Muscle contraction induced arterial shear stress increases endothelial nitric oxide synthase phosphorylation in humans

Elevations in brachial artery shear in response to forearm exercise increased endothelial nitric oxide synthase Ser¹¹⁷⁷ phosphorylation in brachial artery endothelial cells of healthy humans. Our present study provides the first evidence in humans that muscle contraction-induced increases in conduit arterial shear lead to in vivo posttranslational modification of endothelial nitric oxide synthase activity in endothelial cells.

Abstract

We determined if local increases in brachial artery shear during repetitive muscle contractions induce changes in protein expression of endothelial nitric oxide synthase (eNOS) and/or phosphorylated (p-)eNOS at Ser¹¹⁷⁷, the primary activation site on eNOS, in endothelial cells (ECs) of humans. Seven young male subjects (25 ± 1 yr) performed 20 separate bouts (3 min each) of rhythmic forearm exercise at 20% of maximum over a 2-h period. Each bout of exercise was separated by 3 min of rest. An additional six male subjects (24 ± 1 yr) served as time controls (no exercise). ECs were freshly isolated from the brachial artery using sterile J-wires through an arterial catheter at baseline and again after the 2-h exercise or time control period. Expression of eNOS or p-eNOS Ser¹¹⁷⁷ in ECs was determined via immunofluorescence. Brachial artery mean shear rate was elevated compared with baseline and the time control group throughout the 2-h exercise protocol (P < 0.001). p-eNOS Ser¹¹⁷⁷ expression was increased 57% in ECs in the exercise group [0.06 ± 0.01 vs. 0.10 ± 0.02 arbitrary units (au), P = 0.02] but not in the time control group (0.08 ± 0.01 vs. 0.07 ± 0.01 au, P = 0.72). In contrast, total eNOS expression did not change in either the exercise (0.13 ± 0.04 vs. 0.12 ± 0.03 au) or time control (0.12 ± 0.03 vs. 0.11 ± 0.03 au) group (P > 0.05 for both). Our novel results suggest that elevations in brachial artery shear increase eNOS Ser¹¹⁷⁷ phosphorylation in the absence of changes in total eNOS in ECs of young healthy male subjects, suggesting that this model is sufficient to alter posttranslational modification of eNOS activity in vivo in humans.

NEW & NOTEWORTHY Elevations in brachial artery shear in response to forearm exercise increased endothelial nitric oxide synthase Ser¹¹⁷⁷ phosphorylation in brachial artery endothelial cells of healthy humans. Our present study provides the first evidence in humans that muscle contraction-induced increases in conduit arterial shear lead to in vivo posttranslational modification of endothelial nitric oxide synthase activity in endothelial cells.

the endothelial lining of blood vessels is subject to numerous hemodynamic forces, including blood flow induced shear stress, which has a powerful influence on endothelial phenotype and function (9). Mechanoreceptors located along the endothelium are capable of detecting changes in shear stress and activating various cell signal transduction pathways (4). In turn, the pattern of blood flow and the resulting shear stress in arteries are hypothesized to influence endothelial cell function and vascular health. In vitro studies using endothelial cells (ECs) and isolated perfused arterioles have suggested that low shear stress or disturbed flow patterns are linked to a proatherogenic EC phenotype (6, 8, 10, 12). Conversely, ECs and isolated arterioles exposed to elevated shear stress and/or intraluminal flow demonstrate increased expression and phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at its activation site, Ser¹¹⁷⁷ [phosphorylated (p-)eNOS Ser¹¹⁷⁷] (7, 26, 27), which contributes to greater NO bioavailability and improved vascular function (17).

One major stimulus for increasing shear stress in the arterial circulation is the rhythmic muscle contractions that occur during exercise. Muscle blood flow is closely matched to the metabolic demands of contraction and therefore increases as the intensity/force of contraction increases (14). Depending on the type of exercise and amount of muscle mass involved, skeletal muscle blood flow can increase 50- to 100-fold during exercise (1, 14, 18). Along with the drastic increase in blood flow to the active muscles during exercise, the corresponding vasculature is exposed to extremely high levels of frictional and shear forces, which are primary activators for multiple EC signal transduction pathways. Along these lines, exercise training is associated with an increased eNOS protein expression and phosphorylation of Ser¹¹⁷⁷ in animals and humans (11, 15, 16).

