Abstract
Hypercholesterolemia impairs endothelial function [e.g., the nitric oxide (NO)-cyclic GMP-phosphodiesterase 5 (PDE5) pathway], limits shear stress-induced vasodilation, and is therefore expected to reduce exercise-induced vasodilation. To assess the actual effects of hypercholesterolemia on endothelial function and exercise-induced vasodilation, we compared the effects of endothelial NO synthase (eNOS) and PDE5 inhibition in chronically instrumented Yucatan (Control) and Rapacz familial hypercholesterolemic (FH) swine, at rest and during treadmill exercise. The increases in systemic vascular conductance produced by ATP (relative to nitroprusside) and exercise were blunted in FH compared with Control swine. The vasoconstrictor response to eNOS inhibition, with nitro-l-arginine (NLA), was attenuated in FH compared with Control swine, both at rest and during exercise. Furthermore, whereas the vasodilator response to nitroprusside was enhanced slightly, the vasodilator response to PDE5 inhibition, with EMD360527, was reduced in FH compared with Control swine. Finally, in the pulmonary circulation, FH resulted in attenuated vasodilator responses to ATP, while maintaining the responses to both NLA and EMD360527. In conclusion, hypercholesterolemia reduces exercise-induced vasodilation in the systemic but not the pulmonary circulation. This reduction appears to be the principal result of a decrease in NO bioavailability, which is mitigated by a lower PDE5 activity.
Keywords: arginine, endothelium, exercise, PDE inhibitors
hypercholesterolemia is characterized by high total cholesterol levels (>7.5 mmol/l) and elevated LDL levels (>4.9 mmol/l) (6) and is an important risk factor for the development of atherosclerosis (19). Familial hypercholesterolemia (FH) is a well-known genetic disorder with high circulating levels of total cholesterol and particularly LDL, resulting in excessive atherosclerosis. An early consequence of elevated cholesterol is endothelial dysfunction, as evidenced by reduced nitric oxide (NO)-mediated vasodilator responses to L-arginine (L-Arg) and acetylcholine in the forearm of FH patients (3, 22). Endothelial function is particularly important during exercise, when dilation of the vasculature is essential to supply the exercising muscle with adequate oxygen (O2) and nutrients (25). Therefore, we hypothesize that exercise-induced, endothelium-mediated systemic vasodilation is reduced in subjects with FH.
During exercise, it is not only important that the skeletal muscle vasculature dilates to facilitate delivery of O2 and nutrients but also that the consequent increase in cardiac output has to be pumped through the pulmonary vasculature, resulting in an increase in pulmonary artery pressure during exercise. The increase in pulmonary artery pressure with increasing flow is limited by an increase in pulmonary vascular conductance (PVC) through active vasodilation and passive distension. Endothelial dysfunction in the pulmonary vasculature is accompanied by a decreased PVC, which may limit exercise capacity by increasing right ventricular afterload. Since the pulmonary vasculature is also exposed to elevated levels of cholesterol, we hypothesize that next to systemic endothelial dysfunction, pulmonary endothelial dysfunction is present in subjects with FH and that exercise-induced pulmonary vasodilation is thus reduced.
In light of these considerations, we investigated how FH affects the vasodilator influence of different parts of the NO- cyclic GMP-phosphodiesterase 5 (cGMP-PDE5) pathway, which is an important signaling pathway in the regulation of vascular tone in various vascular beds. For this purpose, we chronically instrumented Yucatan (Control) and Rapacz FH swine (7) and assessed the effects of hypercholesterolemia on endothelial and smooth muscle function, as well as vascular tone at rest and during exercise in the systemic and pulmonary vascular beds.
METHODS
Animals
Studies were performed in accordance with the “Principles for the Utilization and Care of Vertebrate Animals Used in Testing Research and Training” and approved by the Institutional Animal Care Committee at the University of Missouri. A total of nine Yucatan (Control) swine (∼18 mo old; 40 ± 3 kg) and seven Rapacz FH swine (∼20 mo old; 84 ± 4 kg) entered the study. The pigs were housed in the animal care facility in the Department of Biomedical Sciences, University of Missouri, in rooms maintained at 20-23°C with a 12-h:12-h light:dark cycle. Control pigs were fed a normal diet (Purina Laboratory 5082 Mini-Pig Breeder Chow); calories provided by fat were 8%. At 14 mo of age, the FH pigs were switched to a high-fat chow (by weight 13% protein, 21.3% fat, 41.4% total carbohydrate, 2% cholesterol) and fed on this diet for 6 mo, as described previously (13).
Surgery
Swine were sedated with telazol (5 mg/kg im) and xylazine (2.2 mg/kg im), intubated and ventilated with 1–3% (vol/vol) isoflurane in air to induce and maintain anesthesia. Swine were instrumented under sterile conditions, as described previously (15). Briefly, a thoracotomy was performed in the fourth intercostal space. Subsequently, a polyvinylchloride catheter was inserted into the aortic arch for the measurement of aortic pressure and blood sampling to determine O2 partial pressure (PO2), carbon dioxide partial pressure, pH (Radiometer ABL 700), O2 saturation (SO2), and hemoglobin (Hb) concentration (Radiometer OSM3). Fluid-filled catheters were implanted for measurement of blood pressure in the left atrium and pulmonary artery. Furthermore, a Transonic flow probe was placed around the aorta for measurement of both cardiac output and stroke volume (14). Two FH swine died shortly after surgery, due to recurrent fibrillation. Electrical wires and catheters were tunneled subcutaneously to the back. The chest was closed, and the animals were allowed to recover.
