Research Article

Essential hypertension is associated with blunted smooth muscle cell vasodilator responsiveness and is reversed by 10-20-30 training in men

Published Online:https://doi.org/10.1152/ajpcell.00047.2020

Abstract

Essential hypertension is associated with impairments in vascular function and sympathetic nerve hyperactivity; however, the extent to which the lower limbs are affected remains unclear. We examined the leg vascular responsiveness to infusion of acetylcholine (ACh), sodium nitroprusside (SNP), and phenylephrine (PEP) in 10 hypertensive men [HYP: age 59.5 ± 9.7 (means ± SD) yr; clinical and nighttime blood pressure: 142 ± 10/86 ± 10 and 141 ± 11/83 ± 6 mmHg, respectively; and body mass index (BMI): 29.2 ± 4.0 kg/m2] and 8 age-matched normotensive counterparts (NORM: age 57.9 ± 10.8 yr; clinical and nighttime blood pressure: 128 ± 9/78 ± 7 and 116 ± 3/69 ± 3 mmHg, respectively; and BMI: 26.3 ± 3.1 kg/m2). The vascular responsiveness was evaluated before and after 6 wk of 10-20-30 training, consisting of 3 × 5 × 10-s sprint followed by 30 and 20 s of low- to moderate-intensity cycling, respectively, interspersed by 3 min of rest. Before training, the vascular responsiveness to infusion of SNP was lower (P < 0.05) in HYP compared with NORM, with no difference in the responsiveness to infusion of ACh and PEP. The vascular responsiveness to infusion of SNP and ACh improved (P < 0.05) with training in HYP, with no change in NORM. With training, intra-arterial systolic blood pressure decreased (P < 0.05) by 9 mmHg in both HYP and NORM whereas diastolic blood pressure decreased (5 mmHg; P < 0.05) in HYP only. We provide here the first line of evidence in humans that smooth muscle cell vasodilator responsiveness is blunted in the lower limbs of hypertensive men. This impairment can be reversed by 10-20-30 training, which is an effective intervention to improve the responsiveness of smooth muscle cells in men with essential hypertension.

INTRODUCTION

Essential hypertension is a well-established risk factor for developing cardiovascular disease. The putative mechanisms underlying the elevated blood pressure are multifactorial and include impairments in peripheral vascular function and sympathetic nerve activity (18, 30, 38, 39, 47). Smooth muscle cell relaxation is primarily modulated by release of nitric oxide (NO) and prostacyclin from endothelial cells (endothelium-dependent vasodilation). The smooth muscle vasodilator responsiveness portion of the endothelium-dependent vasodilator response can be evaluated by infusion of a NO donor such as sodium nitroprusside (SNP), as nitroprusside relaxes smooth muscle largely by activation of the same pathways as endothelium-dependent relaxation (19, 36).

Endothelium-dependent vasodilator responsiveness has been shown to be reduced in the forearm of hypertensive individuals (50), whereas the smooth muscle cell vasodilator responsiveness appears not to be affected by essential hypertension (31). Importantly, due to the upright stature of humans, the hydrostatic pressure is higher in the legs compared with the arms, which leads to increased intima medial thickness that serves to normalize wall stress (9). Intima medial thickness has been found to be more pronounced in essential hypertension (13), indicating that smooth muscle cell structure and function in the lower limbs may be altered in this disease state. Seeing that in some arteries the intima is free of smooth muscle (46), the medial layer is where changes in smooth muscle number and phenotype are expected to be most responsible for normalizing wall stress in response to elevated pressure. Although the leg smooth muscle cell vasodilator responsiveness has never been evaluated in human hypertension, it was blunted in patients with congestive heart failure (21), a disease associated with elevated blood pressure. Moreover, given the greater vasoconstrictor responsiveness in the legs (40) likely representing an adaptive mechanism to the upright posture, it is conceivable that such vascular response is altered also in human hypertension. Hence, given the lack of studies investigating smooth muscle cell vasodilation and vasoconstriction in the lower limbs of hypertensive individuals, studies on this topic are required to unravel the mechanisms underlying essential hypertension-related impairments in leg vascular function.

Exercise is a potent stimulus to elicit marked vascular responses. Indeed, exercise-induced hyperemia, i.e., the increase in blood flow associated with skeletal muscle contraction, promotes a vasodilator response via shear stress-induced release of NO and prostacyclin (18, 37). Such response, when repeated over time, may induce vascular remodeling (51) thereby improving vascular function, an adaptive process possibly contributing to the antihypertensive effects of exercise training (7, 10, 35). Exercise intensity may play a critical role for the magnitude of training-induced vascular adaptations, as moderate to vigorous (70–85% of peak heart rate, HRpeak) (33) but not low-intensity (<60% of HRpeak) (8) exercise training improved vascular function in patients with chronic heart failure. In addition, evidence supports the effectiveness of high-intensity interval training (HIIT) for improving leg vascular function in individuals with essential hypertension (17, 38). An emerging HIIT modality is 10-20-30 training, consisting of repeated 10-s sprints followed by 30 and 20 s of low- and moderate-intensity exercise, eliciting a high average HR response (~90% of HRpeak). Despite their all-out nature, the 10-s sprints are less demanding, and therefore easier to conduct, compared with longer duration (20–30 s) sprints, which may be of importance for long-term adherence in patients with cardiometabolic disease. In various patient groups, cycling-based 10-20-30 training lowered blood pressure, increased maximum oxygen uptake (V̇o2max), and improved body composition in concert with a high compliance (11, 52). It is conceivable that the antihypertensive effects of the 10-20-30 training relates to a high shear stress, a high adrenergic stress response and/or marked metabolic perturbations during the 10-s sprints (12, 16).

