Femoral Artery Occlusion Increases Muscle Pressor Reflex and Expression of Hypoxia-Inducible Factor-1α in Sensory Neurons

Abstract

Hypoxia inducible factor-1 (HIF-1) has an important contribution to pathophysiological changes of homeostasis under conditions of oxygen deprivation as well as ischemia. We examined the effects of femoral artery occlusion on HIF-1α expression in sensory dorsal root ganglion (DRG) neurons of rats. Also, we examined cardiovascular responses to static muscle contraction following femoral occlusion. We hypothesized that hindlimb vascular insufficiency increases the levels of sensory nerves’ HIF-1α and augments autonomic responses induced by activation of muscle afferent nerves. In addition, we examined if the reflex cardiovascular responses were altered as HIF-1α was increased in the DRG neurons. Our data show that HIF-1α was significantly increased in the lumbar DRG neurons 6, 24 and 72 hours after femoral artery ligation as compared with sham control. Administration of dimethyloxalylglycine (DMOG), a stabilizer of HIF-α, significantly increased HIF-1α in the lumbar DRG neurons. Furthermore, femoral occlusion enhanced the reflex pressor response to muscle contraction; however, the response was not altered by injection of DMOG. Overall, our results indicate that 1) femoral artery occlusion increases HIF-1α levels of in DRG neurons and contraction-induced pressor response; and 2) an increase in HIF-1α of DRG neurons per se may not alter the muscle pressor reflex.

I. Introduction

It is common that critical limb ischemia appear in peripheral arterial occlusive disease due to atherosclerosis ^{1, 2}. Insufficient blood flow to metabolic demands of tissue contributes to physiological and metabolic alterations in the tissue thereby leading to the functional impairment ³. Especially, consumption of oxygen is lower in patients with peripheral artery occlusive disease compared with health controls ⁴.

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric protein composed of constitutively expressed HIF-1α and HIF-1β subunits ⁵. In the two subunits, oxygen-sensitive HIF-1α accumulates rapidly under hypoxic conditions and modulates the expression of several target genes in protecting tissues against ischemia and infarction ^6-9. Thus, HIF-1α is considered as a transcription factor that mediates adaptive responses to hypoxia and ischemia ^6-9.

A rodent model of the femoral artery ligation is widely used for the study of hindlimb ischemia-induced responses ^10-14, because the ligated animals exhibit normal flow at rest but impaired limb blood flow reserve capacity with activity ^15-17. Thus, in this study, we first used the western blotting methods to examine expression of HIF-1α protein in sensory neurons-dorsal roots ganglion (DRG) neurons of rats following femoral artery occlusion.

Static muscle contraction induces increases in arterial blood pressure and heart rate (HR) via activation of thin fiber muscle afferent nerves ¹⁸. Thus, we also examined effects of femoral artery occlusion on the reflex cardiovascular responses to muscle contraction. In addition, under normal conditions, the HIF-α subunit is hydroxylated by the enzyme HIF-α prolyl hydroxylase (HIF-PH) which causes ubiquitylation of HIF-α and subsequent destruction. Dimethyloxalylglycine (DMOG) is a cell permeable, competitive inhibitor of HIF-α HIF-PH thereby leading to the stabilization of HIF. Thus, in the current study, DMOG was injected into the hindlimb muscles to increase expression of HIF-1α protein ¹⁹, and the reflex cardiovascular responses to muscle contraction were further examined after DMOG injection. We hypothesized that arterial occlusion increases the levels of sensory nerves’ HIF-1α and augments the reflex cardiovascular responses induced by activation of muscle afferent nerves.

II. Methods

All procedures of this study were approved by the Animal Care Committee of this institution.