Animals received analgesia (fentanyl 50 μg/h transdermal and buprenorphine 0.3 mg im) for 2 days and antibiotic prophylaxis (25 mg/kg amoxicillin and 5 mg/kg gentamycin iv) for 5 days.
Experimental Protocols
Studies were performed 1–3 wk after surgery on a motor-driven treadmill and are illustrated in Fig. 1.
Fig. 1.
Graphic depiction of resting and exercise protocols. Shown are the various steps needed to conduct resting and exercise experiments in awake Yucatan (Control) and Rapacz familial hypercholesterolemic (FH) swine. Nitro-l-arginine (NLA; 20 mg/kg iv) was infused 10 min before the run. EMD360527 (300 μg·kg−1·min−1 iv) was infused starting 10 min before and maintained during the run. SNP, sodium nitroprusside; NO, nitric oxide; PDE5, phosphodiesterase 5.
Vascular smooth muscle and endothelial function.
With swine standing quietly on the treadmill, resting hemodynamic measurements were obtained. NO sensitivity of vascular smooth muscle was assessed by determining the response to the NO donor sodium nitroprusside (SNP; 0.5, 1, 2, 3, 4, 5 μg·kg−1·min−1 iv; Control, n = 4; FH, n = 4). Endothelial function was assessed by intravenous infusion of the endothelium-dependent vasodilator ATP (50, 100, 200, 300 μg·kg−1·min−1 iv; Control, n = 7; FH, n = 4). Each dose of ATP and SNP was infused for 10 min. In general, a plateau was reached after 6–8 min.
Effect of hypercholesterolemia on exercise hyperemia—role of the NO-PDE5 pathway.
To assess the effect of hypercholesterolemia on NO-mediated vasodilation during exercise, two different protocols were performed. With swine (Control, n = 8; FH, n = 4) standing quietly on the treadmill, resting hemodynamic measurements were obtained and blood samples collected. Hemodynamic measurements were repeated, and rectal temperature was measured with animals standing on the treadmill. Subsequently, a four-stage (2–5 mph) treadmill exercise protocol was started for Control swine, whereas a three-stage exercise protocol (1–3 mph) was started for FH swine; each exercise stage lasted 2–3 min. Hemodynamic variables were recorded continuously, and blood samples were collected during the last 60 s of each stage. After completing the untreated exercise protocol, animals were allowed to rest on the treadmill for 90 min. Then, animals received the NO synthase (NOS) inhibitor Nω-nitro-l-arginine (NLA; 20 mg/kg iv) (4) and underwent a second exercise trial.
On a different day, the untreated exercise protocol was performed as described above. After a 90-min resting period, animals (Control, n = 9; FH, n = 5) received infusion of the PDE5 inhibitor EMD360527 (300 μg·kg−1·min−1 iv) (9), and the exercise protocol was repeated. EMD360527 infusion started 10 min before and was maintained during the exercise protocol.
Data Analysis
Hemodynamic data were recorded and digitized live using a five-channel data-acquisition program, WinDaq (Dataq Instruments, Akron, OH), and were stored on a computer for offline analysis using a program written in MatLab (MathWorks, Natick, MA). A minimum of 15 consecutive heart beats was used for analysis of the digitized hemodynamic signals, from which heart rate, mean left atrial pressure, mean aortic pressure, mean pulmonary artery pressure, cardiac output, and stroke volume were determined. Systemic vascular conductance (SVC) and PVC were calculated as cardiac output, divided by mean arterial pressure, and cardiac output, divided by the transpulmonary pressure gradient (pulmonary pressure minus left atrial pressure), respectively. O2 content in ml O2/l was calculated as follows: {[Hb × 0.6206 × (SO2/100)] + (0.00131 × PO2)} × 22.391 (5). Body O2 consumption (BVO2) was used as a measure of exercise intensity and computed as the product of cardiac output and the arteriovenous O2 content difference. Body O2 extraction was calculated using the following formula: [(arterial O2 content − mixed venous O2 content)/arterial O2 content] × 100%. To accommodate for varying body weights between animals and breeds, we indexed cardiac output (cardiac index), stroke volume (stroke volume index), SVC (SVCi), PVC (PVCi), and BVO2 (BVO2i) to body weight.
Statistical Analysis
Statistical analysis of hemodynamic data was performed using one-way (exercise), two-way (breed, exercise), or three-way (treatment, breed, exercise) ANOVA for repeated measures. When significant effects were detected, post hoc testing for the effects of exercise and drug treatment was performed using Scheffe's test. To test for the effects of drug treatment of the relation between exercise (BVO2) and vasomotor tone (vascular conductance), regression analysis was performed with each animal as a dummy variable and with drug treatment, breed, and BVO2 as independent variables. P ≤ 0.05 was considered statistically significant (two-tailed). All data are presented as mean ± SE.