Taken together, it is unknown whether the leg smooth muscle cell vasodilator responsiveness is blunted in essential hypertension and whether a period of 10-20-30 training is effective for improving peripheral vascular function in hypertensive individuals. Thus, the aim of the present study was to compare the vascular responsiveness in the lower limbs of hypertensive versus normotensive men, and to examine the adaptive response of this system to a period of 10-20-30 training. We hypothesized that 1) the leg smooth muscle cell vasodilator responsiveness was blunted in men with essential hypertension compared with normotensive counterparts, and 2) that a period of 10-20-30 training would reverse the blunted leg vasodilator responsiveness associated with essential hypertension. To test these hypotheses, we examined the effect of 6 wk of 10-20-30 training on the leg vascular responsiveness to infusion of the endothelium-dependent vasodilator acetylcholine (ACh), the smooth muscle cell vasodilator SNP, and the α1-receptor specific vasoconstrictor phenylephrine (PEP) in men with essential hypertension compared with age-matched normotensive counterparts.

MATERIALS AND METHODS

Ethical Approval

The study was approved by the Ethics Committee of Copenhagen and Frederiksberg communities (H-4-2014-100) and adhered to the requirements of the United States Federal Policy for the Protection of Human Subjects (45 CFR, Part 46) and to the principles of the latest revision of the Declaration of Helsinki. The study was registered as a clinical trial at ISRCTN.com (ISRCTN11181410), and written informed consent was obtained from all subjects before enrollment in the study. This study is part of a larger project, and data on maximum oxygen uptake (V̇o2max) have been reported elsewhere (11).

Participants

Eighteen sedentary men with a mean age of 59 ± 10 yr (range: 43–73 yr) were included in the study. Based on consecutive clinical and nighttime blood pressure measurements (BOSO TM-2430 PC2, Bosch+Sohn, Jungingen, Germany), 10 were diagnosed with essential hypertension (HYP) and 8 were classified as normotensive controls (NORM) (Table 1). Of the 10 subjects in HYP, 3 were under treatment with angiotensin II receptor antagonists and 1 was under treatment with angiotensin converting enzyme inhibitors (Supplemental Table S1; see https://doi.org/10.6084/m9.figshare.11494404.v1). Four days before the first experimental trial day subjects were instructed to stop medication (both before and after the training-intervention period). Subjects did not abstain from medication again after the experimental day and were medicated during the intervention period. Inclusion criteria were body mass index (BMI) 20–35 kg/m2, clinical and nighttime systolic/diastolic blood pressure values >140/90 and >135/80 mmHg for the hypertensive men, and <130/85 and <125/75 mmHg for the normotensive men, respectively, <2 h of physical activity per week, HbA1c <6.5% equivalent to <48 mmol/mol. Exclusion criteria were smoking, irregular resting ECG, history of cardiovascular, renal or peripheral vascular disease other than essential hypertension, use of medications other than antihypertensive drugs, and inability to perform physical exercise. No subjects in HYP were excluded based on upper limit blood pressure. Clinical blood pressure of some men in NORM were higher than guideline values (41), but all normotensive men had nighttime blood pressure values <125/75 mmHg, confirming that these men were in fact normotensive at the onset of the study. This was further confirmed by intra-arterial blood pressure measurements obtained during pretesting.

Table 1. Baseline characteristics in hypertensive and normotensive male individuals

HYP (n = 10) NORM (n = 8)
Age, yr 59.5 ± 9.7 57.9 ± 10.8
Height, cm 177 ± 8 182 ± 8
Nighttime systolic BP, mmHg 141 ± 11 116 ± 3
Nighttime diastolic BP, mmHg 83 ± 6 69 ± 3
Clinical systolic BP, mmHg 142 ± 10 128 ± 9
Clinical diastolic BP, mmHg 86 ± 10 78 ± 7

Values are means ± SD. HYP, hypertensive; NORM, normotensive; BP, blood pressure.

P < 0.01, different from NORM.

Experimental Design

A cross-sectional and longitudinal two-group design was used to investigate baseline differences between hypertensive and normotensive men as well as training-induced changes in vascular function. Before enrollment in the study, subjects underwent a medical screening with a physician. After the screening, subjects completed a graded exercise test on a cycle ergometer (Monark, Ergomedic 839E, Vansbro, Sweden) to determine V̇o2max. After inclusion, subjects underwent two experimental days before and after a 6-wk training-intervention period. Subjects were instructed to refrain from caffeine, alcohol, and exercise for 24 h before arrival on experimental days.

Screening Procedures

Subjects reported to the laboratory in the morning (8–10 AM) for the screening visit. A 12-lead ECG was conducted, blood samples were collected [red blood cells (RBC), hemoglobin (hb), HbA1c, creatinine, C-reactive protein (CRP), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), gamma glutamyl transpeptidase (GGT), activated partial thromboplastin time (APTT), international normalized ratio (INR), and coagulation factors II+VII+X], a health-questionnaire including questions on physical activity levels was filled out (level of physical activity did not differ between groups), and medically trained personnel completed a physical evaluation. Afterwards, subjects completed a graded exercise test (GXT) on a cycle ergometer (Monark, Ergomedic 839E, Vansbro, Sweden) (50 W for 4 min followed by an increase in workload of 25 W/min until volitional fatigue and/or a drop in revolutions below 55 revolutions per minute despite strong verbal encouragement) to determine V̇o2-max (highest 30-s average with a plateau in V̇o2 despite increase in workload and a respiratory exchange ratio (RER) above 1.10 as objective criteria using a breath-by-breath gas analyzing system (Oxycon Pro, Viasys Healthcare, Hoechberg, Germany). It should be mentioned, that RER values above 1.00 are not indicative of the muscular respiratory quotient and thus does not represent muscle substrate utilization but an excess production of CO2 through bicarbonate buffering of protons. If included in the study, this test served as a habituation trial.