Femoral artery ligation

Twenty-four male Sprague-Dawley rats weighing 400-600 g were anesthetized by inhalation of an isoflurane-oxygen mixture (2-5% isoflurane in 100% oxygen). The bilateral femoral arteries were surgically exposed and immediately isolated distal to the inguinal ligament ^15-17. The right femoral artery was ligated by a ligature (3-0 silk) around ~3mm distal to the inguinal ligament. Sham control limbs underwent the same procedure as described except that a suture was placed below the left femoral artery but was not tied. The rats were allowed to recover 6, 24, and 72 hours after the surgery of femoral artery ligation.

In another group of experiments, in order to examine the effects of HIF-1α on the reflex pressor response to static muscle contraction, DMOG (40mg/kg in 0.1 ml of saline) was injected into the hindlimb muscle in eleven rats after they received an isoflurane-oxygen mixture. In nine control rats, the same volume of saline was injected into the hindlimb muscles. A period of 24 hours was allowed before the muscle contraction experiments were performed. Previous studies have shown that DMOG at this range of dosages can induce stabilize or increase expression of HIF-1α ^{12, 19, 20}.

Western blots analysis

The rats were deeply anesthetized with isoflurane and euthanized at the time points of 6, 24, and 72 h following the femoral artery ligation or 24 hours following injection of DMOG. The bilateral DRGs at the lumbar levels (L4-L6) and cervical DRGs were then removed. Note that the DRGs on the leg with the femoral occlusion and DMOG injection were used experimental group; and DRGs on another leg of the same rat with sham-control procedures and saline injection were used as control. The bilateral DRG tissues from the same rat were used for the western blot analysis. This procedure offered us an opportunity to minimize differences likely caused by individual animal. After removal of the DRGs, they were immediately homogenized in ice-cold lysis buffer containing 20mM HEPES, 1.5mM MgCl₂, 0.2mM EDTA, 0.1M NaCl, 0.2mM DTT supplemented with protease inhibitors (Sigma-Aldrich, St. Louis, MO), followed by addition of NaCl to a final concentration of 0.45M. The supernatant was collected after the lysates were centrifuged at 15,000 × g for 15 min at 4°C and glycerol was added to a final concentration of 20%. The concentration of protein was determined using BCA protein assay kit (Pierce Biotech, Rockford, IL) and bovine serum albumin as standard. Equal amounts of protein (100-200μg) were subjected onto NuPAGE Bis-Tris (4-20%) gel electrophoresis (Invitrogen, Carlsbad, CA) and electrotransferred to a hydrophobic polyvinylidene difluoride membrane (GE Water & Process Technologies, Gloucester, MA). After blocking with 10% non-fat milk in PBS containing 0.1% Tween 20 (PBST) for 1 hr at room temperature, the membrane was incubated overnight at 4°C with mouse monoclonal anti-HIF-1α (1:500, Novus biological, Littleton, CO) or β-actin (Sigma-Aldrich, St. Louis, MO) as the loading control. After washing twice with PBST, the membrane was incubated at room temperature for 3 hours with secondary anti-mouse IgG horseradish peroxidase-conjugated antibodies (1:1000, Amersham Biosciences, Piscataway, NJ). Antigen-antibody complexes were visualized by SuperSignal®West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL), and exposed onto an x-ray film. Then, the film was scanned and the optical density of the bands was analyzed using the Scion image software.

Experiment to examine the muscle reflex

The rats with experimental treatments, and control rats were anesthetized with a mixture of 2%-5% insoflurane in 100% oxygen. The trachea was cannulated and then the lungs were artificially ventilated with a respirator (model 683, Harvard). The right jugular vein and common carotid artery were inserted by polyethylene catheters-50 for the delivery of fluids and the measurement of arterial blood pressure. The carotid arterial was connected to a pressure transducer (AD Instruments). Heart rate was obtained by EKG recording. Body temperature was maintained between 37°C and 38°C by a heating pad, fluid balance was stabilized by a continuous infusion of saline ²¹.

A laminectomy was performed to expose the lower lumbar and upper scaral portions of the spinal cord after the rats were placed in a spinal unit (Kopf Instruments). The spinal roots were exposed and the right L4&5 ventral roots visually identified with assistance of an anatomical microscope (Cooper Surgical, Inc). The peripheral ends of the transected L4&5 ventral roots were then placed on bipolar platinum stimulating electrodes. A pool was formed by using the skin and muscle on the back and the exposed spinal region was filled with warmed (37°C) mineral oil.

