Hypoxic induction of T‐type Ca2+ channels in rat cardiac myocytes: role of HIF‐1α and RhoA/ROCK signalling

Key points

• T‐type Ca²⁺ channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are downregulated as development progresses. However, these channels are re‐expressed in adult cardiomyocytes under pathological conditions.
• Hypoxia induces the upregulation of the T‐type Ca²⁺ channel Ca_v3.2 mRNA in cardiac myocytes, whereas Ca_v3.1 mRNA is not significantly altered.
• The effect of hypoxia on Ca_v3.2 mRNA requires hypoxia inducible factor‐1α (HIF‐1α) stabilization and involves the small monomeric G‐protein RhoA and its effector ROCKI.
• Our results suggest that the hypoxic regulation of the Ca_v3.2 channels may be involved in the increased probability of developing arrhythmias observed in ischemic situations, and in the pathogenesis of diseases associated with hypoxic Ca²⁺ overload.

Abstract

T‐type Ca²⁺ channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are normally downregulated as development progresses. Interestingly, however, these channels are re‐expressed in adult cardiomyocytes under pathological conditions. We investigated low voltage‐activated T‐type Ca²⁺ channel regulation in hypoxia in rat cardiomyocytes. Molecular studies revealed that hypoxia induces the upregulation of Ca_v3.2 mRNA, whereas Ca_v3.1 mRNA is not significantly altered. The effect of hypoxia on Ca_v3.2 mRNA was time‐ and dose‐dependent, and required hypoxia inducible factor‐1α (HIF‐1α) stabilization. Patch‐clamp recordings confirmed that T‐type Ca²⁺ channel currents were upregulated in hypoxic conditions, and the addition of 50 μm NiCl₂ (a T‐type channel blocker) demonstrated that the Ca_v3.2 channel is responsible for this upregulation. This increase in current density was not accompanied by significant changes in the Ca_v3.2 channel electrophysiological properties. The small monomeric G‐protein RhoA and its effector Rho‐associated kinase I (ROCKI), which are known to play important roles in cardiovascular physiology, were also upregulated in neonatal rat ventricular myocytes subjected to hypoxia. Pharmacological experiments indicated that both proteins were involved in the observed upregulation of the Ca_v3.2 channel and the stabilization of HIF‐1α that occurred in response to hypoxia. These results suggest a possible role for Ca_v3.2 channels in the increased probability of developing arrhythmias observed in ischaemic situations, and in the pathogenesis of diseases associated with hypoxic Ca²⁺ overload.

Abbreviations

Ca_V channels

voltage‐gated Ca²⁺ channels

DRB

5,6‐dichloro‐1‐β‐d‐ribofuranosylbenzimidazole

DMOG

dimethyloxalylglycine

DPI

diphenyliodonium

HIF

hypoxia inducible factor

NMDG

N‐methyl‐d‐glucamine

NRVMs

neonatal rat ventricular myocytes

P_O2

partial pressure of oxygen

ROCK

Rho‐associated kinase

ROS

reactive oxygen species

siRNA

small interfering RNA

     

What limits performance during whole‐body incremental exercise to exhaustion in humans?

Key points

• At the end of an incremental exercise to exhaustion a large functional reserve remains in the muscles to generate power, even at levels far above the power output at which task failure occurs, regardless of the inspiratory O₂ pressure during the incremental exercise.
• Exhaustion (task failure) is not due to lactate accumulation and the associated muscle acidification; neither the aerobic energy pathways nor the glycolysis are blocked at exhaustion.
• Muscle lactate accumulation may actually facilitate early recovery after exhaustive exercise even under ischaemic conditions.
• Although the maximal rate of ATP provision is markedly reduced at task failure, the resynthesis capacity remaining exceeds the rate of ATP consumption, indicating that task failure during an incremental exercise to exhaustion depends more on central than peripheral mechanisms.

Abstract

To determine the mechanisms causing task failure during incremental exercise to exhaustion (IE), sprint performance (10 s all‐out isokinetic) and muscle metabolites were measured before (control) and immediately after IE in normoxia (P_IO2: 143 mmHg) and hypoxia (P_IO2: 73 mmHg) in 22 men (22 ± 3 years). After IE, subjects recovered for either 10 or 60 s, with open circulation or bilateral leg occlusion (300 mmHg) in random order. This was followed by a 10 s sprint with open circulation. Post‐IE peak power output (W _peak) was higher than the power output reached at exhaustion during IE (P < 0.05). After 10 and 60 s recovery in normoxia, W _peak was reduced by 38 ± 9 and 22 ± 10% without occlusion, and 61 ± 8 and 47 ± 10% with occlusion (P < 0.05). Following 10 s occlusion, W _peak was 20% higher in hypoxia than normoxia (P < 0.05), despite similar muscle lactate accumulation ([La]) and phosphocreatine and ATP reduction. Sprint performance and anaerobic ATP resynthesis were greater after 60 s compared with 10 s occlusions, despite the higher [La] and [H⁺] after 60 s compared with 10 s occlusion recovery (P < 0.05). The mean rate of ATP turnover during the 60 s occlusion was 0.180 ± 0.133 mmol (kg wet wt)⁻¹ s⁻¹, i.e. equivalent to 32% of leg peak O₂ uptake (the energy expended by the ion pumps). A greater degree of recovery is achieved, however, without occlusion. In conclusion, during incremental exercise task failure is not due to metabolite accumulation or lack of energy resources. Anaerobic metabolism, despite the accumulation of lactate and H⁺, facilitates early recovery even in anoxia. This points to central mechanisms as the principal determinants of task failure both in normoxia and hypoxia, with lower peripheral contribution in hypoxia.