Experimental Trial Days

Experimental trial day 1.

A schematic presentation of the experimental trial day is depicted in Fig. 1. Subjects reported to the laboratory in the morning between 8 and 9 AM, 2 h after breakfast. Subjects were instructed to record the content of the breakfast to replicate it after the intervention period. After administration of local anesthesia (lidocaine, 20 mg/mL; Astra Zeneca, Denmark), two catheters (20 gauge; Arrow Int., Reading, PA) were placed in the femoral artery and vein of the experimental leg (i.e., the leg infused with vasoactive substances) and one in the femoral artery of the nonexperimental leg in the supine position. After insertion of catheters subjects rested in the supine position for ~45 min during which the experimental leg volume was measured and mass was calculated based on measurements of circumferences and partial lengths of the leg (20). Based on estimations of leg mass, infusion rates were calculated. Afterwards, subjects received femoral arterial infusion of acetylcholine (ACh; 10, 25, and 100 μg·min−1·kg leg mass−1; Miochol-E, Bausch & Lomb, Bridgewater, NJ), sodium nitroprusside (SNP; 3, 6, and 9 μg·min−1·kg leg mass−1; Nitropress, Hospira, Lake Forest, IL), and phenylephrine (PEP; 3 μg·min−1·kg leg mass−1; Herlev Pharmacy, Copenhagen, Denmark). In HYP, one catheter clogged during infusion of SNP at 9 µg·min−1·kg leg mass−1, and another clogged during infusion of PEP; therefore, vascular measurements reported in HYP are based on n = 9 during infusion of SNP at 9 µg·min−1·kg leg mass−1 and n = 8 during infusion of PEP. Femoral arterial leg blood flow (LBF) was measured using Ultrasound Doppler (Vivid E9; GE Healthcare, Denmark) equipped with a linear probe operating at an imaging frequency of 4.0/8.0 MHz and Doppler frequency of 4.3 MHz. The site of measurements in the common femoral artery was distal to the inguinal ligament but above the bifurcation into the superficial and deep femoral branches to avoid turbulence from the bifurcation. All recordings were obtained at the lowest possible insonation angle and always <60°. Sample volume was maximized according to the width of the vessel and kept clear of the vessel walls. Doppler traces and B-mode images were recorded continuously before and from the 3rd to the 5th min during all infusions, and Doppler traces were averaged over ~30 s. Arterial diameter measures (in triplicate) were assessed during systole from arterial B-mode images with the vessel parallel to the transducer before and after each Doppler recording.

Fig. 1.

Fig. 1.Experimental overview. The leg vasodilator and vasoconstrictor responsiveness was evaluated to intra-arterial infusion of vasodilators acetylcholine (ACh) and sodium nitroprusside (SNP) and the vasoconstrictor phenylephrine (PEP) on the first experimental trial day, and body composition and maximum oxygen uptake (V̇o2max) was evaluated on the second experimental trial day before and after 6 wk of 10-20-30 training in hypertensive (n = 7–10) and normotensive (n = 8) men. Moreover, ambulatory blood pressure was measured following the first experimental trial day before the intervention period.


Heart rate and mean, systolic and diastolic femoral arterial- and venous blood pressure were continuously measured during the experimental trial. Measurements were obtained with pressure transducers (Pressure Monitoring Kit, Baxter Deerfield, IL) positioned at the level of the heart. Arterial and venous blood samples (3 mL) were sampled at 3 min during low and high infusion rates (Fig. 1). Baseline measurements of arterial and venous blood samples and LBF were obtained 5 min before initiation of infusions, each infusion protocol was separated by at least 30 min of rest, and infusion of PEP was performed last because of the long-term effects (plasma half-life of ≥3 h). On the subsequent day and night, while hypertensive subjects were off medication, nighttime blood pressure was measured in 30-min intervals. Sleep time was recorded manually by switching the apparatus between day- and nighttime by a simple one button procedure (when going to sleep/waking up). Subsequently, on the day before nighttime measurements, daily activities, working hours, and physical activity levels were recorded.

Experimental trial day 2.

Subjects reported to the laboratory between 8 and 9 AM after an overnight fasting 48–96 h after the first experimental trial day. First, subjects completed a whole body dual-energy X-ray absorptiometry (DXA) scan (Prodigy, GE Healthcare) after 10 min in the supine position. After the scan, subjects consumed a standardized meal consisting of 75 g of carbohydrates, 25 g of fats, 15 g of proteins, and water ad libitum. After ingestion of the meal (90 min) subjects completed a GXT to exhaustion on a cycle ergometer to determine V̇o2-max (Oxycon Pro, Viasys Healthcare, Hoechberg, Germany). The GXT protocol consisted of two 5-min submaximal bouts at 49 ± 9 and 92 ± 10 W, based on their screening GXT followed by 3 min of recovery. Thereafter, the GXT protocol continued with an incremental ramp test to exhaustion including increments of 20 W/min starting at the highest submaximal workload (92 ± 10 W). V̇o2max was determined as the highest value achieved during a 30-s period. Criteria used for achievement of V̇o2max were a plateau in V̇o2 despite an increase in workload and a respiratory exchange ratio above 1.10. Maximal workload (W) and time to exhaustion (s) were recorded, and heart rate was monitored throughout the test and maximal heart rate established as the highest value achieved cleansed for spikes.

