eIF4G with eIF4E) was increased twofold by IGF-I in the perfusate. In addition, phosphorylation of eIF4G and ribosomal protein S6 kinase-1 (S6K1) was also enhanced by IGF-I. Activation of mammalian target of rapamycin, an upstream kinase implicated in phosphorylating both 4E-BP1 and S6K1, was also observed. Thus the ability of IGF-I to accelerate protein synthesis during sepsis may be related to a stimulation of signaling to multiple steps in translation initiation with an ensuing increased phosphorylation of eIF4G, eIF4E availability, and S6K1 phosphorylation.     

Angiotensin II AT1 receptors preserve vasodilator reactivity in skeletal muscle resistance arteries

Resistance arteries (100-150 microm) were isolated from the gracilis muscle of normotensive Sprague-Dawley rats placed on a high-salt (HS) diet (4.0% NaCl) for 3-7 days. Exposure to the HS diet eliminated vascular relaxation in response to hypoxia (PO2 reduction to 35-40 Torr) and iloprost, a stable analog of prostacyclin. Vasodilator responses were restored in arteries isolated from chronically instrumented HS rats receiving a continuous intravenous infusion of either angiotensin II (ANG II; 5-6 ng x kg(-1) x min(-1)) or ANG II plus the AT2 receptor blocker PD-123319 (5 microg x kg(-1) x min(-1)) for 3 days before the isolated vessel studies. In contrast, coinfusion of the AT1 receptor blocker losartan (20 microg x kg(-1) x min(-1)) or coinfusion of both receptor blockers with ANG II eliminated the protective effect of ANG II to restore dilator responses to hypoxia and iloprost. Neither a HS diet nor ANG II infusion affected the dilation of gracilis arteries in response to direct activation of adenylyl cyclase by forskolin, suggesting that the effect of both the HS diet and the ANG II on the vasculature is mediated upstream from second messenger systems. These findings indicate that the protective effect of ANG II to maintain vasodilator reactivity in resistance arteries of rats on a HS diet is mediated via the AT1 receptor subtype.     

Chronic intermittent hypoxia alters NE reactivity and mechanics of skeletal muscle resistance arteries.

Although arterial dilator reactivity is severely impaired during exposure of animals to chronic intermittent hypoxia (CIH), few studies have characterized vasoconstrictor responsiveness in resistance arteries of this model of sleep-disordered breathing. Sprague-Dawley rats were exposed to CIH (10% inspired O2 fraction for 1 min at 4-min intervals; 12 h/day) for 14 days. Control rats were housed under normoxic conditions. Diameters of isolated gracilis muscle resistance arteries (GA; 120-150 microm) were measured by television microscopy before and during exposure to norepinephrine (NE) and angiotensin II (ANG II) and at various intraluminal pressures between 20 and 140 mmHg in normal and Ca2+-free physiological salt solution. There was no difference in the ability of GA to constrict in response to ANG II (P = 0.42; not significant; 10(-10)-10(-7) M). However, resting tone, myogenic activation, and vasoconstrictor responses to NE (P < 0.001; 10(-9)-10(-6) M) were reduced in CIH vs. controls. Treatment of rats with the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol; 1 mM) in the drinking water restored myogenic responses and NE-induced constrictions of CIH rats, suggesting that elevated superoxide production during exposure to CIH attenuates vasoconstrictor responsiveness to NE and myogenic activation in skeletal muscle resistance arteries. CIH also leads to an increased stiffness and reduced vessel wall distensibility that were not correctable with oral tempol treatment.     

Mechanotransduction pathways in skeletal muscle hypertrophy

In the last decade, molecular biology has contributed to define some of the cellular events that trigger skeletal muscle hypertrophy. Recent evidence shows that insulin like growth factor 1/phosphatidyl inositol 3-kinase/protein kinase B (IGF-1/PI3K/Akt) signaling is not the main pathway towards load-induced skeletal muscle hypertrophy. During load-induced skeletal muscle hypertrophy process, activation of mTORC1 does not require classical growth factor signaling. One potential mechanism that would activate mTORC1 is increased synthesis of phosphatidic acid (PA). Despite the huge progress in this field, it is still early to affirm which molecular event induces hypertrophy in response to mechanical overload. Until now, it seems that mTORC1 is the key regulator of load-induced skeletal muscle hypertrophy. On the other hand, how mTORC1 is activated by PA is unclear, and therefore these mechanisms have to be determined in the following years. The understanding of these molecular events may result in promising therapies for the treatment of muscle-wasting diseases. For now, the best approach is a good regime of resistance exercise training. The objective of this point-of-view paper is to highlight mechanotransduction events, with focus on the mechanisms of mTORC1 and PA activation, and the role of IGF-1 on hypertrophy process.     

Signaling pathways controlling skeletal muscle mass

Abstract

The molecular mechanisms underlying skeletal muscle maintenance involve interplay between multiple signaling pathways. Under normal physiological conditions, a network of interconnected signals serves to control and coordinate hypertrophic and atrophic messages, culminating in a delicate balance between muscle protein synthesis and proteolysis. Loss of skeletal muscle mass, termed “atrophy”, is a diagnostic feature of cachexia seen in settings of cancer, heart disease, chronic obstructive pulmonary disease, kidney disease, and burns. Cachexia increases the likelihood of death from these already serious diseases. Recent studies have further defined the pathways leading to gain and loss of skeletal muscle as well as the signaling events that induce differentiation and post-injury regeneration, which are also essential for the maintenance of skeletal muscle mass. In this review, we summarize and discuss the relevant recent literature demonstrating these previously undiscovered mediators governing anabolism and catabolism of skeletal muscle.     