Once the surgical preparation was completed, decerebration was performed as previously described and anesthesia was removed from the inhalant mixture ^{21, 22}. The calcaneal bone of right hindlimb was cut and its tendon was attached to a force transducer (Grass FT10), and the knee joints were secured by clamping the patellar tendon to a spinal unit.

A recovery of 60 min was allowed following the end of the decerebration. Static muscle contractions were then induced by electrical stimulation of the L4 and L5 ventral roots (30 seconds, 3 times motor threshold with a duration of 0.1 ms at 40 Hz). The reflex pressor and HR responses to contraction were examined.

Data Analysis

Arterial blood pressure and HR were displayed continuously on a Dell computer using a PowerLab system (AD Instruments). Mean arterial pressure (MAP) was obtained by integrating the arterial signal with a time constant of 4 seconds. Control values were determined by analyzing at least 30 seconds of the data immediately before the stimulation. The peak response of each variable was determined by the peak change from control.

All experimental data were expressed as mean ± S.E.M. One-way ANOVA with repeated measures was performed to assess significant changes in the data of ΔMAP (mmHg), ΔHR (beats/min) and developed tension (g) between groups. As appropriate, Tukey’s post hoc tests were used to determine differences. An unpaired t-test was performed to analyze the optical density for the western blotting experiments. Statistical significances were considered at P<0.05.

III. Results

Expression of HIF-1α protein in DRG neurons

First, we examined effects of femoral artery ligation on expression of HIF-1α protein in L4-L6 of DRG neurons. Figure 1 shows that HIF-1α was significantly increased 6, 24 and 72 hours after femoral artery occlusion as compared with sham-control. HIF-1α expression tended to decline with time of the femoral ligation. The optical densities were 3.4±0.5, 3.1±0.4 and 2.3±0.4 at 6, 24 and 72 hours after the ligation (n=4 in each group). However, there was no significant difference in response of HIF-1α in the DRG neurons in different time courses.

Fig. 1.

The Western blot assays were performed to analyze expression of hypoxia inducible factor-1α (HIF-1α) protein in the dorsal root ganglia (DRG) 6, 24 and 72 hours after the femoral artery ligation. β-actin was used as a loading control. The femoral occlusion was performed on one leg; and sham-control procedures were performed on another leg of the same rat. The bilateral lumbar 4-6 DRGs (from ischemic/ISC and nonischemic/NIS legs) were collected for the blotting analysis. Results represent means±SEM of n=4 (each time course). *P<0.05, vs. NIS

Next, we examined if infusion of DMOG in the hindlimb muscles can increase expression of HIF-1α protein in the DRG neurons. L4-L6 and cervical 1-7 of DRG neurons were used for analyzing the levels of HIF-1α protein 24 hours after intramuscular injection of DMOG. Figure 2 shows that HIF-1α expression in the lumbar DRG neurons was significant increased after administration of DMOG compared with control (optical density: 5.0±0.3 after DMOG vs 1.3±0.2 in control, P<0.05, n=3). However, no significant changes in HIF-1α were seen in the cervical DRGs after DMOG (optical density: 1.5±0.1 after DMOG vs 1.4±0.1 in control, P>0.05, n=3). These results indicate that DMOG injected in the hindlimb muscles did not affect expression of HIF-1α outside the lumbar DRG.

Fig. 2.

Expression of hypoxia inducible factor-1α (HIF-1α) protein in the dorsal root ganglia (DRG) 24 hours after intramuscular injection of dimethyloxalylglycine (DMOG). β-actin was used as a loading control. DMOG (40mg/kg body weight) was injected on one leg; and the same volume of saline was injected on another leg as control. The bilateral lumbar 4-6 DRGs and cervical DRGs were collected for the western blot analysis. Average data and typical bands show that HIF-1α protein was increased in lumbar 4-6 DRG neurons after DMOG, but this was not seen in control and cervical DRG neurons. Results represent means±SEM of n=3. *P<0.05, compared with lumbar control.