Training Intervention

The 10-20-30 training intervention in the present study was conducted as cycling to eliminate the risk of injury. The 10-20-30 training was conducted for 2 × 20 min the first 2 wk and for 3 × 28 min the following 4 wk. In short, 10-20-30 training consists of five consecutive 1-min intervals divided into 30, 20, and 10 s at an intensity corresponding to ~30%, ~50%, and ~100% of maximal intensity, respectively. For the first 2 wk of training subjects completed a 6-min low-intensity warm up followed by two 5-min bouts interspersed by 3 min of recovery. From week 3, training volume was increased to three 5-min bouts per training session interspersed by 3 min of recovery. Subjects were instructed to push maximally during the 10-s sprints. The total amount of training planned for each subject was 16 training sessions over the 6-wk intervention period comprising 44 consecutive 5-min bouts or 220 10-s sprints. The participants in HYP and NORM completed 97 ± 5 and 98 ± 4% of the 16 training sessions planned during the 6-wk intervention period. All training sessions were supervised, and subjects wore heart rate monitors (TEAM2 Wearlink+, Polar, Kempele, Finland) during training sessions to record training load. Average heart rate during training sessions was similar in HYP and NORM (81 ± 4 vs. 79 ± 3% of HRmax) with 58 ± 4 vs. 59 ± 12% of training time spent between 80 and 90% of HRmax and 13 ± 2 vs. 12 ± 3% of total training time spent above 90% of HRmax, respectively.

Methodology

Measurements of leg vascular conductance.

The leg vascular conductance (LVC) was calculated as femoral arterial LBF divided by the difference between the mean femoral arterial and venous blood pressure. The changes in LVC induced by infusions of ACh, SNP, and PEP were calculated by subtracting the baseline LVC of the current infusion from the LVC value measured during the respective infusion rate.

Blood analyses.

Blood samples were drawn into 3-mL tubes containing EDTA and kept on ice before being centrifuged at 3,500 g for 10 min at 4°C. Plasma was subsequently collected and stored at −80°C until further analysis. Plasma concentration of norepinephrine was measured using an ELISA plasma kit (Plasma ELISA High Sensitive kit, LDN, Nordhorn, Germany). Plasma concentration of the stable metabolite of prostacyclin, 6-keto-prostaglandin F, (PGF), was measured with an immunoassay kit (EIA; Cayman Chemical, Ann Harbor, MI). Exchange of PGF across the leg was calculated as the arterial-venous difference multiplied by the leg blood flow, which was assumed to be the same at the arterial and venous sampling site.

Statistical Analyses

A priori power analysis was conducted based on previous results from our laboratory on the effects of training on endothelium-dependent vasodilator responsiveness in essential hypertension (38), and it was observed that inclusion of 10 hypertensives was sufficient to detect changes in leg vascular conductance with training. In addition, a more homogenous cohort (only men) was included in the present study, suggesting of a lower variability on the primary study outcome.

Between group differences before (Pre) and after (Post) training were determined using a linear mixed model with group (HYP; NORM) and time (Pre, Post) as fixed factors and subject as random factor. In addition, given that age and V̇o2max may affect vascular function, age and baseline V̇o2max (expressed as mL·min−1·kg−1) were included as covariates to limit confounding influence of other factors than essential hypertension. The effect of training within each group was determined using a linear mixed model with time as fixed factor and subject as random factor, with age, baseline V̇o2max, and baseline value of the outcome variable included as covariates. To estimate between-group differences in the training-induced changes, as well as between-group (HYP vs. NORM) and within-group (Pre vs. Post) differences in leg vascular function-related variables, a linear mixed model was used with group, time, and infusion rate interaction as fixed factors, with subject as random factor for a full factorial design, and with age, baseline V̇o2max, and the baseline value of the outcome variable included as covariates. Moreover, for between-group differences in the training-induced changes, as well as between- and within-group differences for outcome variables not related to leg vascular function, i.e., subject characteristics and intra-arterial blood pressure and performance variables, a mixed linear model was used with group and time interaction as fixed factors and subject as random factor for a full factorial design with age, baseline V̇o2max, and the baseline value of the outcome variable included as covariates.

Model checking was based on Shapiro Wilk’s tests and Q-Q plots. In case of heteroscedasticity (i.e., unequal variance), log transformation was applied before analysis. Model-based Student’s t tests with no multiplicity adjustments were used in pairwise comparisons to identify between- and within-group differences. The level of significance for all analyses was defined as P < 0.05. Statistical analyses were carried out in R ver. 3.4.1 (https://www.r-project.org/) applying extension packages lme4 and multcomp. Data are presented as means ± SD unless stated otherwise.

RESULTS

Baseline Leg Vascular Conductance and Blood Flow

Before, but not after, training, baseline LVC (average of LVC before infusion of ACh, SNP and PEP, respectively) was lower (P < 0.05) in HYP than in NORM. With training, baseline LVC was increased (P < 0.01) in HYP, with no change in NORM, and the training-induced change in baseline LVC was greater (P < 0.05) in HYP than in NORM.

Leg Vascular Conductance and Blood Flow in Response to Infusion of Acetylcholine

Before training, no difference in LVC in response to infusion of ACh was observed between HYP and NORM, but after training, LVC was higher (P < 0.05) in HYP than in NORM at 25 µg·min−1·kg leg mass−1 (Fig. 2A). With training, the change in LVC in response to infusion of ACh was increased in HYP only at 25 (P < 0.01) and 100 (P < 0.05) µg·min−1·kg leg mass−1, respectively, and the change with training was greater (P < 0.05) in HYP than in NORM at 25 µg·min−1·kg leg mass−1 (group by time interaction) (Fig. 2D). With training, no change in LVC in response to infusion of ACh was observed in NORM (Fig. 2A). Before training, LBF before infusion of ACh was similar between groups, whereas after training it was higher (P < 0.05) in HYP than in NORM at 10 and 25 µg·min−1·kg leg mass−1, respectively (Fig. 2B). With training, LBF increased (P < 0.05) in HYP before and during infusion at 25 µg·min−1·kg leg mass−1, with no change in NORM (Fig. 2B).