Drugs
EMD360527 was a kind gift from E. Merck (Darmstadt, Germany). SNP, ATP, and NLA were obtained from Sigma (St. Louis, MO) and dissolved in 0.9% saline solution. EMD360527 was dissolved in 0.9% saline solution and sodium bicarbonate in a 1:1 ratio.
RESULTS
Plasma Lipid Profiles
The plasma lipid profiles for Control and FH pigs are shown in Table 1. Total cholesterol, LDL, HDL, and the total cholesterol:HDL ratio were increased significantly in FH swine.
Table 1.
Lipid profiles of Control and FH swine at death
| Control | FH | |
|---|---|---|
| Total cholesterol, mmol/l | 1.86 ± 0.14 | 17.92 ± 3.12* |
| LDL cholesterol, mmol/l | 0.66 ± 0.09 | 15.05 ± 2.74* |
| HDL cholesterol, mmol/l | 0.87 ± 0.07 | 1.45 ± 0.28* |
| Total cholesterol/HDL cholesterol | 2.82 ± 0.30 | 12.38 ± 3.22* |
Values presented as mean ± SE. Yucatan swine (Control), n = 9; Rapacz familial hypercholesterolemic swine (FH), n = 5.
P < 0.05 vs. Control.
Effects of FH on Vascular Smooth Muscle and Endothelial Function
Smooth muscle and endothelial function were assessed by intravenous infusion of SNP and ATP, respectively. The systemic vasodilator response to SNP was enhanced in FH swine, indicating that smooth muscle responsiveness to NO was enhanced in FH (Fig. 2). In contrast, the vasodilator response to ATP was not statistically different between groups (Fig. 2). To correct for the enhanced vascular smooth muscle responsiveness to NO, we plotted the ATP-induced changes in SVCi as a function of the SNP-induced changes in SVCi (Fig. 3). The significant clockwise rotation of this relation in FH compared with Control swine reflects reduced NO production and hence, endothelial dysfunction. In the pulmonary circulation, the vasodilator response to ATP was reduced significantly in FH swine, whereas the response to SNP was unaltered (Fig. 2).
Fig. 2.
Systemic and pulmonary dose-response bar graphs for the vasodilators SNP and ATP in Control and FH swine. Shown are the systemic and pulmonary dose-response curves for the NO donor SNP and the endothelium-dependent vasodilator ATP. SNP Control, n = 4; FH, n = 4. ATP Control, n = 6; FH, n = 4. SVCi, systemic vascular conductance index; PVCi, pulmonary vascular conductance index.
Fig. 3.
Systemic and pulmonary vasodilator responses to the vasodilators SNP and ATP in Control and FH swine. Shown are maximal changes (Δ) in SVCi and PVCi as a result of ATP and SNP infusions plotted against each other. Control, n = 6; FH, n = 4. *P < 0.05 response to ATP corrected for SNP compared with Control.
Exercise Hyperemia in Control and FH Swine
Under resting conditions, mean arterial pressure, heart rate, and SVCi were similar in both breeds (Fig. 4). Cardiac index and stroke volume index were lower in FH swine, despite elevated left ventricular filling pressures, as evidenced by the higher mean left atrial pressure (Fig. 4). The increased atrial pressure was transmitted backwards into the pulmonary circulation, resulting in an increased mean pulmonary arterial pressure, whereas the transpulmonary pressure gradient and PVCi were similar in FH and Control swine. BVO2i and body O2 extraction were significantly higher in FH swine, resulting in lower mixed venous O2 content, whereas arterial O2 content was essentially unaltered (Fig. 5).
Fig. 4.
Hemodynamic differences between Control and FH swine at rest and during exercise. Cardiac index (CI) was lower at rest and during exercise in FH swine compared with Control swine, whereas mean arterial pressure (MAP) showed an exaggerated increase in FH swine during exercise at similar levels of body oxygen consumption index (BVO2i). Consequently, SVCi showed blunted exercise-induced increases in FH swine. PVCi was essentially unaltered. Control, n = 9; FH, n = 5. *P < 0.05 baseline difference due to FH; †P < 0.05 slope difference due to FH. HR, heart rate; SVi, stroke volume index; PAP, pulmonary arterial pressure; LAP, left atrial pressure.
Fig. 5.
Metabolic differences between Control and FH swine at rest and during exercise. At similar BVO2 (BVO2i), FH swine demonstrated a higher body O2 extraction (BO2ex) compared with Control swine, as mixed venous (mv) O2 content was decreased, whereas arterial (art) O2 content was unaltered. Furthermore, hemoglobin (Hb) showed an exaggerated exercise-induced increase in FH swine. Control, n = 9; FH, n = 5. *P < 0.05 baseline difference due to FH; †P < 0.05 slope difference due to FH. PO2, O2 partial pressure.