Cardiovascular responses to static muscle contraction

Table 1 shows that there is no significant difference in baseline MAP and HR values in control rats (n=7) and rats with 24 hours of femoral artery occlusion (n=5). Effects of femoral occlusion on MAP and HR responses to static muscle contraction were examined. Figure 3 demonstrates that muscle contraction significantly increased MAP and HR in control rats and rats with 24 hours of the femoral occlusion. As compared with control, femoral occlusion augmented contraction-induced MAP response. Note that there were no significant differences in developed tension in two groups.

Table 1. Baseline MAP and HR and their responses to muscle contraction in control rats and rats with 24 hours of femoral artery occlusion.

	Control (n=7)		Occlusion (n=5)
	Baseline	Response	Baseline	Response
MAP, mmHg	88±6	101±7*	90±12	119±10*
HR, beats/min	520±15	539±12*	505±15	532±18*

Values are means ± SE. MAP, mean arterial pressure; HR, heart rate. There is no significant difference among basal values.

^*P<0.05, vs. baseline.

Fig. 3.

Effects of femoral artery ligation on mean arterial pressure (MAP) and heart rate (HR) responses induced by static muscle contraction. Muscle contraction was induced by electrical stimulation of the lumbar 4-5 ventral roots 24 hours after the femoral ligation. MAP response to muscle contraction was significantly enhanced in ligated rats as compared with control rats. The duration of stimulation was 30 seconds. Note that there were no significant differences in HR response during muscle contraction and developed tension in two experimental groups. Data represent means±SEM. *P<0.05, vs. control. The number of animals = 7 in control; and 5 after the femoral occlusion.

In order to examine the effects of HIF-1α on the reflex pressor response to static muscle contraction, DMOG was given via intramuscular injection. Table 2 shows that baseline values for MAP and HR in control rats (n=9) and in rats with DMOG injection (n=8). Note that there is no significant difference in baseline MAP and HR values in two experimental groups. Figure 4 demonstrates that muscle contraction induced significant MAP and HR responses in both control and DMOG groups; however, there were no significant differences in MAP, HR and peak muscle tension in two groups (P>0.05). These data suggest that DMOG-induced HIF-1α in sensory neurons per se may not manipulate the reflex pressor response during muscle contraction.

Table 2. Baseline MAP and HR and their responses to muscle contraction in control rats and rats with prior injection of DMOG.

	Control (n=9)		DMOG (n-8)
	Baseline	Response	Baseline	Response
MAP, mmHg	89±5	102±5*	102±12	113±11*
HR, beats/min	520±17	538±16*	512±15	531±15*

Values are means ± SE. MAP, mean arterial pressure; HR, heart rate. There is no significant difference among basal values.

^*P<0.05, vs. baseline.DMOG: dimethyloxalylglycine.

Fig. 4.

Effects of intramuscular injection of dimethyloxalylglycine (DMOG) on mean arterial pressure (MAP) and heart rate (HR) responses induced by static muscle contraction. Muscle contraction was induced by electrical stimulation of the lumbar 4-5 ventral roots 24 hours after injection DMOG. The duration of stimulation was 30 seconds. There are no significant differences in MAP and HR responses, and peak muscle tension in control and after DMOG. Data represent means±SEM. The number of animals = 9 in control; and 8 after application of DMOG.

IV. Discussion

Static muscle contraction induces increases in arterial blood pressure and HR via activation of thin fiber muscle afferent nerves. The purpose of this study was to examine if arterial occlusion increases the levels of HIF-1α in sensory neurons; and if engagement of HIF-1α is responsible for enhancement in the reflex cardiovascular responses induced by activation of muscle afferent nerves.