Fig. 2.

Fig. 2.Leg vasodilator responsiveness before (0) and during arterial infusion of acetylcholine (ACh) at infusion rates of 10, 25, and 100 µg·min−1·kg leg mass−1 before (Pre) and after (Post) 6 wk of 10-20-30 training (AC), respectively, and change with training (DF; calculated as Post minus Pre) on leg vascular conductance (A and D), blood flow (B and E), and mean intra-arterial blood pressure (C and F) in hypertensive (HYP; n = 10) and normotensive (NORM; n = 8) men. LM, leg mass. *Different (P < 0.05) from Pre. †Different (P < 0.05) from NORM. ‡Two-way interaction effect (group by time; P < 0.05). §Three-way interaction effect (group by time by infusion rate; P < 0.05).


Leg Vascular Conductance in Response to Infusion of Sodium Nitroprusside

Before training, LVC in response to infusion of SNP was lower at 3 (P < 0.05), 6 (P < 0.01), and 9 (P < 0.05) µg·min−1·kg leg mass−1, respectively, with no difference between groups after training (Fig. 3A). With training, LVC improved in HYP before (P < 0.01) and in response to infusion of SNP at 3 (P < 0.001) and 9 (P < 0.05) µg·min−1·kg leg mass−1, respectively, with no change in NORM (Fig. 3A). The training-induced change in LVC in response to infusion of SNP was greater (P < 0.05) in HYP than in NORM at 3 µg·min−1·kg leg mass−1 (group by time interaction) (Fig. 3D). A significant (P < 0.001) three-way interaction was observed in LVC in response to infusion of SNP (group by time by infusion rate) (Fig. 3D). Before training, LBF was lower (P < 0.05) in HYP than in NORM at 3 and 9 µg·min−1·kg leg mass−1, respectively, with no difference between groups after training (Fig. 3B). With training, LBF increased (P < 0.05) in HYP at 3 and 9 µg·min−1·kg leg mass−1, respectively, with no change in NORM (Fig. 3B). The training-induced change was greater (P < 0.05) in HYP than in NORM (group by time interaction) (Fig. 3E).

Fig. 3.

Fig. 3.Leg vasodilator responsiveness before (0) and during arterial infusion of sodium nitroprusside (SNP) at infusion rates of 3, 6, and 9 µg·min−1·kg leg mass−1 before (Pre) and after (Post) 6 wk of 10-20-30 training (A–C), respectively, and change with training (DF; calculated as Post minus Pre) on leg vascular conductance (A and D), blood flow (B and E), and mean intra-arterial blood pressure (C and F) in hypertensive (HYP; n = 10 and n = 9 during infusion rates of 3 and 6 vs. 9 µg·min−1·kg leg mass−1, respectively) and normotensive (NORM; n = 8) men. LM, leg mass. *Different (P < 0.05) from Pre. †Different (P < 0.05) from NORM. ‡Two-way interaction effect (group by time; P < 0.05). §Three-way interaction effect (group by time by infusion rate; P < 0.05).


Leg Vascular Conductance in Response to Infusion of Phenylephrine

Before, but not after, training, LVC before infusion of PEP was lower (P < 0.05) in HYP than in NORM (Fig. 4A). With training, LVC before infusion of PEP was higher (P < 0.05) in HYP, with no change in NORM. In addition, no training-induced change in LVC was observed in HYP and NORM in response to infusion of PEP (Fig. 4A). The training-induced change in HYP before infusion of PEP was greater (P < 0.05) than in NORM (group by time interaction) (Fig. 4D). Before and after training, LBF was similar between groups (Fig. 4B). With training, LBF before, but not during, infusion of PEP was lower (P < 0.05) in HYP, with no change in NORM (Fig. 4B).

Fig. 4.

Fig. 4.Leg vasodilator responsiveness before (0) and during arterial infusion of phenylephrine (PEP) at an infusion rate of 3 µg·min−1·kg leg mass−1 before (Pre) and after (Post) 6 wk of 10-20-30 training (AC) and change with training (DF; calculated as Post minus Pre) on leg vascular conductance (A and D), blood flow (B and E), and mean intra-arterial blood pressure (C and F) in hypertensive (HYP; n = 8) and normotensive (NORM; n = 8) men. LM, leg mass. *Different (P < 0.05) from Pre. †Different (P < 0.05) from NORM. ‡Two-way interaction effect (group by time; P < 0.05).


Intra-Arterial, Clinical, and Nighttime Blood Pressure and Resting Heart Rate

Before and after training, intra-arterial blood pressure was higher (P < 0.001) in HYP than in NORM (Fig. 5A). With training, intra-arterial systolic blood pressure decreased (P < 0.05) similarly in HYP and NORM (9 ± 11 vs. 9 ± 14 mmHg, respectively) (Fig. 5B). In addition, intra-arterial diastolic and mean blood pressure decreased (P < 0.05) by 5 ± 9 and 7 ± 8 mmHg in HYP only (Fig. 5A). Before training, nighttime blood pressure was higher (P < 0.01) and clinical systolic, but not diastolic, blood pressure was higher (P < 0.05) in HYP than in NORM (Table 1). Before training, intra-arterial heart rate at rest was not different between HYP and NORM (61.7 ± 7.1 vs. 57.6 ± 6.7 beats/min, respectively). With training, clinical and intra-arterial resting heart rate did not change in neither HYP nor NORM (−1.3 ± 4.6 vs. 0.8 ± 4.7 beats/min, respectively).