Although evidence suggests that experimentally induced increases in shear stress enhance eNOS protein expression and phosphorylation in vitro (16, 25), it is unclear whether results obtained from cell culture and isolated vessels from rodents can be extrapolated to ECs lining a blood vessel that is subjected to an increase in blood flow in vivo. This is particularly important when considering that many of the in vitro studies have used a steady laminar flow to increase shear stress without exposure to arterial pressure, whereas ECs in vivo are exposed to pulsatile flow and pressure that often presents with more of an oscillatory flow pattern (20). With this information as background, we determined if local increases in conduit artery shear during repetitive muscle contractions induce acute changes in the expression of total eNOS and/or p-eNOS in humans. We hypothesized that elevatation of brachial artery shear during rhythmic forearm contractions would increase the in vivo expression of p-eNOS Ser¹¹⁷⁷, the primary phosphorylation site responsible for the activation of eNOS in humans.

METHODS

Subjects.

Fourteen young (24 ± 1) male subjects volunteered to participate in this study. Subjects gave written informed consent and were sedentary to recreationally active, nonobese, nonsmokers, free of any diagnosed cardiovascular or metabolic complications, and not taking any medications. Experiments were performed after an overnight fast and after the subjects refrained from exercise and caffeine for at least 24 h. All study protocols were approved by the Institutional Review Board of the University of Iowa. At study entry, subjects were randomly assigned to either an exercise group (n = 8) or a time control group (n = 6).

Arterial catheterization and cell collection.

A 20-gauge, 5-cm catheter (model RA-04020, Arrow, Reading, PA) was placed in the brachial artery of the left (experimental) arm under aseptic conditions after local anesthesia (1% lidocaine) as previously described (5, 21). Shortly after catheter placement, two separate flexible 0.018- or 0.021-in. mesh 3-mm guide wires with a J-shaped tip (Daig, Minnetonka, MN) were advanced ~3–4 cm beyond the tip of the catheter and retracted. The distal portion of each J-wire and the guide wire used for EC collection were transferred to dissociation buffer. Cells were immediately taken to the laboratory for processing (see below). Cell collection was repeated immediately after the completion of the forearm exercise protocol or time control period. After the initial cell collection, the catheter was connected to a pressure transducer positioned at heart level (model PX600F, Edwards Lifescience, Irvine, CA) to allow the measurement of arterial pressure.

Forearm exercise protocol.

Subjects in the exercise group performed rhythmic forearm exercise with a handgrip device in the left arm at 20% maximal voluntary contraction (MVC; mean: 47 ± 2 kg, range: 43−55 kg). The weight was lifted 4−5 cm over a pulley at a duty cycle of 1-s contraction and 2-s relaxation (20 contractions/min) using a metronome to ensure correct timing. The average weight used for forearm exercise was 9.4 ± 0.4 kg. Subjects performed 20 separate exercise bouts (3 min each). Each bout was separated by 3 min of rest. The on-and-off approach (switching between exercise and rest) allowed for each subject to complete the full 2-h protocol without fatiguing while the brachial artery shear rate remained elevated above baseline throughout the protocol. Pilot work from our laboratory demonstrated that mean shear in the brachial artery during 20% MVC forearm exercise is elevated ~5- to 6-fold across the 20 exercise bouts and remains ~2- to 3-fold higher during the rest periods (in between exercise bouts) compared with baseline values.

Brachial artery blood flow.

Brachial artery mean blood velocity and diameter were determined with a 12-MHz linear-array Doppler probe (model M12L, Vivid 7, General Electric, Milwaukee, WI). Blood velocity was measured with a probe insonation angle previously calibrated to 60°. Sample volume was adjusted to cover the width of the brachial artery to encompass the entire lumen of the vessel and the cursor was set at midvessel. Measured velocity waveforms were synchronized to a data-acquisition system (WinDaq, DATAQ Instruments, Akron, OH) via a Doppler audio transformer (13). Brachial artery diameter measurements were obtained at end diastole during the last 15 s of each measured exercise and rest bout, respectively.

EC protein expression via immunofluorescence.

Cells were recovered from the dissociation buffer [0.5% BSA, 2mM EDTA, and 18 U/ml heparin in PBS (pH 7.4)] by centrifugation. The pellet was then treated with a erythrocyte lysing kit (R&D Systems). The remaining cells were resuspended in EBM-2 (Lonza) and applied to chambered glass slides pretreated with poly-l-lysine (Sigma). Gentle centrifugation in a microplate swinging-bucket rotor (450 rpm/~28 g, 10 s with deceleration set to 0, Thermo Scientific) was used to adhere the cells to the slides. Cells were fixed with 4% paraformaldehyde (Santa Cruz Biotechnology), washed in PBS, dried for 5 min, and frozen at −80°C until staining and analysis (22).