The first insight we gained in this study by using western blot analysis showed that HIF-1α protein expression is significantly increased in DRG neurons 6-72 hours after femoral artery ligation as compared with non-ligated controls. This result suggests that femoral occlusion induces HIF-1α response in sensory nerves.

DMOG, an inhibitor of prolyl hydroxylase, has been shown to stabilize or increase HIF-1α protein and also enhance the expression downstream target genes ^{12, 19}. Milkiewicz et al ¹² reported that inhibition of endogenous HIF inactivation by DMOG induces angiogenesis in ischemic skeletal muscles of mice. In the current study, we further examined expression of HIF-1α protein in DRG neurons induced by intramuscular injection of DMOG. HIF-1α protein expression was significantly increased in lumbar DRG neurons 24 hours after injection of DMOG into the hindlimb muscles as compared with sham-controls. In contrast, DMOG injection induced no significant changes in HIF-1α protein expression in cervical DRG neurons. Our data suggest that injection of DMOG into the hindlimb muscles did not affect outside lumbar DRG neurons.

In this report, we also examined effects of femoral occlusion on the reflex cardiovascular responses evoked by activation of muscle afferent nerves. Our data have shown that 24 hours of femoral artery occlusion significantly increased MAP response induced by static muscle contraction. In order to determine if HIF-1α has a potential effect on the muscle reflex, we injected DMOG into the hindlimb muscles. Then, MAP and HR responses induced by static muscle contraction were examined 24 hours after DMOG injection. Our result shows that there were no significant differences in increases of the reflex MAP and HR responses after DMOG as compared with controls.

HIF-1α is considered as a central factor to play a role in cardioprotection ²³. Accumulation of HIF-1α induced by DMOG seems to protect tissue against myocardial ischemia injury within 3 hours ²⁴ and to induce angiogenesis in ischemic skeletal muscles of mice after 11 days ¹² or in human critical limb ischemia over two weeks ²⁵. Thus, it is noted that time course for injection of DMOG might affect the effects of HIF-1α on responses of MAP and HR induced by static muscle contraction. In this study, increased expression of HIF-1α was seen in DRG neurons 24 hours after DMOG injection. Nevertheless, DMOG did not significantly alter MAP response to muscle contraction. As a result, these data suggest that DMOG-induced HIF-1α in sensory neurons per se may not directly affect the reflex pressor response during muscle contraction.

A limitation of this study was that we did not determine if attenuation of HIF-1α in DRG neurons affects the cardiovascular responses induced by muscle contraction given that currently there are no availabilities for chemicals to restrain the levels of HIF-1α. If inhibition of arterial occlusion-induced HIF-1α response in sensory neurons could attenuate blood pressure response to muscle contraction, this would suggest that there is a possibility that HIF-1α is engaged in the muscle pressor reflex.

Interestingly, HIF-1α has been reported to play an important role in exercising skeletal muscle. One study ²⁶ showed that levels of HIF-1α mRNA and protein are greater in glycolytic muscles than the levels seen in oxidative muscles. Moreover, acute exercise leads to increased levels of HIF-1α protein in muscles and increases in HIF-1α mRNA are linked to changes in endothelial growth factor (VEGF) ^{27, 28}. In a rat study, muscle contraction induced by electrical stimulation of the sciatic nerve increases HIF-1α protein in muscle by ~six-fold and this response occurs with increases of VEGF mRNA and protein levels ²⁹. In HIF-1α knockout mice, exercise-induced expression of related genes in the skeletal muscle as well as exercise capacity are decreased ³⁰.

