effect of 6 weeks of high-intensity one-legged cycling on

University of Southern Denmark

Effect of 6 wk of high-intensity one-legged cycling on functional sympatholysis and ATPsignaling in patients with heart failure

Munch, Gregers Wibe; Iepsen, Ulrik Winning; Ryrsø, Camilla Koch; Rosenmeier, JayaBirgitte; Pedersen, Bente Klarlund; Mortensen, Stefan P.

Published in:American Journal of Physiology: Heart and Circulatory Physiology

DOI:10.1152/ajpheart.00379.2017

Publication date:2018

Document version:Accepted manuscript

Citation for pulished version (APA):Munch, G. W., Iepsen, U. W., Ryrsø, C. K., Rosenmeier, J. B., Pedersen, B. K., & Mortensen, S. P. (2018).Effect of 6 wk of high-intensity one-legged cycling on functional sympatholysis and ATP signaling in patients withheart failure. American Journal of Physiology: Heart and Circulatory Physiology, 314(3), H616-H626.https://doi.org/10.1152/ajpheart.00379.2017

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1

1

Effect of 6 weeks of high-intensity one-legged cycling on functional 2

sympatholysis and ATP signaling in patients with heart failure 3

4

Munch, Gregers W.1; Iepsen, Ulrik W.

1; Ryrsø, Camilla K.

1; Rosenmeier, Jaya B.

1,2; 5

Pedersen, Bente K.1 and Mortensen, Stefan P.

1,3 6

7

1 The Centre of Inflammation and Metabolism and the Centre for Physical Activity Research, 8

Rigshospitalet, Copenhagen, Denmark 9

2Department of Cardiology Y, Copenhagen University Hospital Bispebjerg and Frederiksberg 10

3Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, 11

University of Southern Denmark, Odense, Denmark 12

13

Running title: exercise training and heart failure 14

Key words: vascular function, vasodilation, 15

16

Corresponding author: 17

Address for correspondence: 18

Gregers W. Munch 19

Rigshospitalet 7641 20

Centre of Inflammation and Metabolism (CIM) and 21

The Centre for Physical Activity Research (CFAS) 22

Blegdamsvej 9, 23

DK-2100 Copenhagen, Denmark 24

Tel. +45 2623 5720 25

Fax +45 3545 7644 26

Email: [email protected] 27

http://www.inflammation-metabolism.dk and http://aktivsundhed.dk 28

29

Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.226.087.174) on December 14, 2017.

mailto:[email protected]

http://www.inflammation-metabolism.dk/

http://aktivsundhed.dk/

2

ABSTRACT 30

Breathlessness during daily activities is the primary symptom in patients with heart failure (HF). 31

Poor correlation between the hemodynamic parameters of left ventricular performance and 32

perceived symptoms suggests that other factors such as skeletal muscle function plays a role in 33

determining exercise capacity. We investigated the effect of six weeks of high-intensity one-legged 34

cycling (HIC; 8x4 at 90% one-legged cycling max) on: 1) the ability to override sympathetic 35

vasoconstriction (arterial infusion of tyramine) during one-legged knee-extensor exercise (KEE); 2) 36

vascular function (arterial infusion of ACh, SNP, tyramine and ATP); 3) exercise capacity in HF 37

patients with reduced ejection fraction (n=8) compared with healthy individuals (n=6). Arterial 38

tyramine infusion lowered leg blood flow and leg vascular conductance at rest and during KEE 39

before the training intervention in both groups (P<0.05), but not during KEE after the training 40

intervention. There was no difference between groups. The peak vasodilatory response to ATP was 41

blunted in the HF patients. (P<0.05), whereas there was no difference in ACh- and SNP- induced 42

vasodilation between HF patients and healthy individuals. ACh induced vasodilation increased in 43

the HF patients after the training intervention (P<0.05). HIC improved aerobic capacity in both 44

groups (P<0.05), whereas only the HF patients improved six min walking distance (P<0.05). These 45

results suggest that exercise hyperemia and functional sympatholysis is not altered in HF patients, 46

and that functional sympatholysis is improved with HIC in both HF patients and healthy 47

individuals. Moreover, these results suggest that the peak vasodilatory response to ATP is blunted 48

in HF. 49

New & Noteworthy 50

The ability to override sympathetic vasoconstrictor activity (by arterial tyramine infusion) during 51

exercise is not different between heart failure patients and healthy individuals and is improved by 52


3

high-intensity one-legged cycling training. The peak vasodilatory response to ATP is reduced in 53

heart failure patients. 54

55


4

INTRODUCTION 56

Chronic heart failure (HF) is associated with exercise intolerance primarily because of 57

breathlessness and muscle fatigue (54, 60). These symptoms often lead to reduced physical activity, 58

which progressively worsen the exercise intolerance. Exercise intolerance does not seem to be 59

related to left ventricular performance (12), indicating that other factors play a major role. 60

Abnormal skeletal muscle perfusion and metabolism, as well as abnormal autonomic reflexes 61

originating in the skeletal muscles have been observed (4, 21, 45, 46). When exercising with a small 62

muscle mass, and thereby removing any central hemodynamic limitation to skeletal muscle 63

perfusion (1), HF patients are able to achieve equally high peak muscle perfusion rates as healthy 64

individuals (11, 31, 32, 59). Therefore, single leg training might be optimal for improving exercise 65

capacity in HF patients (8, 31, 58), but the underlying mechanisms of how improved muscle 66

function reduce symptoms of HF are still unclear. 67

Skeletal muscle blood flow is regulated by a complex interaction between vasodilator and 68

vasoconstrictor substances (16), including nitric oxide (NO) and prostacyclin (3, 36, 39). HF is 69

associated with endothelial NO dysfunction (10, 26, 27) and exercise training has been shown to 70

improve endothelial NO function both locally and systemically (14, 23, 28, 62). In recent years, 71