After being thawed, cells were permeabilized with 0.1% Triton X-100 (Sigma) and blocked with 0.5% BSA (Fisher Scientific). Cells were incubated overnight at 37°C with monoclonal antibodies for eNOS or p-eNOS Ser¹¹⁷⁷ (dilution: 1:100, BD Biosciences, San Diego, CA). Proteins were fluorescently labeled using Alexa Fluor 594-conjugated secondary antibody for 1 h at 37°C (1:100, Life Technologies, Carlsbad, CA), and slides were treated with VECTASHIELD Antifade Mounting Media with DAPI (Vector Laboratories). Cells were costained with FITC-conjugated anti-von Willebrand factor antibody (Abcam). Slides were viewed on a fluorescence microscope (Eclipse 810i, Nikon), and 20 individual ECs were imaged using a digital camera (Diagnostic Instruments). In addition to von Willebrand factor, the granular morphology of Weibel-Palade bodies coupled with a single intact nucleus was used to reliably select ECs (over granulocytes) for analysis. We have verified this approach by confirming that ECs are CD45 negative and granulocytes are CD45 positive (data not shown). EC images were analyzed using ImageJ (National Institutes of Health, Bethesda, MD) to quantify the intensity of the eNOS/p-eNOS staining (i.e., average pixel intensity) (21). Values are reported as ratios of sample ECs to cultured human aortic EC (HAEC) average pixel fluorescence intensity to reduce variability between staining batches.

Data analysis and statistics.

Data were collected at 250 Hz, stored on a computer, and analyzed offline with signal processing software (WinDaq, DATAQ Instruments). Mean arterial pressure (MAP) was determined from the brachial artery pressure waveform. Diameter and velocity measures were used to estimate brachial artery shear rates and to calculate forearm blood flow (FBF). Mean shear rate (in s⁻¹) was defined as 4 × V_m/D, where V_m is mean blood velocity (in cm/s) and D is arterial diameter (in cm) (5). FBF was calculated as the product of mean blood velocity (in cm/s) and brachial artery cross-sectional area (in cm²) and expressed as milliliters per minute. Forearm vascular conductance (FVC) was calculated as the ratio between forearm blood flow and MAP and expressed as milliliters per minute per 100 mmHg. Brachial artery velocity measurements were acquired at rest (baseline) and throughout five specific exercise/recovery cycles. These included cycle 1 (0–6 min), cycle 5 (24–30 min), cycle 10 (54–60 min), cycle 15 (84–90 min), and cycle 20 (114–120 min). Each cycle consisted of the 3 min of exercise and 3 min of rest/recovery that followed the exercise bout (Fig. 1). Measurements of FBF, FVC, and MAP reflect an average over each 3-min period of exercise or rest. Mean shear values are presented as an average over the entire cycle (6-min period). For the time control group, all measured and calculated hemodynamic variables reflect 6 min of rest within each cycle, as there were no contractions.

Fig. 1.

Schematic diagram of the experimental protocol. Each cycle (C) consisted of 3 min of exercise and 3 min of rest/recovery. Gray shaded boxes indicate cycles in which brachial artery velocity measurements were acquired.

All values are expressed as means ± SE. Brachial artery hemodynamic and shear variables as well as endothelial protein expression were compared via two-way repeated-measures ANOVA to detect differences across and between conditions. Tukey’s post hoc analysis determined where statistical differences occurred. Additionally, linear regression analysis was performed and Pearson correlation coefficients were calculated to assess the relationship between changes in mean shear and endothelial protein expression in the seven subjects that performed forearm exercise. All statistical analyses were performed using SigmaPlot software version 11.0 (Systat Software, San Jose, CA). Statistical difference was set a priori at P < 0.05.

RESULTS

All 14 subjects completed the protocol. However, an insufficient amount of ECs was collected during the postmeasurement in one subject. Therefore, brachial artery hemodynamic and EC protein expression data are presented for 13 subjects (7 exercise and 6 time control subjects). Those subjects included in the group analysis were 25 ± 2 yr of age, 181 ± 2 cm in height, and weighed 79 ± 3 kg (body mass index: 24.3 ± 0.7 kg/m²). There were no differences between groups for any of the demographic variables.

Brachial artery hemodynamics.