Exercise up to 5 mph led to a fivefold increase in BVO2i in Control swine, which was met by a near tripling of cardiac index (Fig. 4) and an increase in whole-body O2 extraction from 44 ± 2% to 75 ± 1% (Fig. 5). Despite the marked increase in cardiac index, mean aortic pressure increased by only 25% (from 95 ± 3 at rest to 118 ± 4 mmHg during exercise), due to a doubling of SVCi. In contrast, PVCi increased only slightly as the transpulmonary pressure gradient, and cardiac index increased to a similar extent (Fig. 4).
FH swine, in general, would not exercise beyond 3 mph. Although the increases in both heart rate and cardiac index during exercise were similar in FH and Control swine, the increase in mean arterial pressure was exacerbated in FH swine at similar levels of BVO2i (Fig. 4). Exercise-induced systemic vasodilation was reduced in FH compared with Control swine, as evidenced by a blunted increase in SVCi with incremental exercise intensity. The blunted increase in SVCi was accompanied by an exercise-induced increase in Hb, as well as a significantly higher whole-body O2 extraction at rest and during exercise (Fig. 5). Consequently, mixed venous O2 content reached similar levels in FH swine at 3 mph, as was seen in Control swine at 5 mph (FH, 37 ± 5 ml O2/l; Control, 39 ± 2 ml O2/l; Fig. 5).
Both mean pulmonary arterial pressure and mean left atrial pressure were significantly higher in FH swine; however, PVCi was essentially unchanged. Moreover, the exercise-induced increase in PVCi was similar in FH and Control swine. Taken together, these data indicate that hypercholesterolemia had no effect on endogenous control of pulmonary vascular tone.
Alteration of the NO-cGMP Axis During Exercise Hyperemia in FH
Systemic circulation.
The endothelial NOS (eNOS) inhibitor NLA increased mean arterial pressure, while simultaneously decreasing heart rate and cardiac index in both Control and FH swine (Tables 2 and 3). However, the decrease in SVCi was significantly less pronounced in FH swine compared with Control swine, both at rest and during exercise (Fig. 6). Interestingly, following NLA, the relationship between BVO2i and SVCi was identical in Control and FH swine, indicating that a reduced NO production was responsible for the lower baseline SVCi in FH swine (Fig. 6).
Table 2.
Systemic and pulmonary hemodynamic responses to NLA and EMD360527 in Control swine
| Rest |
Exercise, mph |
|||||
|---|---|---|---|---|---|---|
| 0 | 2 | 3 | 4 | 5 | ||
| HR, beats/min | Untreated | 94 ± 4 | 165 ± 6* | 206 ± 8* | 239 ± 7* | 256 ± 6* |
| NLA | 76 ± 4† | 136 ± 5*† | 171 ± 8*† | 211 ± 9*† | 236 ± 6*† | |
| Untreated | 99 ± 3 | 167 ± 8* | 208 ± 8* | 237 ± 6* | 250 ± 6* | |
| EMD360527 | 108 ± 5† | 179 ± 8*† | 219 ± 6*† | 248 ± 3* | 260 ± 5*† | |
| CI, ml·min−1·kg−1 | Untreated | 117 ± 8 | 199 ± 15* | 252 ± 20* | 292 ± 19* | 310 ± 18* |
| NLA | 82 ± 5† | 153 ± 11*† | 188 ± 14*† | 230 ± 18*† | 253 ± 18*† | |
| Untreated | 123 ± 9 | 200 ± 12* | 250 ± 16* | 276 ± 13* | 305 ± 15* | |
| EMD360527 | 136 ± 7 | 220 ± 14*† | 269 ± 17*† | 311 ± 20*† | 337 ± 20*† | |
| SVi, ml/kg | Untreated | 1.23 ± 0.07 | 1.21 ± 0.08 | 1.22 ± 0.08 | 1.22 ± 0.07 | 1.21 ± 0.06 |
| NLA | 1.09 ± 0.06† | 1.13 ± 0.07† | 1.11 ± 0.06† | 1.11 ± 0.06† | 1.09 ± 0.06† | |
| Untreated | 1.25 ± 0.08 | 1.21 ± 0.07 | 1.21 ± 0.08 | 1.18 ± 0.08 | 1.23 ± 0.07 | |