ATP has been proposed to contribute to exercise hyperemia by inducing local vasodilation and 72

overriding sympathetic vasoconstrictor activity (25, 50, 51). However, it remains unknown if ATP 73

signaling is altered in HF patients. 74

In healthy humans, the vasoconstrictor effect of increased sympathetic nerve activity (SNA) during 75

muscle contraction is blunted, termed “functional sympatholysis” (49) and this mechanism is 76

thought to play an important role in securing adequate delivery of O2 to contracting muscle. HF is 77

associated with increased levels of SNA (31, 34, 47, 53), and an impaired functional sympatholysis 78

could therefore accelerate vasoconstrictor activity in these patients. Whether functional 79


5

sympatholysis is impaired in HF patients remains unknown. The ability for functional 80

sympatholysis is heavily dependent on training status of the exercising muscle (35, 37, 56) and 81

exercise training could therefore improve functional sympatholysis in HF. 82

The aim of the study was to evaluate the effect of high-intensity one-legged cycling (HIC) training 83

on leg hemodynamics and oxygen uptake, functional sympatholysis and endothelial function. To 84

investigate this, we determined endothelial function and leg hemodynamics during arterial ATP 85

infusion and one-legged knee extensor exercise (KEE), with and without co-infusion of tyramine 86

before and after six weeks of HIC in heart failure patients with reduced ejection fraction and healthy 87

individuals. 88

89

90


6

METHODS 91

Fourteen subjects, 8 heart failure (HF) patients New York Heart Association Classification (NYHA-92

class II) with mildly reduced ejection fraction and 6 healthy individuals (Table 1) were studied 93

before and after six weeks of HIC. Patients were recruited from Hospitals in the Region of 94

Copenhagen whereas the healthy individuals were recruited from advertisements. All subjects were 95

screened with a medical history interview, physical examination, blood chemistry analysis, and 96

resting 12-lead electrocardiogram. Stable HF patients (with a reduced ejection fraction 37±2%, at 97

the time of diagnosis) in stable pharmacological treatment (ACE-inhibitors and beta-blockers) with 98

and without implantable cardioverter defibrillator were recruited. Patients with intermittent 99

claudication, aneurysm in a. femoralis, moderate to severe heart valve disease and arrhythmias were 100

excluded. Other exclusion criteria were chronic obstructive pulmonary disease (forced expiratory 101

volume in 1 sec <60%) and renal failure (creatinine >2.5 mg dL-1

). The healthy individuals were 102

non-smokers and none of the subjects had been diagnosed with cardiovascular disease, renal 103

dysfunction, insulin resistance, diabetes, or hypercholesterolaemia. Before the start of the training 104

intervention standard echocardiography was performed (Vivid 9, GE Healthcare, Pittsburgh, PA, 105

USA). 106

The study was approved by the Ethics Committee of the Capital Region of Denmark (H-3-2013-107

048) and conducted in accordance with the guidelines of the Declaration of Helsinki. Written 108

informed consent was obtained from all subjects before enrolment into the study. 109

110

Initial testing 111

Before the experimental day all subjects visited the laboratory to be accustomed to the KEE (1). On 112

the same day the subjects performed: (1) an incremental bicycle test on a bicycle ergometer 113

(Monark 839E; Monark, Varberg, Sweden) to determine peak pulmonary oxygen uptake (l/min, 114


7

VO2peak) (Cosmed CPET, Rome, Italy) and peak workload (Wattpeak), (2) an incremental KEE test, 115

to determine peak workload, and (3) distance during a six minute walk (6MWD) test (54). Each test 116

was separated by >30 min of rest. On the same day body composition was assessed by whole-body 117

dual-energy X-ray absorptiometry scanning (Lunar Prodigy Advance; GE Healthcare, Madison, 118

WI). 119

120

Experimental protocol 121

All subjects refrained from caffeine, alcohol and exercise 24 hours before the experimental day. 122

Medication was gradually withdrawn from the HF patients (ACE-inhibitors were withdrawn 48 123

hours prior to the experimental day, whereas beta-blockers, anti-platelet and diuretic drugs were 124

withdrawn 24 hours before the experimental day). On the experimental day all subjects arrived at 125

the laboratory after a light breakfast. After local anesthesia (Lidocaine (20 mg dL-1

)), catheters were 126

placed in the femoral artery and vein of the experimental leg and in the left brachial artery. In the 127

experimental leg, two microdialysis probes (63 MD Catheter, M Dialysis AB, Stockholm, Sweden) 128

membrane length of 30 mm and a cut-off at 20 KDa were inserted into m. vastus lateralis. After 129

insertion of catheters the experimental leg was passively moved for ~5 min in supine position, with 130

the purpose of minimizing the tissue response to insertion trauma. 131

Following 30-60 min of rest, the subjects received a femoral arterial infusion of: 1) Tyramine 132

1µmol/min/kg leg mass (Sigma Aldrich, St Louis MO); 2) sodium nitroprusside (SNP) 2 µg/min/kg 133

leg mass (Nitropress, Hospira, Lake Forrest. IL); 3) ACh 100 µg/min/kg leg mass-1

(Miochol-E, 134

Bausch + Lomb, Berlin, Germany); 4) ATP 0.05 (ATP1) µmol/min/kg leg mass (Sigma Aldrich) 135

with and without co-infusion of tyramine 1µmol/min/kg leg mass; 5) ATP 0.3 (ATP2) and 3.0 136

(ATP3) µmol/min/kg leg mass. Each dose was infused for 2.5 min and measurements were obtained 137

after 2.0 min. All infusions were separated by ~20 min. After additional 60-90 min of supine rest 138


8

the subjects completed two exercise bouts separated by ~20 min: 1) 2 min one-legged passive leg 139

movement (PLM); 2) 11 min of KEE at an absolute workload of 10 watt. During the 11 min of 140