VEGF has been reported to regulate angiogenic process in ischemic limb muscles ³¹. There is a general agreement that increased HIF-1α contributes to atherosclerosis through alteration of smooth muscle cell proliferation and migration, angiogenesis, and lipid metabolism ³². Thus, it would be interesting to investigate if exercise training can improve the levels of HIF-1α and VEGF in exercising muscle and if this is linked to exercise capacity in patients with peripheral arterial disease largely due to atherosclerosis. In addition, a prior report suggested that exercise training can increase the levels of β₂-adrenergic receptor (β₂ AR and reverse the impairments to and lipid metabolism observed in the skeletal muscle of rats who have genetic low intrinsic running capacity ³³. This is interesting because β₂-AR has been reported to play a critical role in endothelial cell proliferation and function including revascularization in neoangiogenesis in response to ischemia ³⁴. Also, the reduced availability of glucose downregulates HIF-1 in part through the inhibition of HIF-1α mRNA translation under hypoxia observed in pathophysiological situations such as ischemic diseases ³⁵. Thus, it is speculated that exercise training likely improve angiogenesis to increase muscle blood supply to active muscle as well as glucose and lipid metabolism via a mechanism of HIF-1α in patients with peripheral arterial disease. Nevertheless, another limitation of this study was that we did not determine the effects of exercise training on the levels of HIF-1α, VEGF and β₂-AR in DRG and muscle tissues.

In addition, our published studies have demonstrated that femoral artery ligation induces greater expression of metabolic receptors i.e. transient receptor potential vanilloid type 1 (TRPV1), purinergic P2X3 and acid sensing ion channel (ASIC) ^36-41. Also, response of DRG neurons with activation of those receptors is enhanced in rats with the femoral occlusion ^{36, 39, 40, 42}. These alternations in expression and response of those metabolic receptors lead to augments in afferent nerve-mediated sympathetic responsiveness. Prior reports suggest that nerve growth factor (NGF) can increase TRPV1, P2X and ASIC receptors expression in DRG neurons ^43-46. NGF is likely to play an important role in arterial occlusion-augmented metabolic responses ^{37, 38, 42}. First, the levels of NGF are increased in DRG neurons of rats with 24 hours of femoral occlusion ⁴². Second, infusion of NGF into the hindlimb muscles increases sympathetic and blood pressure and DRG responses with stimulation of TRPV1, P2X and ASIC receptors ^{37, 38, 42}. NGF selectively affects a subpopulation of DRG neurons that supply metabolically sensitive muscle afferent nerves ³⁸.

It is noted that the time courses are very similar in increased HIF-1α expression, and elevated NGF and amplitude of DRG response to stimulation of TRPV1, P2X3 and ASIC3 receptors after ischemic insult induced by the femoral artery occlusion ^{36, 39, 40, 42}. The similarity may indicate that there is a close relationship between NGF and HIF-1α responses in the DRG neurons in the processing of the muscle ischemia. Interestingly, published work shows that increasing HIF-1α or inhibiting HIF-1α prolyl hydroxylases can attenuate NGF deprivation induced-effects on neurons, suggesting that HIF-1α plays a regulating role in effects of NGF ^{20, 47, 48}. Thus, we postulate that HIF-1α may contribute to effects of NGF on augmented TRPV1 response in the DRG neurons after arterial occlusion.

Figure 5 summarizes the potential mechanisms by which HIF-1α modulates blood pressure response to stimulation of muscle metabolic receptors and blood supply to active muscle under the conditions of muscle ischemia observed in peripheral arterial disease.

Fig. 5.

The schematic diagram summarizes the regulatory mechanisms of HIF-1α in blood pressure response to stimulation of muscle metabolic receptors and blood supply to active muscle after muscle ischemia induced by femoral artery ligation (a model of peripheral arterial disease). Refer to abbreviations in the text.

V. Conclusions

Our data show that femoral occlusion augments expression of HIF-1α protein in DRG neurons, and enhances the muscle afferent-mediated reflex pressor response. DMOG injected into the hindlimb muscles increases expression of HIF-1α protein in DRG neurons without altering the reflex cardiovascular responses during muscle contraction. Additional investigations are required to clarify if HIF-1α is responsible for enhancement in the reflex cardiovascular responses induced by activation of muscle afferent nerves under conditions of the hinlimb vascular inefficiency seen in peripheral artery occlusive disease. However, our current data suggest that an increase in HIF-1α of DRG neurons per se may not alter the muscle reflex.