| EMD360527 | 1.27 ± 0.07 | 1.24 ± 0.07 | 1.23 ± 0.08 | 1.25 ± 0.08 | 1.30 ± 0.07 | |
| MAP, mmHg | Untreated | 95 ± 3 | 103 ± 4 | 107 ± 4 | 112 ± 4* | 115 ± 5* |
| NLA | 138 ± 3† | 137 ± 2† | 143 ± 3† | 147 ± 3† | 148 ± 2*† | |
| Untreated | 95 ± 4 | 108 ± 5* | 115 ± 4* | 119 ± 5* | 120 ± 4* | |
| EMD360527 | 86 ± 3 | 95 ± 4* | 100 ± 4*† | 105 ± 4*† | 108 ± 5*† | |
| PAP, mmHg | Untreated | 14 ± 1 | 20 ± 2* | 24 ± 2* | 28 ± 2* | 31 ± 2* |
| NLA | 21 ± 1† | 33 ± 2*† | 37 ± 1*† | 41 ± 1*† | 40 ± 2*† | |
| Untreated | 15 ± 1 | 23 ± 1* | 25 ± 2* | 26 ± 2* | 28 ± 3* | |
| EMD360527 | 10 ± 1† | 18 ± 2*† | 25 ± 3*† | 28 ± 2*† | 28 ± 2*† | |
| LAP, mmHg | Untreated | 1 ± 1 | 5 ± 1 | 7 ± 1* | 8 ± 1* | 7 ± 1* |
| NLA | 5 ± 1† | 6 ± 1 | 7 ± 1 | 7 ± 2 | 6 ± 1* | |
| Untreated | 2 ± 1 | 5 ± 1* | 8 ± 1* | 8 ± 1* | 10 ± 2* | |
| EMD360527 | 0 ± 1 | 6 ± 1* | 8 ± 1* | 11 ± 2* | 9 ± 1* | |
| SVCi, ml·min−1·kg−1·mmHg−1 | Untreated | 1.23 ± 0.11 | 1.89 ± 0.07* | 2.30 ± 0.09* | 2.59 ± 0.07* | 2.69 ± 0.06* |
| NLA | 0.59 ± 0.04† | 1.10 ± 0.07*† | 1.30 ± 0.08*† | 1.55 ± 0.09*† | 1.69 ± 0.09*† | |
| Untreated | 1.32 ± 0.11 | 1.87 ± 0.11* | 2.21 ± 0.16* | 2.38 ± 0.18* | 2.55 ± 0.13* | |
| EMD360527 | 1.59 ± 0.09† | 2.32 ± 0.11*† | 2.69 ± 0.14*† | 2.96 ± 0.15*† | 3.14 ± 0.18*† | |
| PVCi, ml·min−1·kg−1·mmHg−1 | Untreated | 8 ± 1 | 10 ± 1* | 11 ± 1* | 12 ± 1* | 11 ± 1* |
| NLA | 6 ± 1† | 6 ± 1† | 7 ± 1† | 8 ± 1*† | 9 ± 1*† | |
| Untreated | 9 ± 1 | 10 ± 1* | 12 ± 1* | 14 ± 1* | 14 ± 1* | |
| EMD360527 | 16 ± 1† | 18 ± 1† | 17 ± 1† | 19 ± 2† | 19 ± 3† | |
| BVO2i, ml·min−1·kg−1 | Untreated | 6 ± 1 | 15 ± 1* | 24 ± 3* | 33 ± 3* | 36 ± 3* |
| NLA | 5 ± 1 | 15 ± 1* | 21 ± 2* | 28 ± 3* | 32 ± 2*† | |
| Untreated | 6 ± 1 | 16 ± 1* | 24 ± 1* | 31 ± 3* | 36 ± 2* | |
| EMD360527 | 6 ± 1 | 15 ± 1*† | 25 ± 1* | 31 ± 2* | 34 ± 3* | |
Data are presented as mean ± SE. NLA, nitro-l-arginine; HR, hear rate; CI, cardiac index; SVi, stroke volume index; MAP, mean arterial pressure; PAP, pulmonary artery pressure; LAP, left atrial pressure; SVCi, systemic vascular conductance index; PVCi, pulmonary vascular conductance index; BVO2i, body oxygen consumption index. NLA, n = 8; EMD360527, n = 9.
P < 0.05 vs. Rest;
P < 0.05 vs. Control.
Table 3.
Systemic and pulmonary hemodynamic responses to NLA and EMD360527 in FH swine
| Rest |
Exercise, mph |
||||
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | ||
| HR, beats/min | Untreated | 134 ± 5 | 144 ± 8 | 160 ± 4* | 200 ± 5* |
| NLA | 101 ± 12† | 128 ± 8 | 146 ± 9 | 172 ± 2*† | |
| Untreated | 128 ± 5 | 145 ± 9* | 165 ± 8* | 196 ± 9* | |
| EMD360527 | 136 ± 11 | 155 ± 7* | 187 ± 5*† | 215 ± 9*† | |
| CI, ml·min−1·kg−1 | Untreated | 119 ± 9 | 134 ± 8* | 155 ± 10* | 187 ± 14* |
| NLA | 78 ± 9† | 110 ± 13 | 118 ± 15† | 139 ± 12*† | |
| Untreated | 110 ± 8 | 127 ± 3* | 147 ± 8* | 170 ± 12* | |
| EMD360527 | 120 ± 12 | 140 ± 11* | 167 ± 7*† | 181 ± 10*† | |
| SVi, ml/kg | Untreated | 0.90 ± 0.08 | 0.95 ± 0.08 | 0.98 ± 0.09 | 0.94 ± 0.08 |
| NLA | 0.79 ± 0.08 | 0.86 ± 0.07 | 0.80 ± 0.05 | 0.81 ± 0.07 | |
| Untreated | 0.86 ± 0.06 | 0.89 ± 0.06 | 0.90 ± 0.07 | 0.87 ± 0.06 | |
| EMD360527 | 0.88 ± 0.05 | 0.90 ± 0.05 | 0.90 ± 0.05 | 0.84 ± 0.05 | |
| MAP, mmHg | Untreated | 95 ± 5 | 94 ± 6 | 102 ± 6 | 115 ± 8* |
| NLA | 126 ± 8† | 128 ± 15† | 137 ± 15† | 149 ± 10† | |