KEE, tyramine (1µmol/min/kg leg mass) was infused for 3 minutes (5-8 mins of exercise). Leg 141

blood flow was measured and arterial and venous blood samples (1-5 ml) were drawn 142

simultaneously before and during (after: 1 min during PLM; and 3.5, 7 and, 10 during the 11 min of 143

KEE) each trail. 144

Exercise training 145

The subjects performed supervised HIC on a cycle ergometer (Monark 839E) modified with a fixed 146

flywheel. The subjects completed six weeks of training with three training sessions per week. Each 147

training session consisted of 10 min two-legged warmup at 50 watts where after 8x4 min intervals 148

at ~90% of one-legged Wattmax were completed (four intervals with each leg). Each interval was 149

separated by 1.5-2 min rest. The workload during the intervals was determined from a graded one-150

legged cycling test during the first training session. Heart rate was monitored during each training 151

session (Polar WearLink, Polar Electro Denmark Aps, Denmark) and intervals were rated from the 152

Borg scale. 153

154

Microdialysis 155

Microdialysate was collected for ~10 min during resting conditions, during infusion of tyramine, 156

ACh, ATP+tyramine and ATP and KEE. After collection of samples, the microdialysate was 157

weighed, and the actual flow rate was calculated to estimate any loss of fluid or abnormal decrease 158

in perfusion rate. The microdialysis probes were perfused with isotonic saline (9 g NaCl/l) at a rate 159

of 5 µl min-1

and to determine the relative exchange of substances, a small amount (2.7 nM) of [2.8-160

3H] ATP (<0.1 μCi/ml) was added to the perfusate for calculation of probe recovery. The molecular 161


9

probe recovery (PR) was calculated as [PR = (dpminfusat – dpmdialysate/ dpminfusat], where dpm denotes 162

disintegrations per min (22, 52). The [2.8-3H] ATP activity (in dpm) was measured on a liquid 163

scintillation counter (Tri-Carb 2910 TR, Perkin Elmer) after an addition of the infusate and 164

dialysate (5 µl each) were added to 3 ml Ultima Gold scintillation liquid (Perkin Elmer, MA). 165

166

Measurements and calculations 167

Femoral arterial blood flow was measured with ultrasound Doppler (Logic E9, GE Healthcare, 168

Pittsburgh, PA) equipped with a linear probe operating an imaging frequency of 9MHz and Doppler 169

frequency of 4.2–5.0MHz. All recordings were assessed using an insonation angle ≤60◦. The sample 170

volume was maximized according to the width of the vessel, and kept clear of the vessel walls. A 171

low-velocity filter (velocities <1.8ms−1) rejected noises caused by turbulence at the vascular wall. 172

Doppler tracings and B-mode images were recorded continuously and Doppler tracings were 173

averaged over 8 heart cycles at the time of blood sampling. Arterial diameter was determined during 174

the systole from arterial B-mode images under a perpendicular insonation angle at supine and seated 175

rest, assuming negligible changes in vessel-diameter during exercise. 176

Intra-arterial (brachial artery) and venous pressures were monitored with transducers (Pressure 177

Monitoring Kit, Baxter, IL) positioned at the level of the experimental leg. Leg mass was calculated 178

from the whole-body dual energy x-ray absorptiometry scanning. Blood gasses, hemoglobin, and 179

lactate concentrations were measured using ABL825 flex analyzer (Radiometer, Copenhagen 180

Denmark). Leg vascular conductance (LVC) was calculated as leg blood flow (LBF)/(mean arterial 181

pressure (MAP) – femoral venous pressure (FVP)). Functional sympatholysis was calculated as 182

percentage change in LBF and LVC after 2 min of arterial tyramine infusion during exercise 183

compared to exercise without tyramine infusion (1.5 min before tyramine infusion and 2 min after 184


10

the stop of tyramine infusion). The hemodynamic measurements in the healthy individuals before 185

the training have been published (18). 186

187

Analysis of nitrite, nitrate, prostacyclin, endothelin-1 and norepinephrine 188

Stable metabolites of NO, NO2- and NO3 – (NOx) were measured using colorimetric assay kit 189

(Cayman Chemical Co., Ann Arbor, MI, USA). The stable metabolite of prostacyclin (PGI2) 6-keto 190

prostaglandin F1α, was measured using an enzyme immunoassay kit (Cayman Chemical, Ann 191

Arbor, MI) following manufacturer’s instructions. Plasma endothelin-1 was measure using 192

immunoassay (QuantiGlo, R&D systems, Minneapolis, MI) following manufacturer’s instructions. 193

Plasma [NE] was determined with a radioimmunoassay (LDN, Nordhorn, Germany). Dialysate K+ 194

was measured by flame photometry (FLM3, Radiometer, Denmark) using lithium as internal 195

standard. Dialysate lactate was measured with an enzymatic–spectrophotometric method using a 196

Cobas Mira analyser (Roche, Branchburg, NJ). 197

198

Statistical analysis 199

One-way analysis of variance (ANOVA) was used to compare between-group baseline 200

characteristics, and within groups for changes in interstitial variables and plasma [NA]. A two-way 201

repeated measures analysis of variance (ANOVA) was performed to test significance within and 202

between trials and before and after the training intervention within the two groups. A two-way 203

ANOVA was performed to test significance between the two groups. Following a significant F test, 204

all pair-wise differences were identified using Tukey’s honestly significant difference (HSD) post 205

hoc procedure. A significant interaction indicates that the outcome variable is different within or 206

between groups before and after the training intervention. All data are presented as means ± SEM. 207

All analyses were conducted using SIGMAPLOT (13) and statistical significance was accepted 208