By design, brachial artery shear rate was elevated throughout the protocol in the exercise group (P < 0.001), whereas it remained unchanged in the time control group (Fig. 2). In the exercise group, FBF and FVC were increased above baseline during both the exercise and rest phases of each cycle (P < 0.001; Table 1). MAP was increased above baseline during the exercise phase in cycles 5, 10, 15, and 20 as well as the rest phase of cycle 20 (P < 0.05; Table 1). Brachial artery diameter increased starting at cycle 5 and remained above baseline throughout the remainder of the protocol in the exercise group (P < 0.001; Table 1). FBF, FVC, brachial artery diameter, and MAP did not change at any time point in the time control group.

Fig. 2.

Mean shear rate across exercise/recovery cycles for subjects that performed rhythmic forearm exercise as well as subjects that served as time controls (no exercise). Values are means ± SE. *P < 0.001 vs. baseline; †P < 0.001 vs. the time control group.

Table 1.

Brachial artery hemodynamics

		Cycle 1		Cycle 5		Cycle 10		Cycle 15		Cycle 20
	Baseline	Exercise	Rest	Exercise	Rest	Exercise	Rest	Exercise	Rest	Exercise	Rest
Exercise (n = 7)
FBF, ml/min	43 ± 8	217 ± 18*	204 ± 16*†	279 ± 21*	185 ± 14*†	302 ± 18*	172 ± 16*†	304 ± 19*	163 ± 21*†	314 ± 20*	147 ± 11*†
FVC, ml⋅min−1⋅100 mmHg−1	49 ± 10	235 ± 20*	243 ± 22*†	292 ± 23*	212 ± 19*†	317 ± 24*	197 ± 22*†	310 ± 24*	178 ± 25*†	317 ± 25*	159 ± 14*†
Brachial artery diameter, cm	0.40 ± 0.01	0.42 ± 0.02	0.43 ± 0.02	0.46 ± 0.02*	0.45 ± 0.01*	0.46 ± 0.02*	0.46 ± 0.02*	0.46 ± 0.02*	0.46 ± 0.02*	0.47 ± 0.02*	0.46 ± 0.02*
MAP, mmHg	88 ± 2	92 ± 2	85 ± 2	95 ± 3*	88 ± 2	96 ± 2*	89 ± 2	98 ± 2*	91 ± 2	99 ± 2*	93 ± 2*
Time control (n = 6)
FBF, ml/min	53 ± 12		56 ± 13		47 ± 9		46 ± 6		47 ± 5		46 ± 6
FVC, ml⋅min−1⋅100 mmHg−1	62 ± 15		66 ± 16		54 ± 11		52 ± 8		53 ± 6		52 ± 7
Brachial artery diameter, cm	0.43 ± 0.02		0.44 ± 0.02		0.44 ± 0.02		0.44 ± 0.02		0.43 ± 0.02		0.43 ± 0.02
MAP, mmHg	88 ± 1		86 ± 1		88 ± 1		88 ± 2		90 ± 2		91 ± 3

Values are means ± SE. FBF, forearm blood flow; FVC, forearm vascular conductance; MAP, mean arterial pressure.

^*P < 0.05 vs. baseline;

^†P < 0.001 vs. time control.

eNOS and p-eNOS Ser¹¹⁷⁷ expression.

Brachial artery EC expression of total eNOS did not change in either group after each respective trial (group × time interaction, P = 0.72; Fig. 3A). Conversely, endothelial levels of p-eNOS Ser¹¹⁷⁷ increased by 57% in the exercise group (P < 0.05), with no change in the time control group (P = 0.72; Fig. 3B). Additionally, there were moderate yet nonsignificant correlations between the relative changes in mean shear and endothelial expression of eNOS (r = 0.57, P = 0.18) and p-eNOS Ser¹¹⁷⁷ (r = 0.65, P = 0.11) in the seven subjects that performed forearm exercise.

Fig. 3.

Brachial artery endothelial protein expression of total endothelial nitric oxide synthase (eNOS; A) and phosphorylated (p-)eNOS Ser¹¹⁷⁷ (B) before (Pre) and after (Post) 2 h of intermittent forearm contractions (exercise) or rest (time control). Mean responses are represented by vertical bars; individual responses are represented by symbols. Values are means ± SE. *P < 0.05 vs. Pre. Representative immunofluorescent images of 4′,6′-diamidino-2-phenylindole (DAPI; blue)- and von Willebrand factor (vWF; green)-stained endothelial cells as well as eNOS and p-eNOS Ser¹¹⁷⁷ proteins (red) are shown for the exercise group only.