| Untreated | 88 ± 7 | 90 ± 7 | 98 ± 7* | 108 ± 10* | |
| EMD360527 | 93 ± 6 | 85 ± 8* | 96 ± 8 | 99 ± 9 | |
| PAP, mmHg | Untreated | 25 ± 2 | 26 ± 3 | 29 ± 3 | 33 ± 2* |
| NLA | 35 ± 3† | 40 ± 2† | 52 ± 6† | 53 ± 4*† | |
| Untreated | 21 ± 1 | 25 ± 2* | 27 ± 2* | 31 ± 2* | |
| EMD360527 | 25 ± 3 | 20 ± 1† | 24 ± 2† | 26 ± 3† | |
| LAP, mmHg | Untreated | 9 ± 1 | 10 ± 2 | 12 ± 2 | 13 ± 2* |
| NLA | 19 ± 5 | 16 ± 4 | 21 ± 6 | 21 ± 6 | |
| Untreated | 6 ± 2 | 11 ± 1* | 13 ± 1* | 13 ± 2* | |
| EMD360527 | 15 ± 1† | 11 ± 2 | 14 ± 2 | 14 ± 2 | |
| SVCi, ml·min−1·kg−1·mmHg−1 | Untreated | 1.27 ± 0.13 | 1.46 ± 0.16* | 1.55 ± 0.17* | 1.65 ± 0.18* |
| NLA | 0.63 ± 0.10† | 0.93 ± 0.2† | 0.92 ± 0.21† | 0.96 ± 0.14† | |
| Untreated | 1.28 ± 0.14 | 1.44 ± 0.11 | 1.52 ± 0.11* | 1.60 ± 0.10* | |
| EMD360527 | 1.30 ± 0.14 | 1.67 ± 0.14 | 1.77 ± 0.10*† | 1.86 ± 0.10* | |
| PVCi, ml·min−1·kg−1·mmHg−1 | Untreated | 8 ± 2 | 9 ± 2* | 10 ± 2* | 10 ± 1* |
| NLA | 5 ± 1 | 5 ± 1 | 5 ± 1 | 5 ± 1† | |
| Untreated | 8 ± 1 | 10 ± 1* | 10 ± 1* | 10 ± 1* | |
| EMD360527 | 13 ± 2† | 17 ± 3† | 17 ± 1*† | 17 ± 2*† | |
| BVO2i, ml·min−1·kg−1 | Untreated | 9 ± 1 | 11 ± 1* | 14 ± 1* | 20 ± 1* |
| NLA | 7 ± 1 | 11 ± 1* | 13 ± 1† | 17 ± 1* | |
| Untreated | 8 ± 1 | 11 ± 1* | 14 ± 1* | 19 ± 1* | |
| EMD360527 | 8 ± 1 | 10 ± 1 | 14 ± 1* | 18 ± 1* | |
Data are presented as mean ± SE. NLA, n = 4; EMD360527, n = 5.
P < 0.05 vs. Rest;
P < 0.05 vs. Control.
Fig. 6.
Effect of endothelial NO synthase (eNOS) and PDE5 inhibition on systemic vasomotor tone in Control (C) and FH swine. eNOS inhibitions, using NLA, resulted in decreased SVCi, which was significantly less pronounced in FH swine compared with Control swine, both at rest and during exercise. Whereas PDE5 inhibition with EMD360527 resulted in systemic vasodilation in Control swine, its effects were nearly abolished in FH swine. NLA: Control, n = 8; FH, n = 4; EMD360527: Control, n = 9; FH, n = 5. *P < 0.05 FH vs. Control; †P < 0.05 effect of NLA or EMD360527 vs. Untreated; ‡P < 0.05 effect of NLA or EMD360527 altered in FH swine compared with Control swine.
In Control swine, infusion of the PDE5 inhibitor EMD360527 decreased mean arterial pressure at rest and during exercise, which was accompanied by an increase in cardiac index (Table 2), resulting in an increased SVCi (Fig. 6). In contrast, in FH swine, PDE5 inhibition produced only minimal increases in SVCi and cardiac index, so that mean arterial pressure remained essentially unchanged in response to EMD360527 (Table 3).
Together, our data indicate that NO signaling in the systemic vasculature of FH swine appears to be preserved, in part, by downregulation of the PDE5-mediated breakdown of cGMP. This interpretation is consistent with the increased systemic vasodilator response to SNP and the decreased response to ATP, relative to nitroprusside, in FH swine.
Pulmonary circulation.
Administration of NLA resulted in an increase in mean pulmonary arterial pressure in Control and FH swine, whereas mean left atrial pressure showed a trend to increase in FH (Tables 2 and 3). However, the transpulmonary gradient was increased by NLA to a similar extent in FH and Control swine, so that PVCi was decreased similarly in both swine breeds (Fig. 7). The effect of exercise on PVCi after NLA was not attenuated in FH or Control swine (FH, P = 0.15; Control, P = 0.89).