11

when P<0.05. For technical reasons and/or catheter displacement during the post test, infusion and 209

blood samples could not be obtained in two HF patients. 210

211


12

RESULTS 212

Baseline characteristics 213

Table 1 shows baseline characteristics for HF and the healthy individuals. There were no 214

differences in age, sex or anthropometric measurements between groups. The ejection fraction was 215

lower in the HF patients compared to the healthy individuals (P<0.05). We found no difference in 216

resting leg blood flow, MAP, or LVC between groups. 217

218

Leg hemodynamic responses to SNP infusion 219

Arterial infusion of SNP increased LBF and LVC (P<0.05), whereas MAP decreased in both groups 220

(P<0.05). There was no difference in LBF, LVC or MAP before and after the training intervention 221

in both group, and no difference between groups (Figure 1). 222

223

Leg hemodynamic responses to ACh infusion 224

In the healthy individuals, arterial infusion of ACh increased LBF and LVC (P<0.05), and lowered 225

MAP (P<0.05) similarly before and after the training intervention (Figure 1). ACh increased LBF 226

and LVC in the HF patients both before and after the training intervention (P<0.05), but the 227

increase in LBF and LVC was higher after the training intervention (P<0.05). After the training 228

intervention, MAP was lower during infusion of ACh in the HF patients (P<0.05). There was no 229

difference between groups in LBF, LVC and MAP, before and after the training intervention 230

(Figure 1). 231

232

Leg hemodynamic responses to tyramine, and ATP with and without co-infusion of tyramine 233

ATP: Both groups increased LBF and LVC in response to ATP (P<0.05; Figure 2), but in the HF 234

patients the effect was blunted at the highest dose (ATP3). At the highest infusion rate, LBF tended 235


13

(P=0.06) to be higher before the training intervention and both LBF and LVC were higher (P<0.05) 236

after the training intervention in the healthy individuals. Within the healthy individuals LBF tended 237

(P=0.055) to be, and LVC (P<0.05) was higher after the training intervention during the highest 238

ATP infusion (ATP3). Tyramine: In both groups, LBF and LVC decreased similarly during 239

tyramine infusion (P<0.05) before and after the training intervention (Figure 3) and there was no 240

difference between groups. ATP+tyramine: Both before and after the training intervention, LBF 241

and LVC decreased in both groups (P<0.05) when tyramine was co-infused with ATP (Figure 3), 242

whereas MAP increased in both groups (P<0.05). The decrease in LBF was greater before (P<0.05) 243

and with a tendency (P=0.083) to be greater after the training intervention in the healthy individuals 244

when compared to the HF patients. The decrease in LVC tended to be greater both before and after 245

training (Pre: P=0.053; Post: P=0.096) in the healthy individuals when compared to the HF patients 246

(Figure 3). 247

248

Leg hemodynamic responses during exercise with and without infusion of tyramine 249

LBF, LVC and MAP increased (P<0.05) in both groups during exercise (10 W) and there was no 250

difference between groups. Before the training intervention, LBF and LVC was lowered in both 251

groups when tyramine was infused during exercise (P<0.05) and increased after the termination of 252

tyramine infusion (P<0.05; Figure 4). After the training intervention, LBF, LVC and MAP 253

remained similar before, during, and after tyramine infusion. 254

Leg (a-v) O2 and leg VO2 were similar with and without tyramine in both groups before and after 255

the training intervention, this despite a higher O2 extraction in the HF patients reflected by a lower 256

femoral venous O2 saturation (P<0.05; Table 2). Before the training intervention, leg VO2 increased 257

in both groups when tyramine infusion was terminated (P<0.05), but not after the training 258

intervention. In the healthy individuals leg VO2 was lower after the training intervention when 259


14

tyramine infusion was terminated (P<0.05). Between groups, leg VO2 was higher in the HF patients 260

after the training intervention after tyramine infusion (P<0.05). 261

262

Exercise capacity 263

Six weeks of HIC increased absolute and relative VO2peak in both groups (P<0.05), with no 264

difference between groups. Training increased cycling Wattpeak only in the healthy individuals 265

(P<0.05), whereas Wattpeak during KEE only increased in the HF patients (P<0.05). There was no 266

difference in Wattpeak between groups during both cycling and KEE before and after the training 267

intervention. 6 MWD was longer in the healthy individuals before the training intervention. 268

Exercise training increased 6 MWD in the HF patients only (P<0.05), and there was no difference 269

in 6 MWD after the training intervention between groups. Variables are listed in table 3. 270

271

Plasma norepinephrine at rest and during exercise 272

Before and after the training intervention resting plasma [NE] was not different between groups 273

(Healthy: pre 0.7±0.2 mmol/l vs post 1.0±0.2 mmol/l; HF: pre 1.3±0.5 mmol/l vs post 1.1±0.5 274

mmol/l, respectively). In both groups, [NE] increased during tyramine infusion (P<0.05), and within 275

the healthy group, [NE] was higher during tyramine infusion after the training (P<0.05) (Healthy: 276

pre 1.3±0.2 mmol/l vs post 2.2±0.5 mmol/l; HF: 1.9±0.7 mmol/l vs post 3.0±1.2 mmol/l, 277

respectively). Both before and after the training intervention, infusion of tyramine during exercise 278

increased [NE] similarly in both groups (P<0.05; Figure 5). 279

280


15

Plasma endothelin-1 281

There was no difference in [ET-1] between the two groups before and after training (Healthy rest: 282

pre 1.1±0.2 vs post 1.4±0.4; HF rest: pre 1.3±0.2 vs post 1.6±0.2). In the healthy individuals resting 283

[ET-1] was higher (P<0.05) after training. 284

285

Plasma NOx at rest, during arterial ACh infusion, and during PLM and KEE 286

Plasma [NOx] was not different within or between groups at baseline and during ACh infusion both 287

before and after the training intervention. Before and after the training intervention, plasma NOx 288

was unaltered during PLM and KEE and there was no difference between groups. 289

290

Interstitial variables during rest and exercise 291

Before the training intervention, interstitial [lactate] levels increased with exercise in both groups 292