Fig. 5.

Fig. 5.Intra-arterial mean (MAP), systolic (SYS), and diastolic (DIA) blood pressure before (Pre) and after (Post) 6 wk of 10-20-30 training (A) and change with training (B; calculated as Post minus Pre) in hypertensive (HYP; n = 10) and normotensive (NORM; n = 8) men. *Different (P < 0.05) from Pre. †Different (P < 0.05) from NORM.


Maximum Oxygen Uptake and Performance

Before and after training, V̇o2max and performance were not different between groups and increased (P < 0.05) similarly with training in HYP and NORM (Table 2).

Table 2. Subject characteristics and performance variables before and after 6 wk of 10-20-30 training in hypertensive and normotensive male individuals

HYP (n = 10)
NORM (n = 8)
Pre Post Pre Post
Weight, kg 91.7 ± 11.5 91.2 ± 12.2 86.9 ± 12.7 85.6 ± 12.6*
BMI, kg/m2 29.2 ± 4.0 29.0 ± 4.2 26.3 ± 3.1 26.0 ± 4.9*
Fat mass, kg# 29.5 ± 5.4 27.9 ± 6.4* 27.7 ± 4.5 26.1 ± 4.9*
Fat, % of body wt# 33.1 ± 3.6 31.4 ± 4.4* 33.1 ± 4.0 31.7 ± 4.2*
Fat free mass, kg# 59.1 ± 6.5 60.1 ± 6.5* 56.2 ± 9.8 56.4 ± 9.4
Visceral fat, kg# 2.1 ± 0.8 1.9 ± 1.0* 1.6 ± 0.6 1.5 ± 0.6*
Bone mineral density, g/cm2# 1.4 ± 0.1 1.4 ± 0.1 1.3 ± 0.1 1.3 ± 0.1
o2max, L/min 3.0 ± 0.5 3.1 ± 0.6 3.0 ± 0.6 3.2 ± 0.7*
o2max, mL·kg−1·min−1 32.9 ± 4.9 33.9 ± 5.5* 34.3 ± 3.3 37.0 ± 4.3*
GXT peak power output, W 232 ± 47 248 ± 58* 263 ± 56 294 ± 65*
GXT time to exhaustion, s 479 ± 122 528 ± 135* 549 ± 168 641 ± 122*

Values are means ± SD. HYP, hypertensive; NORM, normotensive; Pre, before 6 wk of 10-20-30 training; Post, after 6 wk of 10-20-30 training; BMI, body mass index; V̇o2max, maximum oxygen uptake; GXT, graded exercise test.

#n = 7 in HYP.

*P < 0.05, different from Pre.

P < 0.05, different from NORM.

Body Composition

Before training, total fat mass, fat percentage, visceral fat, and fat free mass were not different between groups (Table 2). With training, total fat mass, fat percentage, and visceral fat decreased (P < 0.05) similarly in HYP and NORM, whereas fat free mass increased (P < 0.05) only in HYP and BMI decreased (P < 0.05) only in NORM (Table 2).

Plasma Biomarkers

Before training, venous plasma levels of norepinephrine were similar between HYP and NORM. After training, plasma levels of norepinephrine were lower (P < 0.05) in NORM than in HYP (Table 3). With training, plasma norepinephrine levels during infusion of ACh and SNP did not change in HYP but decreased (P < 0.05) with training in NORM in response to infusion of SNP (Table 3). Plasma levels of norepinephrine during infusion of PEP did not change with training in neither HYP nor NORM (Table 3).

Table 3. Plasma norepinephrine levels before and during infusion of acetylcholine, sodium nitroprusside, and phenylephrine at different infusion rates in hypertensive and normotensive men before and after 6 wk of 10-20-30 training

HYP (n = 10)
NORM (n = 8)
Infusion rate, µg·min−1·kg leg mass−1 Pre Post Pre Post
ACh 0, nM 1.13 ± 0.81 1.46 ± 1.74 0.76 ± 0.99 0.76 ± 0.63
ACh 10, nM 1.04 ± 0.51 1.12 ± 0.75 0.82 ± 0.67 0.56 ± 0.45
ACh 100, nM 1.22 ± 0.89 1.40 ± 1.03 0.92 ± 0.33 0.68 ± 0.63
SNP 0, nM 1.33 ± 1.08 1.12 ± 0.76 0.90 ± 0.55 0.49 ± 0.23*
SNP 3, nM 1.46 ± 1.22 1.12 ± 0.47 1.20 ± 0.62 0.65 ± 0.26*
SNP 6, nM 3.22 ± 2.53 2.24 ± 1.11 1.80 ± 1.68 1.05 ± 0.51
PEP 0, nM 1.40 ± 1.02 2.04 ± 2.02 1.48 ± 2.40 1.06 ± 0.99
PEP 3, nM 1.41 ± 0.79 1.10 ± 0.98 0.70 ± 0.34 1.38 ± 0.98

Values are presented as means ± SD. ACh, acetylcholine; SNP, sodium nitroprusside; PEP, phenylephrine HYP, hypertensive; NORM, normotensive; Pre, before 6 wk of 10-20-30 training; Post, after 6 wk of 10-20-30 training.

*P < 0.05, different from Pre.

P < 0.05, different from NORM.

Before and after training, release of PGF during infusion of ACh was not different between groups (Supplemental Table S2; see https://doi.org/10.6084/m9.figshare.11494404.v1). With training, release of PGF during infusion of ACh was not different within HYP and NORM (Supplemental Table S2). In both HYP and NORM, release of PGF was higher (P < 0.001) during infusion of ACh at 100 compared 10 µg·min−1·kg leg mass−1 (Supplemental Table S2).