DISCUSSION

The major novel finding of the present study was that contraction-induced elevations in conduit artery shear results in increased phosphorylation of eNOS in humans. Specifically, we found that elevation of brachial artery shear via forearm exercise over a 2-h period increased levels of p-eNOS Ser¹¹⁷⁷ in ECs, in the absence of any change in total eNOS expression in young male subjects. To our knowledge, this is the first evidence demonstrating that short-term experimental elevations in shear stress via exercise lead to posttranslational alterations in eNOS activity in vivo.

Over the past couple decades, the importance of shear stress as a signal in regulating eNOS mRNA and eNOS protein expressions has become well recognized (2). However, these studies have been mainly restricted to cultured ECs or isolated vessels and in many cases performed under conditions of steady laminar and nonpulsatile flow and shear patterns. Conversely, conduit arteries in vivo are constantly exposed to pulsatile flow and shear that often present in a bidirectional pattern (antegrade and retrograde) (3). Additionally, since skeletal muscle blood flow can increase 50- to 100-fold during exercise, the magnitude of shear that the ECs are exposed to can be quite high. To address this, Zhang and colleagues (29) assessed various intracellular signaling pathways and the expression of eNOS from isolated blood vessels in mice after 50 min of treadmill running. They found that arteries from exercising mice demonstrated greater eNOS activity and increased eNOS phosphorylation compared with arteries from sedentary mice (29). Thus, these data suggest that an acute bout of exercise and the subsequent increase in shear stress can promote alterations in eNOS phosphorylation in vivo. However, it should be noted that the overall assessment of phosphorylation of the various proteins were derived from a combination of different arteries collected from each mouse and the magnitude of change in shear stress was not quantified during exercise. Therefore, it is unclear 1) what level of shear stress elicited the reported changes in eNOS and 2) whether the changes were due to a local factor (i.e., shear stress in a given artery exposed to high flows) or some systemic factor that occurs during whole body exercise. In the present study, we extended the findings of Zhang et al. (29) to humans and demonstrated that increases in local brachial artery blood flow and shear stress during a single exercise session is a sufficient stimulus for the phosphorylation of the primary site (Ser¹¹⁷⁷) of eNOS activation. In contrast, it is possible that the duration or magnitude of the shear stimulus exposure was not long enough to induce changes in total eNOS expression.

From a functional standpoint, it is clear that acute changes in shear in peripheral conduit arteries of humans directly influence endothelial function and vasodilation. Reductions in mean shear and/or increased retrograde shear negatively impact endothelial dilator function (19, 23, 24), whereas elevating mean shear can enhance endothelium-dependent dilation (24). Moreover and of particular interest to the present study, contraction-induced elevations in shear stress via rhythmic forearm exercise elicits brachial artery vasodilation via NO-mediated mechanisms (28).

In summary, to our knowledge, this is the first study to directly assess potential changes in eNOS activation and expression in response to contraction-induced increases in shear stress in humans. We found that 2 h of elevated brachial artery shear as a result of forearm exercise increases eNOS Ser¹¹⁷⁷ phosphorylation in ECs in vivo, in the absence of any detectable changes in total eNOS expression. The study is limited by studying only a small number of young male subjects (n = 6–7 subjects/group) and only one eNOS phosphorylation site but provides proof of concept that this model can be used to assess local in vivo responses to shear stress in conscious humans. Therefore, future studies should examine 1) the local EC responses to shear stress in female subjects, aged adults, or persons with cardiovascular disease risk factors and 2) other potential phosphorylation and regulatory sites of human eNOS, including Ser¹¹⁴, Ser⁶³³, and Thr⁴⁹⁵. Additionally, establishing the shear stress dose response for changes in human eNOS activation and expression are needed. Nonetheless, the experimental approach used in this study provides direct evidence for how local increases in shear stress during exercise exert beneficial effects on the endothelium.

GRANTS

This research was supported by National Institutes of Health Research Grants HL-105467 (to D. P. Casey) and AG-043722 (to G. L. Pierce) and American Heart Association Grant 13SDG143400012 (to G. L. Pierce).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

D.P.C. and G.L.P. conceived and designed research; D.P.C., K.U., L.J.W.-P., and G.L.P. performed experiments; D.P.C. and L.J.W.-P. analyzed data; D.P.C. and G.L.P. interpreted results of experiments; D.P.C. prepared figures; D.P.C. drafted manuscript; D.P.C., K.U., L.J.W.-P., and G.L.P. edited and revised manuscript; D.P.C., K.U., L.J.W.-P., and G.L.P. approved final version of manuscript.