(P<0.05), and in the HF group it tended (P=0.055) to increase during exercise after the training 293

intervention (Table 4). In the HF patients, [lactate] was higher (P<0.05) before compared to after 294

the training intervention. Before the training intervention, [PGI2] tended to be lower at rest (P=0.08) 295

and during exercise (P=0.07) in the HF patients compared to the healthy individuals. No difference 296

in interstitial [potassium] was observed before and after the training intervention in either group or 297

between groups. 298

299

Muscle interstitial NOx at rest, during arterial ACh infusion, and during PLM and KEE 300

Muscle interstitial [NOx] was not different within or between groups at baseline and during arterial 301

infusion of ACh. In the healthy individuals, [NOx] increased during PLM only after the training 302

intervention (P<0.05), and was higher compared to before the training intervention (P<0.05). In the 303


16

HF patients, [NOx] increased during PLM only before the training intervention (P<0.05). [NOx] did 304

not increase during exercise when compared to baseline (Table 4). 305

306


17

DISCUSSION 307

We evaluated the effect of six weeks of high-intensity one-legged cycling (HIC) in patients with 308

mild heart failure (HF) and healthy individuals. The major findings were that: a) HIC increased 309

aerobic capacity in parallel with improved ability to override sympathetic vasoconstriction (arterial 310

infusion of tyramine) during small muscle mass exercise in both groups; b) the peak vasodilatory 311

responsiveness to arterial ATP infusion was lower in HF patients; c) HIC increased ACh induced 312

vasodilation in the HF patients. These results suggest that exercise hyperemia and the ability for 313

functional sympatholysis during exercise is maintained in HF and that functional sympatholysis is 314

improved with high intensity training in both healthy individuals and HF patients. Moreover, the 315

peak vasodilatory response to ATP appears to be reduced in patients with HF. 316

317

Blood flow and oxygen delivery 318

We found that the leg hemodynamic responses to KEE did not differ between the HF patients and 319

healthy individuals when evaluated at the same workload, and that it was unaltered by exercise 320

training. Despite a higher oxygen extraction, the metabolic demand was not different between 321

groups. These findings agree with observations showing an unaltered blood flow response in HF 322

patients during exercise with a small active muscle (2, 11, 31, 33). However, our findings are in 323

contrast to observations during whole body exercise, where HF patients show an attenuated exercise 324

hyperemia when compared to healthy individuals (31, 54, 55, 61). This discrepancy is most likely 325

explained by the differences in the active muscle mass. The present results may therefore not reflect 326

exercise engaging a large muscle mass with higher levels of sympathetic nerve activity (20, 31, 55). 327

In agreement with the similar exercise hyperemia in the HF patients and healthy individuals, plasma 328

and interstitial NOx, a stable metabolite of NO production, PGI2, as well as plasma endothelin-1 329

were not different between the two groups. Surprisingly, endothelin-1 levels were increased after 330


18

the training intervention in the healthy group, which contrast with previous observations that have 331

demonstrated similar or reduced levels after a period of exercise training (29, 43). Notably, exercise 332

training has been found to reduce the vasoconstrictor effect of endothelin (9), thereby counteracting 333

the effect of increased levels. 334

335

α-adrenergic responsiveness and functional sympatholysis 336

The tyramine induced increase in plasma [NE] and the reduction in LBF and LVC was similar in 337

the two groups indicating that α-adrenergic vasoconstrictor responsiveness is unaltered in HF, and 338

unaffected by training. In support of the present findings, forearm vascular responsiveness to 339

phenylephrine, a selective α1-adrenoceptor agonist, was similar in HF patients and healthy 340

individuals (19), and 5 weeks of training did not alter LVC or [NE] in young healthy individuals 341

(37). However, the present data contrasts recent observations in our lab showing reduced post 342

junctional α-adrenergic vasoconstrictor responsiveness in norm- and hypertensive individuals after 343

training (38). A possible explanation for the different observations may be related to a shorter 344

training intervention and weakly training volume in present study, as altered responsiveness in 345

Mortensen et al., 2014 (38), was observed after eight weeks of training (with three-four training 346

sessions per week). 347

The reduction in LBF and LVC in both groups during exercise, with similar tyramine-induced 348

increase in [NE], indicates that functional sympatholysis is unaltered in HF during exercise with an 349

isolated muscle mass. However, the unaltered exercise hyperemia when tyramine was infused 350

during exercise after training indicates that HIC improves the ability to override SNA (functional 351

sympatholysis) in the leg during exercise in both healthy individuals and HF patients (Figure 6). In 352

sedentary, functional sympatholysis is attenuated in the forearm (7) and in the leg (40, 42), but 353


19

improved in the trained state (37, 40). The present data supports and extends these observations by 354

demonstrating that training improves functional sympatholysis in HF patients when exercising with 355

a small muscle mass. In contrast to the literature (15, 31) we observed similar [NE] during resting 356

conditions and during exercise in the two groups both before and after the training intervention. 357

Whether improved functional sympatholysis during exercise with a local muscle mass can be 358

extrapolated to exercise with large muscle mass remains unclear, as HF patients show an 359

exaggerated sympathetic vasoconstriction when exercising with a large muscle mass (15). However, 360

our observations suggest that impaired functional sympatholysis may be more related to physical 361

state rather than age or diseased state. Therefore, the exercise intolerance often observed in HF 362

patients may not be related to the failing heart. 363

364

ATP response 365

Arterial ATP infusion resulted in a dose dependent vasodilatory response in the healthy individuals, 366

whereas LBF and LVC were blunted at the highest dose in the HF patients. The similar vasodilatory 367

response as the healthy individuals at the two lowest doses of ATP indicate that downstream 368

signaling pathway is preserved in HF. The failure to increase LVC at the highest dose therefore 369

appears to be more related to the vasodilatory capacity of the vasculature than ATP signaling per se. 370