Fig. 7.
Effects of eNOS and PDE5 inhibition on pulmonary vasomotor tone in Control and FH swine. eNOS and PDE5 inhibition elicited comparable responses in both Control and FH swine pulmonary vasculature. NLA: Control, n = 6; FH, n = 4; EMD360527: Control, n = 7; FH, n = 5. †P < 0.05 effect of NLA or EMD360527 vs. Untreated.
PDE5 inhibition with EMD360527 resulted in similar decreases of the transpulmonary pressure gradient and similar increases of cardiac index in both swine breeds (Tables 2 and 3). Consequently, PVCi was increased to a similar extent in Control and HF swine (Fig. 7).
Altogether, these data indicate that in contrast to the systemic circulation, NO-cGMP-PDE5 signaling is not impaired in the pulmonary circulation of FH swine at rest or during exercise.
DISCUSSION
The main findings of the present study were that in swine with FH: 1) exercise-induced systemic vasodilation was impaired, as evidenced by a blunted exercise-induced increase in SVCi; 2) FH swine showed only modest impairment of eNOS-mediated systemic vasodilator influence, which was likely mitigated by attenuation of PDE5 systemic vasoconstrictor influence in FH swine; 3) the latter also explains that compared with Control swine, FH swine showed a small increase in nitroprusside-induced vasodilation and only a trend towards reduction in ATP-induced vasodilation; and 4) FH did not alter the pulmonary vasomotor influence of either NO or PDE5, although the response to ATP was blunted. The implications of these observations are discussed below.
Methodological Considerations
The FH swine model.
The Rapacz FH swine model is characterized by a single missense mutation in the LDL receptor that decreases its affinity for LDL, resulting in elevated LDL and total cholesterol levels (7, 8). Most studies into human hypercholesterolemia define their patients by a high cholesterol and LDL combined with a family history of hypercholesterolemia. This definition does not specify the genetic disorders that lead to FH in these patients. However, FH has several underlying genetic disorders that can result in varying degrees of hypercholesterolemia and cardiovascular disease (1, 10). Junyent et al. (11) showed that missense mutations result in moderate-to-severe femoral intima media thickening and plaque scores, thereby representing the average FH patient. These data indicate that the Rapacz FH swine model is highly relevant to human FH patients.
Yucatan swine as Control to FH swine.
For more than three decades, the FH swine model has been developed by Drs. Rapacz and Hasler-Rapacz (8, 18), for which they screened over 4,000 animals to find the original FH trait. Approximately 15 yr ago, the animals were bred for downsized characteristics to increase their manageability. In our experiments, Yucatan swine were used as a control group, as they are also swine with manageable-size characteristics with well-established growth curves on regular chow. As a result, these animals can be age matched to the FH swine, making them an appropriate control group for the FH swine.
Indexing parameters to body weight.
As a result of age matching between the Control group and the FH group, it was not possible to weight-match the animals. To allow comparison of animals with significant differences in size, cardiac output (and its dependent variables) was indexed to body weight. van Essen et al. (24) showed that growth of farm swine, up to 90 kg, obeys allometric scaling laws and thus that cardiac output increases, according to the quarter scaling laws, commensurate with body weight. These findings indicate that indexing cardiac output for body weight is appropriate for swine in the weight range as studied in the present study.
Effects of FH on Systemic Vasomotor Tone
Under baseline conditions, FH did not influence heart rate at rest or during exercise. In contrast, stroke volume index was significantly smaller in FH, resulting in a reduction of cardiac index at rest and during exercise. Furthermore, exercise-induced vasodilation was reduced in FH compared with Control animals, as evidenced by a reduced increase in SVCi during exercise. As a result, mean arterial pressure increased more in FH animals during exercise. To investigate whether these FH-induced changes in hemodynamics resulted from endothelial dysfunction, we compared responses to infusion of ATP, which produces endothelium-dependent, NO-mediated vasodilation, with nitroprusside, an endothelium-independent NO donor (4), in Control and FH swine. Our results demonstrate that the systemic vasodilator response to a NO donor was enhanced slightly, whereas the response to agonist-induced eNOS activation tended to decrease in FH swine. The endothelial dysfunction in FH swine became especially apparent when normalizing the response of ATP to that of nitroprusside in an attempt to correct for alterations in NO sensitivity of the vasculature. Our results are consistent with several studies performed in hypercholesterolemic subjects, which conclude that vasodilation is impaired as a result of endothelial but not vascular smooth muscle dysfunction (3, 17, 22, 23). These previous studies were performed in the forearm of patients with hypercholesterolemia under resting conditions and show impaired NO signaling using serotonin receptor (22, 23) or M-receptor agonists (3) in combination with L-Arg. Similarly, studies in hypercholesterolemic mice show moderate impairment of NO signaling as a result of hypercholesterolemia (20, 21). Altogether, these studies indicate that impaired NO signaling, resulting from hypercholesterolemia, is not due to an inability of the vascular smooth muscle cells to react to the NO signal but rather, results from reduced NO bioavailability. Whether this reduced NO bioavailability is caused by decreased NO production or increased NO scavenging remains to be determined (26). However, the observation that the vasodilation to nitroprusside was enhanced indicates that the biological half-life of NO was not affected, suggesting that a reduced NO production underlies the observations in the present study.