DISCUSSION

The major findings of the present study were that the leg smooth muscle cell vasodilator responsiveness, evaluated by intra-arterial infusion of SNP, was blunted in men with essential hypertension compared with normotensive counterparts, whereas this was not the case for the leg vasodilator responsiveness to ACh infusion, i.e., endothelium-dependent vasodilation. Six weeks of 10-20-30 training reversed the blunted smooth muscle cell vasodilator responsiveness associated with essential hypertension. These findings provide the first line of evidence in humans, that smooth muscle cell vasodilator responsiveness is reduced in the lower limbs of hypertensive men and that an improved endothelial function can compensate for this impairment.

Differences in Leg Vascular Responsiveness Between Hypertensive and Normotensive Men

Before the intervention period, the leg vasodilator responsiveness to SNP infusion was lower by 29–42% in the hypertensive compared with the normotensive men. This finding is the first evidence of an altered smooth muscle cell vasodilator responsiveness in the lower limbs of hypertensive men and contrasts with observations in the forearm, where no differences were observed between hypertensive and normotensive subjects (48, 49). A putative mechanism possibly explaining the difference between the upper and lower limbs is the higher hydrostatic pressure in the legs compared with the arms, giving rise to a higher transmural pressure and circumferential stress (9, 27). The observation that the leg vasodilator responsiveness to ACh was similar between groups before training is in agreement with previous observations (17, 38). Given the lower responsiveness to SNP in the hypertensive subjects, the preserved vasodilator responsiveness to ACh suggests that a greater endothelial-mediated vasodilator stimulus was compensating for the lower smooth muscle cell responsiveness. Indeed, as infusion of ACh induces vasodilation by stimulating endothelial formation of NO, prostanoids, and endothelial-derived hyperpolarizing factor (19), an enhanced signaling of either of these systems could potentially explain the preserved responsiveness to ACh. It is well established that NO mediates its effects on the smooth muscle cells through the second messenger nucleotide cGMP (19, 36), whereas prostacyclin induces vasodilation through cAMP on the smooth muscle cells (54). Before the intervention period, release of the prostacyclin analogue 6-keto prostaglandin F during infusion of ACh was not different between the hypertensive and normotensive men, which is in agreement with previous observations in essential hypertension (17). This indicates that if enhanced smooth muscle cell cAMP signaling was compensating for the lower smooth muscle cell responsiveness, it was not related to a higher endothelial formation of prostacyclin. Future studies should investigate whether hypertension is associated with altered cGMP-mediated signaling in the smooth muscle cells, such as decreased activity of the smooth muscle cell NO receptor soluble guanylate cyclase (sGC), protein kinase G (PKG), elevated activity of phosphodiesterase V, or depressed sensitivity of large-conductance voltage and Ca2+-dependent K+ (BK) channels, exclusively expressed in smooth muscle cells (24).

Training-Induced Changes in Leg Vascular Responsiveness

The present study is the first to demonstrate enhanced leg smooth muscle cell vasodilator responsiveness with exercise training in hypertensive men. Our finding is in contrast to studies in the forearm showing no (3, 4, 15, 22, 32, 34) or only minor training-induced changes (33) in the vasodilator responsiveness to SNP with moderate-intensity training. This discrepancy between the extremities may reflect a preserved forearm vasodilator response to SNP in hypertensive men, and/or the imposed training stimuli caused by the intense exercise bouts used in the present study. The 10-s sprint bouts would induce a pronounced hyperemia response and thus a high shear-stress response promoting a significant vasodilator response via shear stress-induced release of NO and prostacyclin (18, 37). The pronounced shear stress could induce changes in sGC or downstream kinases to cGMP i.e., PKG or phosphodiesterase V, which could serve as possible mechanisms explaining the more pronounced exercise responsiveness in the leg than in the forearm. In addition, the sprints induce a high adrenergic stress response and marked metabolic perturbations (12, 16), which could also positively affect adaptations in the vascular system of the leg.

HIIT was in older men recently shown to enhance leg skeletal muscle cGMP signaling (42), which may have led to the better smooth muscle cell vasodilator responsiveness observed in the hypertensive men. With training, the endothelium-dependent vasodilation was higher in the hypertensive men, which most likely was not due to a higher capacity of the endothelium cells to form prostacyclin, as release of 6-keto prostaglandin F in response to infusion of ACh did not change with training. Instead, the improvement in endothelium-dependent vasodilation observed with training in the hypertensive men may be explained by the better smooth muscle cell vasodilator responsiveness. When normalizing the vascular responsiveness to infusion of ACh to that of infusion of SNP, it appears that the sensitivity of the smooth muscle cells could explain majority of the training-induced endothelium-dependent responsiveness observed in the hypertensive men. What caused the better smooth muscle cell responsiveness in the hypertensive men is unclear, but improved NO/cGMP/PKG signaling and prostacyclin-mediated cAMP signaling in the smooth muscle cells are possible mechanisms that deserves further investigation. The vasoconstrictor responsiveness to infusion of PEP, which is mediated through smooth muscle cell α1-receptors (53), did not change with training, suggesting that the 10-20-30 training did not affect smooth muscle cell α1-receptor sensitivity.