ATP is a potent vasoactive substance and observations in young healthy individuals have shown 371

peak hemodynamic responses similar to maximal exercise at infusion rates similar to the rates in 372

present study (50). It remains unknown whether the lower ejection fraction in the HF patients is 373

responsible for the lower peak hyperemic response or vice versa. However, in healthy individuals 374

stroke volume is higher at peak vasodilation during ATP infusion when compared to peak 375

vasodilation during exercise, leading the increase in cardiac output observed during ATP infusion 376


20

(13). Training does not seem to alter the peak vasodilatory response to ATP in HF patients in 377

contrast to healthy individuals who are able to increase their peak vasodilatory dose response. The 378

improved vasodilatory response in the healthy individuals is in contrast to previous observations 379

from our lab showing unaltered or even reduced ATP responsiveness with training (37, 40). 380

A surprising observation was that ATP tended to be more sympatholytic with HF compared to 381

healthy individuals despite a diminished vasodilatory response to ATP. In addition, training, or at 382

least HIC, seems not to alter the sympatholytic effect of ATP, despite of the improved peak 383

vasodilatory response observed in the healthy group (Figure 3). The sympatholytic properties of 384

ATP are likely caused by interfering of the post-junctional vascular adrenergic receptors and/or 385

intracellular signaling pathways in the smooth muscle cells (57). A possibility is that the relative 386

short withdrawal period of medication affected the higher sympatholytic properties in HF especially 387

given that certain medications have been shown to improve the ability for functional sympatholysis 388

in hypertensive individuals (48). It has previously been reported that the vasodilatory effect of ATP 389

is maintained in both the forearm and in the leg in elderly healthy individuals conflicting with our 390

observations, whereas our results agree that the sympatholytic properties of ATP is not related to 391

training status (24, 40). Thus, other factors than ATP appear to be important in the ability to abolish 392

SNA, and the sympatholytic properties of ATP seems not to be related to the physical state. 393

394

Endothelial function 395

HIC improved endothelial-dependent vasodilator response to arterial infusion of ACh in HF patients 396

but not in healthy individuals. However there was no difference in the vasodilator response to ACh 397

between groups before and after the training intervention. Exercise training has previously shown to 398

improve endothelium-dependent vasodilation in HF patients with both local (14, 23) and systemic 399


21

effects (28), and also improved endothelium-dependent vasodilation is observed healthy individuals 400

(5). The improved vasodilator response to ACh in the HF patients was not associated with increased 401

[NOx] in the interstitial space or in plasma. 402

403

Exercise capacity 404

Six weeks of HIC improved VO2peak by 9% and Wattpeak by 13% in the healthy individuals and 405

VO2peak by 12% and 6 MWD by 8% in the HF patients. This indicates that HIC is beneficial for 406

both healthy individuals and HF patients with mildly reduced ejection fraction. Before the training 407

intervention, 6 MWD was lower in the HF patients when compared to the healthy individuals, 408

whereas no difference was observed after training. HIC therefore not only increases cycling 409

capacity, but also increase functionality which is of great clinical relevance. Our findings are in 410

agreement with previous observations showing that single leg training improved maximal exercise 411

capacity in HF patients and was well tolerated (8, 30, 58). 412

Experimental considerations 413

This study has led to some experimental considerations in regards to the observed vascular function 414

and exercise hyperemia: a) the HF patients were pharmacological stable and recreational physical 415

active, which might have influenced the level of exercise hyperemia and the response to ACh and; 416

b) the medication could have lowered the vasoconstricting neurohormens and influenced the 417

response to ACh in the HF patients. The patients were gradually withdrawn from their medication 418

prior to the experimental day (ACE-inhibitors 48 hours; beta-blockers, anti-platelet and diuretic 419

drugs 24 hours), and in regards to half-life of the medication (44) the ACE-inhibiting medication 420

might have lowered the release of vasoconstricting nuerohormens and improved the endothelial-421

dependent vasodilation (6, 41) as the NO bioavailability is increased with ACE-inhibitors (17). 422

However, the gradually withdrawn from medication was similar before the experimental day before 423


22

and after training, indicating that training and not the medical treatment resulted in the improved 424

endothelial function in the HF patients. The majority of the patients had only mildly reduced 425

ejection fraction and low NYHA classification and thus do not represent patients with more severe 426

HF where the neurohormonal activation is increased in parallel with severity of stage. 427

428

Conclusion 429

The ability for functional sympatholysis during KEE is closely coupled to the physical state, and 430

does not appear to be altered in mild stage HF. ATP signaling appears to be preserved in HF, but 431

the peak vasodilatory capacity of ATP is reduced. 432

433

434

435


23

Acknowledgements 436

Hanne Villumsen, Ruth Rovsing, Kamilla Munch Winding (Centre for Physical Activity Research, 437

Rigshospitalet) and Mie Rytz Hansen (Department of Cardiovascular and Renal Research, Institute 438

of Molecular Medicine, University of Southern Denmark) are acknowledged for their technical 439

assistance. 440

441

Author contributions 442

Conception and design (GWM and SPM), acquisition, analysis, or interpretation (GWM, UWI, JR, 443

BKP, SPM), drafting the manuscript (GWM and SPM), revising the manuscript critically for 444

important intellectual content (UWI, JR, BKP). 445

All authors approved the final version of the manuscript and agree to be accountable for all aspects 446

of the work in ensuring that questions related to the accuracy or integrity of any part of the work are 447

appropriately investigated and resolved. All persons designated as authors qualify for authorship, 448

and all those who qualify for authorship are listed. 449

450

Funding 451

This study was funded by the Centre for Physical Activity Research (CFAS). CFAS is supported by 452

a grant from TrygFonden. During the study period, the Centre of Inflammation and Metabolism 453