Stimulation characteristics that determine arteriolar dilation in skeletal muscle

To determine the skeletal muscle stimulation parameters that are most important in establishing vasodilation in the microvasculature, I tested whether arteriolar diameter during 2 min of repetitive, short-duration, tetanic skeletal muscle contractions increased with changes in stimulus frequency, stimulation train duration, and contraction frequency. To test this, the diameter of transverse arterioles approximately perpendicular to small bundles of cremaster muscle fibers in situ of anesthetized Golden Syrian hamsters was used as a bioassay system. Arteriolar diameter was measured before and during different stimulation patterns that consisted of a contraction frequency [6, 12, or 24 contractions per minute (cpm)], a stimulation train duration (250, 500, or 750 ms) and a stimulus frequency (4, 8, 10, 15, 20, 30, 40, 60, and 80 Hz). The magnitude of the dilation significantly increased with stimulus frequency but not in a simple linear manner. The average rate of increase was 0.32 +/- 0.02 microm/Hz from 4 to 20 Hz and 0.09 +/- 0.02 microm/Hz from 30 to 80 Hz. The magnitude of the dilation increased significantly with the contraction frequency where the dilation at 6 cpm was significantly smaller than the dilation at 24 cpm across all stimulus frequencies. Changing the train duration from 250 to 750 ms did not significantly affect the magnitude of the dilation. These observations suggest that stimulation parameters are important in determining the magnitude of the microvascular dilation and that the magnitude of the dilation was dependent on both the contraction frequency and stimulus frequency but was independent of train duration.     

Mechanotransduction pathways in skeletal muscle hypertrophy

In the last decade, molecular biology has contributed to define some of the cellular events that trigger skeletal muscle hypertrophy. Recent evidence shows that insulin like growth factor 1/phosphatidyl inositol 3-kinase/protein kinase B (IGF-1/PI3K/Akt) signaling is not the main pathway towards load-induced skeletal muscle hypertrophy. During load-induced skeletal muscle hypertrophy process, activation of mTORC1 does not require classical growth factor signaling. One potential mechanism that would activate mTORC1 is increased synthesis of phosphatidic acid (PA). Despite the huge progress in this field, it is still early to affirm which molecular event induces hypertrophy in response to mechanical overload. Until now, it seems that mTORC1 is the key regulator of load-induced skeletal muscle hypertrophy. On the other hand, how mTORC1 is activated by PA is unclear, and therefore these mechanisms have to be determined in the following years. The understanding of these molecular events may result in promising therapies for the treatment of muscle-wasting diseases. For now, the best approach is a good regime of resistance exercise training. The objective of this point-of-view paper is to highlight mechanotransduction events, with focus on the mechanisms of mTORC1 and PA activation, and the role of IGF-1 on hypertrophy process.     

Microstructural analysis of deformation-induced hypoxic damage in skeletal muscle

Deep pressure ulcers are caused by sustained mechanical loading and involve skeletal muscle tissue injury. The exact underlying mechanisms are unclear, and the prevalence is high. Our hypothesis is that the aetiology is dominated by cellular deformation (Bouten et al. in Ann Biomed Eng 29:153-163, 2001; Breuls et al. in Ann Biomed Eng 31:1357-1364, 2003; Stekelenburg et al. in J App Physiol 100(6):1946-1954, 2006) and deformation-induced ischaemia. The experimental observation that mechanical compression induced a pattern of interspersed healthy and dead cells in skeletal muscle (Stekelenburg et al. in J App Physiol 100(6):1946-1954, 2006) strongly suggests to take into account the muscle microstructure in studying damage development. The present paper describes a computational model for deformation-induced hypoxic damage in skeletal muscle tissue. Dead cells stop consuming oxygen and are assumed to decrease in stiffness due to loss of structure. The questions addressed are if these two consequences of cell death influence the development of cell injury in the remaining cells. The results show that weakening of dead cells indeed affects the damage accumulation in other cells. Further, the fact that cells stop consuming oxygen after they have died, delays cell death of other cells.     

Oxidant stress-induced increase in myogenic activation of skeletal muscle resistance arteries in obese Zucker rats

This study characterized myogenic activation of skeletal muscle (gracilis) resistance arteries from lean (LZR) and obese Zucker rats (OZR). Arteries from OZR exhibited increased myogenic activation versus LZR; this increase was impaired by endothelium denudation or nitric oxde synthase inhibition. Treatment of vessels with 17-octadecynoic acid impaired responses in both strains by comparable amounts. Dihydroethidine microfluorography indicated elevated vascular superoxide levels in OZR versus LZR; immunohistochemistry demonstrated elevated vascular nitrotyrosine levels in OZR, indicating increased peroxynitrite presence. Vessel treatment with oxidative radical scavengers (polythylene glycol-superoxide dismutase/catalase) or inhibition of Ca(2+)-activated K(+) (K(Ca)) channels (iberiotoxin) did not alter myogenic activation in LZR but normalized activation in OZR. Application of peroxynitrite to vessels of OZR caused a greater vasoconstriction versus LZR; the response was impaired in OZR by elevated intraluminal pressure and was abolished in both strains by iberiotoxin. These results suggest that enhanced myogenic activation of gracilis arteries of OZR versus LZR 1) is not due to alterations in cytochrome P-450 contribution, and 2) may be due to elevated peroxynitrite levels inhibiting K(Ca) channels following increased intraluminal pressure.