Training-Induced Changes in Intra-Arterial Blood Pressure and Clinical Relevance of Changes

The training-induced reduction in intra-arterial systolic blood pressure of 9 mmHg observed in the hypertensive men is of clinical relevance as a decrease of 10 mmHg in systolic blood pressure is associated with ~40% lower risk of stroke death and ~30% lower risk of death from ischemic heart disease (28). As the responsiveness to SNP was normalized with training in the hypertensive subjects, it may be speculated that the enhancement in smooth muscle cell vasodilator responsiveness contributed to the lower blood pressure. Another potential mechanism underlying the blood pressure-lowering effect of the 10-20-30 training is reduced sympathetic nerve activity (SNA), as hypertensive subjects have been shown to have elevated muscle SNA (14, 45) that has been shown to be normalized with training (25). However, heart rate at rest was similar between groups before training and did not change with training in neither HYP nor NORM, indicative of an unchanged sympathetic control of vascular tone with training. In addition, the lack of training-induced changes in the leg vascular responsiveness to PEP, and in baseline norepinephrine levels in the hypertensive men, do not support of training-induced alterations in the sympathetic control of vascular tone. Nevertheless, the training-induced decreases in intra-arterial systolic blood pressure in both groups, and decrease in diastolic blood pressure in the hypertensive men, suggest the 10-20-30 training as a time-efficient exercise modality that may be suitable as a prevention and management strategy for essential hypertension.

The magnitude of training-induced reductions in blood pressure has been reported to associate with training-induced increments in V̇o2max (6). In the present study, V̇o2max and systolic blood pressure improved similarly with training between groups, suggesting that 10-20-30 training is a powerful stimulus for improving central hemodynamics (44). Furthermore, the observed increase in V̇o2max is of clinical relevance since it is a strong predictor for all-cause mortality (23), and each 1 mL·kg−1·min−1 increase in V̇o2max has been shown to be associated with an ~9% relative risk reduction in all-cause mortality (26) and a 45‐day increase in longevity (5).

Limitations

A limitation of the present study was a limited sample size due to the invasive nature of the experimental design. Nevertheless, such limitation appeared not to affect study outcomes, as the a priori intention of the study was realized. Moreover, the present study included a short-term intervention period of only 6 wk. Whether a longer intervention duration would have affected between-group outcomes is unclear, but 3 mo of biweekly football training (60 min per training session) lowered mean arterial pressure (MAP) and induced significant structural [left ventricular (LV) volume, tissue tracking (TT) displacement, and tricuspid annular plane systolic excursion (TAPSE)] and functional [early/late atrial ventricular filling (E/A) ratio, E’, and global isovolumic relaxation time (IVRTglobal)] cardiac adaptations in sedentary men with mild to moderate hypertension, with no further improvements from 3 to 6 mo (1). In addition, it is worth mentioning that medication usage in 4 of the 10 hypertensive men may have affected the training-induced responses; however, no training-induced differences in vascular measurements were observed when comparing medicated versus nonmedicated hypertensive men. Further limitations to the study were exclusion of women and lack of a hypertensive control group exercising according to existing guidelines; i.e. 3 × 50 min of moderate-intensity continuous cycle training (2). Future studies should include men and women, evaluate the effectiveness of different exercise modalities on vascular outcomes in hypertensive individuals, and use a long-term training period.

Summary

The leg smooth muscle cell vasodilator responsiveness, as assessed by infusion of SNP, was blunted in men with essential hypertension compared with age-matched normotensive controls, whereas this was not the case for endothelium-dependent leg vasodilator responsiveness, assessed by infusion of ACh. Six weeks of 10-20-30 training normalized the blunted leg smooth muscle cell vasodilator responsiveness, which could explain the improved leg endothelium-independent vasodilator responsiveness observed in the hypertensive men. These data provide the first line of evidence that leg smooth muscle cell vasodilator responsiveness is reduced in hypertensive men and that better endothelial cell vasodilator responsiveness can compensate for this impairment. In addition, 10-20-30 training appears as a time-efficient exercise modality to improve mechanisms underlying the impaired leg vascular function characterizing essential hypertension in men.

Perspectives

Based on the observed impairment in leg smooth muscle cell vasodilator responsiveness future studies should assess whether hypertension is associated with altered cGMP mediated signaling in the smooth muscle cells, focusing on downstream targets of sGC such as activity of PKG and phosphodiesterase V and sensitivity of BK channels to phosphorylation by PKG. In addition, future studies should measure NO release in the lower limb in concert with a comprehensive assessments of the NO regulatory system to clarify the potential role of NO bioavailability in the pathology of hypertension. To enhance the understanding of the mechanisms underlying functional alterations, assessment of vascular structure at a tissue as well as a single cell level should be performed. These future studies will improve our understanding of the role of smooth muscle cell function and structure in essential hypertension and may lead to novel therapeutic strategies to counter vascular dysfunction in hypertensive individuals. Based on the present findings, the 10-20-30 training may be considered an attractive exercise modality for men with essential hypertension, as both time spent during training and risk of injury is low and the constant change of pace during the 10-20-30 training is motivating (personal communication with study participants). These aspects may have high relevance when prescribing exercise as a prevention and management strategy for patients with cardiometabolic disease, considering that lack of time, low motivation for regular exercise, and fear of injury are reported barriers in these patients (29, 43).

GRANTS

This study was supported by the Danish Ministry of Culture and Aase and Ejnar Danielsens Fond.

DISCLOSURES

J.B. and T.P.G. have authored a book on the effect of 10-20-30 training on performance and health. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

T.P.G., M.N., and J.B. conceived and designed research; T.P.G., T.S.E., M.F., and M.N. performed experiments; T.P.G., T.S.E., M.F., and M.N. analyzed data; T.P.G., T.S.E., M.N., and J.B. interpreted results of experiments; T.P.G. and M.F. prepared figures; T.P.G. drafted manuscript; T.P.G., T.S.E., M.F., M.N., and J.B. edited and revised manuscript; T.P.G., T.S.E., M.F., M.N., and J.B. approved final version of manuscript.

ACKNOWLEDGMENTS

Jens Jung Nielsen is greatly acknowledged for excellent technical assistance.

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