(CIM) was supported by a grant from the Danish National Research Foundation (DNRF55). 454

CIM/CFAS is a member of DD2 - the Danish Center for Strategic Research in Type 2 Diabetes (the 455

Danish Council for Strategic Research, grant no. 09-067009 and 09-075724). 456


24

Table 1. Baseline characteristics before and after six weeks of training. 457

Values are means ± SEM. P-values at baseline characteristics refer to 1-way ANOVA between 458

groups (Healthy n = 8; Heart failure n = 6). P-values within and between the 2 groups before and 459

after training (Healthy n = 8; Heart failure n = 6) refer to 2-way repeated measures ANOVA.* 460

P<0.05 different from before training within groups. #P<0.05 difference between groups. Blood 461

pressures, femoral arterial blood flow and diameter were obtained after catheterization, but before 462

the beginning of the experimental protocol. 463

Healthy individuals

Heart failure patients

n (male/female) 5/1 7/1

NYHA class II 0 8

Age 66±2 58±4

Ejection fraction (%) 59±1# 45±5

Etiology (Isch/Non-isch) 0/0 7/1

Medication

ACE-inhibitors 0 8

Diuretics 1 2

Beta-blocker 0 7

Antiplatlet drugs 0 8

Statins 0 8

Total cholesterol (mmol/l) 5.2±0.8 3.5±0.8

HDL (mmol/l) 1.54±0.3 1.25±0.3

LDL (mmol/l) 3.19±0.7 1.91±0.9

Triglycerides (mmol/l) 1.32±0.8 1.39±0.7

Before training After training Before training After training

Height (cm) 176±3 176±2 176±4 176±5

Body weight (kg) 77±5 77±5 89±6 88±6

Body Mass Index 25±2 25±2 29±2 29±2

Body fat (%) 25±4 26±5 32±3 31±3

Lean body mass (kg) 54±3 54±3 58±4 58±4

Heart rate (beats min-1) 52±2 56±3 63±3 61±3

Systolic blood pressure

(mmHg)

141±7 140±5 138±5 141±4

Diastolic blood pressure

(mmHg)

66±2 68±3 73±4 74±3

Mean arterial pressure

(mmHg)

92±5 92±3 96±5 97±4

Experimental leg mass (kg) 11.7±0.7 11.7±0.8 12.4±0.7 12.3±1.0

Leg blood flow (ml/min/kg

leg mass)

18±2 22±3 16±2 17±2

464


25

465

Table 2. Blood variables during rest and one-legged knee-extensor exercise with and without co-

infusion of tyramine

a. femoral arterial. v. femoral venous. Values are means ± SEM. P-values refer to 2-way repeated

measures ANOVA within and between the 2 groups (Healthy n = 6; Heart failure n = 6). * P<0.05

different from baseline #P<0.05 difference between groups.

Healthy

individuals

Blood variable Before training After training

Rest 10 W

10 W +

tyramine 10 W

Rest 10 W

10 W +

tyramine 10 W

PO2 (mmHg)

a

88±5 94±4 94±3 93±5

96±5 101±5 99±4 103±8

v

35±4 26±2* 24±1* 25±1*

34±3 26±1* 24±1* 26±2*

Hemoglobin

(g/dl)

A

14.1±0.5 14.3±0.5 14.3±0.4 14.4±0.5

13.8±0.3 14.1±0.3 14.1±0.3 14.1±0.3

V

13.8±0.5 14.3±0.4 14.1±0.3 14.3±0.4

13.2±0.5 14.1±0.3 13.9±0.3 14.1±0.3

O2 saturation

(%)

a

97±0 97±0 98±0 97±0

98±0 98±0 98±0 98±0

v

62±5 40±5* 34±4* 36±4*

60±6 41±2* 36±2* 40±4*

Lactate

(mmol/l)

a

1.2±0.2 2.0±0.3 2.1±0.4 2.0±0.4

0.9±0.1 1.3±0.2 1.2±0.1 1.2±0.2

v 1.4±0.2 2.7±0.5 2.4±0.5 2.7±0.8 0.9±0.1 1.6±0.4 1.6±0.4 1.5±0.2

Heart

failure

patients

Before training After training

Rest 10 W 10 W +

tyramine 10 W

Rest 10 W

10 W +

tyramine 10 W

PO2 (mmHg)

a

86±4 91±5 100±7 89±6

82±4 86±5 92±6 86±2

v

30±3 21±1*# 20±1*# 21±1*#

30±1 23±1*# 20±1*# 18±4*#

Hemoglobin

(g/dl)

a

14.1±0.5 14.2±0.5 14.4±0.5 14.3±0.6

13.5±0.4 14.6±0.2 14.4±0.2 14.6±0.7

v

13.5±0.5 14.2±0.4 14.0±0.4 14.1±0.4

13.4±0.4 13.9±0.5 14.7±0.3 13.9±0.4

O2 saturation

(%)

a

97±0 97±0 97±1 97±1

96±1 96±0 97±0 97±0

v

51±6 27±2*# 24±3*# 27±2*#

49±2 31±2*# 24±3*# 27±1*#

Lactate

(mmol/l)

a

1.2±0.1 1.7±0.3 1.6±0.4 1.6±0.4

0.8±0.1 1.5±0.3 1.6±0.4 1.2±0.1

v 1.3±0.2 2.1±0.6 2.3±0.7 1.7±0.5 0.9±0.1 1.8±0.6 2.2±0.9 1.2±0.2

466


26

Table 3. Exercise capacity before and after the training intervention 467 Values are means ± SEM. P-values refer to 2-way repeated measures ANOVA within and between 468

the 2 groups (Healthy n = 8; Heart failure n = 6). * P<0.05 different from before training within 469