Agonist-induced activation of eNOS occurs via different signal-transduction pathways rather than exercise-induced, shear stress-mediated activation of eNOS. The present study is the first to investigate the effect of FH on control of vasomotor tone during exercise. Exercise results in a significant increase in BVO2, necessitating increased perfusion of the exercising muscles to match O2 and nutrient supply and demand (12). To accommodate this increased O2 demand, cardiac output, SVC, and whole-body O2 extraction increase. In FH swine, whole-body O2 extraction was already elevated by ∼40% at rest compared with Control swine (Fig. 5), whereas cardiac index and SVCi were lower than in Control animals. Furthermore, the exercise-induced increase in SVCi was blunted, indicating that FH hampers exercise-induced vasodilation. Following eNOS inhibition with NLA, the relation between BVO2i and SVCi in FH swine was identical to that in Control swine, consistent with the concept that dysfunction of the NO pathway underlies the baseline difference between FH and Control swine. Moreover, the PDE5 vasoconstrictor influence was significantly lower in FH swine. Interestingly, Champion et al. (2) showed that in eNOS knockout mice, PDE5A protein expression and activity were significantly lower than in normal mice and that transfection with eNOS resulted in a normalization of PDE5A protein expression and activity. These data suggest that the observed attenuation in PDE5 activity occurs as a compensatory mechanism to maintain vasodilation in the presence of reduced NO bioavailability. Such reduction of PDE5 activity also explains the observed increase in nitroprusside-induced vasodilation, as an attenuation of cGMP degradation amplifies the vasodilator response to nitroprusside.
Effects of FH on Pulmonary Vasomotor Tone
To our knowledge, this is the first study into the effect of FH on the pulmonary circulation in vivo. In pulmonary small arteries, isolated from Zucker rats, both NO responsiveness and agonist-induced NO production were unaltered (16), whereas pulmonary artery vasodilation, in response to metacholine, was enhanced in rabbits given a 2% cholesterol diet over a period of 2 wk (17). Neither NO production nor NOS activity was altered in the pulmonary arteries of these rats or rabbits, suggesting that the pulmonary vasculature is less sensitive to the deleterious effects of hypercholesterolemia compared with the systemic vasculature. Indeed, although FH resulted in an increase in pulmonary artery pressure in the present study, this occurred secondary to the increase in left atrial pressure (Fig. 4), as the relation between BVO2i and PVCi remained essentially unchanged. Also, the effects of eNOS inhibition and PDE5 inhibition were unaltered in the pulmonary circulation of FH swine compared with Controls. Infusion of SNP had negligible vasodilator effects on pulmonary vasomotor tone in either Control or FH swine, whereas ATP-induced pulmonary vasodilation was markedly reduced in the FH compared with Control swine. These data suggest that disruptions in agonist-induced pulmonary vasodilation may be the earliest marker of pulmonary endothelial dysfunction in FH. However, whether this endothelial dysfunction is a direct effect of FH or occurs secondary to the increase in pulmonary venous pressure remains to be determined.
Conclusions
The present study demonstrates that FH results in blunting of the exercise-induced systemic, but not pulmonary, vasodilation. Whereas we obtained evidence for endothelial dysfunction in the systemic circulation, the vasodilator influence of eNOS is preserved partially by a downregulation of the PDE5-mediated vasoconstrictor influence in FH swine. In contrast, no evidence was found for alterations in endogenous eNOS-cGMP-PDE5-mediated vascular control in the pulmonary vascular bed.
GRANTS
Support for the authors' work was provided by grants from the National Heart, Lung, and Blood Institute (grants HL-52490 to M. H. Laughlin, D. L. Tharp, and D. K. Bowles and HL-36088 to M. H. Laughlin), the Netherlands Heart Foundation (grant 2000T042 to D. Merkus and the European Commission (FP7-Health-2010; MEDIA-261409) to D. J. Duncker and D. Merkus. Support for this work was also partially provided by the use of facilities at the Harry S. Truman Memorial Veterans' Hospital in Columbia, MO (to S. B. Bender).
DISCLOSURES
The authors have no conflicts of interest to disclose.
AUTHOR CONTRIBUTIONS
Author contributions: V.J.d.B., D.M., S.B., D.K.B., D.J.D., and M.H.L. conception and design of research; V.J.d.B., D.M., S.B., D.L.T., D.K.B., D.J.D., and M.H.L. performed experiments; V.J.d.B., D.M., S.B., and D.K.B. analyzed data; V.J.d.B., D.M., S.B., D.L.T., D.K.B., D.J.D., and M.H.L. interpreted results of experiments; V.J.d.B. and D.J.D., prepared figures; V.J.d.B and D.J.D., drafted manuscript; D.M., S.B., D.L.T., D.K.B., D.J.D., and M.H.L. edited and revised manuscript; V.J.d.B., D.M., S.B., D.L.T., D.K.B., D.J.D., and M.H.L. approved final version of manuscript.
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