groups. #P<0.05 difference between groups. 470

471

472

Table 4. Interstitial variables from the vastus lateralis during

rest and one-legged knee-extensor exercise

Values are means ± SEM. P-values refer to 1-way ANOVA within

groups (Healthy n = 6; Heart failure n = 6). * P<0.05 different from

rest. # P<0.05 different from before training

Healthy

individuals

Blood variable Before training After training

Rest 10 W

Rest 10 W

NOx (µmol/l) 18±3 27±4

11±2 14±2

6-keto PGF1α 394±176 273±93

193±96 256±145

Potassium (mM) 23.2±6.9 24.9±15.1

15.3±5.2 16.5±7.2

Lactate (mmol/l) 1.1±0.2 2.1±0.2*

0.6±0.2 1.1±0.2

Heart failure

patients

Before training After training

Rest 10 W

Rest 10 W

NOx (µmol/l) 13±3 19±5

16±4 18±5

6-keto PGF1α 191±95 157±86

157±75 125±51

Potassium (mM) 12.3±3.2 16.1±8.2

16.1±10.9 28.5±15.9

Lactate (mmol/l) 0.7±0.1 2.1±0.4*

0.9±0.2 1.4±0.3#

473

474

475

476

Healthy individuals Heart failure patients

Before training After training Before training After training

VO2peak (l/min) 2.4±0.2 2.6±0.2* 2.1±0.1 2.4±0.2*

VO2peak relative (ml/kg/min) 31±2 34±3* 25±3 27±3*

Wattpeak 2-legged cycling 158±10 178±12* 144±13 153±16

Wattpeak 1-legged knee-

extensor exercise

39±4 54±5 34±6 50±6*

6 minute walk test 663±23# 677±32 549±35 594±43*

Borg scale during training 15±1 16±0


27

Figure legends 477

Figure 1. Leg vascular conductance during arterial SNP and ACh infusion. P-values refer to 2-way 478

repeated measures ANOVA within and between the 2 groups (Healthy n = 6; Heart failure n = 6). 479

*P<0.05 different from baseline. $ P<0.05 different from before training. 480

481

Figure 2. Leg blood flow and leg vascular conductance during arterial ATP infusion. P-values refer 482

to 2-way repeated measures ANOVA within and between the 2 groups (Healthy n = 6; Heart failure 483

n = 6). *P<0.05 different between infusion rates. $ P<0.05 different from before training. # P<0.05 484

difference between groups. 485

486

Figure 3. Changes in leg blood flow and leg vascular conductance during arterial tyramine infusion 487

and during arterial co-infusion of ATP and tyramine. P-values refer to 2-way repeated measures 488

ANOVA within and between the 2 groups (Healthy n = 6; Heart failure n = 6). * P<0.05 different 489

from baseline. $ P<0.05 different from ATP infusion. # P<0.05 difference between groups. 490

491

Figure 4. Leg hemodynamics during exercise with and without arterial infusion of tyramine. P-492

values refer to 2-way repeated measures ANOVA within and between the 2 groups (Healthy n = 6; 493

Heart failure n = 6). * P<0.05 different from exercise without infusion of tyramine. $ P<0.05 494

different from before training. 495

496

Figure 5. Change in femoral venous norepinephrine during exercise with infusion of tyramine. P-497

values refer to 1-way ANOVA within groups (Healthy n = 6; Heart failure n = 6). *P<0.05 different 498

from without infusion of tyramine. 499

500

501

Figure 6. Relative changes in leg blood flow and leg vascular conductance during tyramine 502

infusion, tyramine and ATP infusion, and exercise and tyramine infusion. P-values refer to 2-way 503

repeated measures ANOVA within and between the 2 groups (Healthy n = 6; Heart failure n = 6). $ 504

P<0.05 different from before training. # P<0.05 difference between groups. 505

506

507


28

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Figure 1.

Arterial ACh infusion(100 μg/min/kg leg mass)

Leg

vasc

ular

con

duct

ance

(ml/m

in/m

mm

Hg/k

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g m

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Arterial SNP infusion(2 μg min kg leg mass)

Leg

vasc

ular

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duct

ance

(ml/m

in/m

mm

Hg/k

g le

g m

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0.2

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1.4Healthy preHealthy postHeart failure preHeart failure post

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Figure 2.

ATP infusion rate(μg/min/kg leg mass)

0 0.05 0.3 3

Leg

vasc

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(ml/m

in/m

mH

g/kg

leg

mas

s)

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2

4

6

8

Leg

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d flo

w(m

l/min

/kg

leg

mas

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0

100

200

300

400

500

600Healthy preHealthy postHeart failure preHeart failure post *

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$ #

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Figure 3. Ty

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g bl

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g m

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-5

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Healthy preHealthy postHeart failure preHeart failure post

Tyramine infusion at rest

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Tyramine infusion during ATP infusion

Leg

vasc

ular

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Figure 4. Le

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3.0Healthy preHealthy postHeart failure preHeart failure post

Leg

vasc

ular

con

duct

ance

(ml/m

in/m

mHg

)

0

5

10

15

20

25

10 Watt 10 Watt +tyramine infusion

10 Watt

Leg

(a-v

) O2 d

iffer

ence

(ml/l

)

0

20

40

60

80

100

120

140

160

180

Leg

oxyg

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l/min

)

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50

100

150

200

250

300

350

10 Watt 10 Watt +tyramine infusion

10 Watt

* *

* ** *

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Figure 5:

Tyramine infusion during exercise

Tyra

min

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duce

d ch

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infe

mor

al v

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s no

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rine

(mm

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0

1

2

3

4


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Figure 6: Le

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(% c

hang

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-80

-60

-40

-20

0


Leg

vasc

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duct

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(% c

hang

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-80

-60

-40

-20

0

Tyramine ATP+Tyramine

Exercise+Tyramine

#

$$

# $#

$

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effect of 6 weeks of high-intensity one-legged cycling on

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