Abbreviations

Cr

creatine

d.w.

dry weight

F_IO2

inspired oxygen fraction

HR

heart rate

HR_peak

peak heart rate

Hyp

hypoxia

IE

incremental exercise to exhaustion

La

lactate

Mb

myoglobin

Nx

normoxia

PCr

phosphocreatine

P _{ETC O2}

end‐tidal CO₂ pressure

P _{ET O2}

end‐tidal O₂ pressure

P_IO2

partial pressure of inspired O₂

RER

respiratory exchange ratio

S_pO2

haemoglobin oxygen saturation measured by pulse‐oximetry

TOI

tissue oxygenation index

V˙CO2

CO₂ production

V˙CO2 peak

peak CO₂ production

V˙E

minute ventilation

V˙O2

O₂ consumption

V˙O2 max

maximal O₂ uptake

V˙O2 peak

peak O₂ uptake

WBIE

whole‐body incremental exercise

W_peak‐i

instantaneous peak power output

W_peak‐1

peak power output using 1 s averages

W_max

peak power output at exhaustion during the incremental exercise test

W_mean

mean power output during the 10 s sprints

w.w.

wet weight

     

Left ventricular atrioventricular plane displacement is preserved with lifelong endurance training and is the main determinant of maximal cardiac output

AbstractAge‐related decline in cardiac function can be prevented or postponed by lifelong endurance training. However, effects of normal ageing as well as of lifelong endurance exercise on longitudinal and radial contribution to stroke volume are unknown. The aim of this study was to determine resting longitudinal and radial pumping in elderly athletes, sedentary elderly and young sedentary subjects. Furthermore, we aimed to investigate determinants of maximal cardiac output in elderly. Eight elderly athletes (63 ± 4 years), seven elderly sedentary (66 ± 4 years) and ten young sedentary subjects (29 ± 4 years) underwent cardiac magnetic resonance imaging. All subjects underwent maximal exercise testing and for elderly subjects maximal cardiac output during cycling was determined using a dye dilution technique. Longitudinal and radial contribution to stroke volume did not differ between groups (longitudinal left ventricle (LV) 52–65%, P = 0.12, right ventricle (RV) 77–87%, P = 0.16, radial 7.9–8.6%, P = 1.0). Left ventricular atrioventricular plane displacement (LVAVPD) was higher in elderly athletes and young sedentary compared with elderly sedentary subjects (14 ± 3, 15 ± 2 and 11 ± 1 mm, respectively, P < 0.05). There was no difference between groups for RVAVPD (P = 0.2). LVAVPD was an independent predictor of maximal cardiac output (R ² = 0.61, P < 0.01, β = 0.78). Longitudinal and radial contributions to stroke volume did not differ between groups. However, how longitudinal pumping was achieved differed; elderly athletes and young sedentary subjects showed similar AVPD whereas this was significantly lower in elderly sedentary subjects. Elderly sedentary subjects achieved longitudinal pumping through increased short‐axis area of the ventricle. Large AVPD was a determinant of maximal cardiac output and exercise capacity.

Abbreviations

AVPD

atrioventricular plane displacement

BSA

body surface area

CO

cardiac output

EDV

end‐diastolic volumes

ESV

end‐systolic volumes

ICG

indocyanine green

LA

left atrium

LV

left ventricle

LVM

left ventricular mass

MRI

magnetic resonance imaging

RA

right atrium

RV

right ventricle

SV

stroke volume

SV_long

longitudinal contribution to stroke volume

THV

total heart volume

     

Impact of sympathetic nervous system activity on post‐exercise flow‐mediated dilatation in humans

AbstractTransient reduction in vascular function following systemic large muscle group exercise has previously been reported in humans. The mechanisms responsible are currently unknown. We hypothesised that sympathetic nervous system activation, induced by cycle ergometer exercise, would contribute to post‐exercise reductions in flow‐mediated dilatation (FMD). Ten healthy male subjects (28 ± 5 years) undertook two 30 min sessions of cycle exercise at 75% HR_max. Prior to exercise, individuals ingested either a placebo or an α₁‐adrenoreceptor blocker (prazosin; 0.05 mg kg⁻¹). Central haemodynamics, brachial artery shear rate (SR) and blood flow profiles were assessed throughout each exercise bout and in response to brachial artery FMD, measured prior to, immediately after and 60 min after exercise. Cycle exercise increased both mean and antegrade SR (P < 0.001) with retrograde SR also elevated under both conditions (P < 0.001). Pre‐exercise FMD was similar on both occasions, and was significantly reduced (27%) immediately following exercise in the placebo condition (t‐test, P = 0.03). In contrast, FMD increased (37%) immediately following exercise in the prazosin condition (t‐test, P = 0.004, interaction effect P = 0.01). Post‐exercise FMD remained different between conditions after correction for baseline diameters preceding cuff deflation and also post‐deflation SR. No differences in FMD or other variables were evident 60 min following recovery. Our results indicate that sympathetic vasoconstriction competes with endothelium‐dependent dilator activity to determine post‐exercise arterial function. These findings have implications for understanding the chronic impacts of interventions, such as exercise training, which affect both sympathetic activity and arterial shear stress.

Abbreviations

BF

blood flow

CO

cardiac output

FMD

flow‐mediated dilatation

HR

heart rate

LBNP

lower body negative pressure

MAP

mean arterial pressure

MSNA

muscle sympathetic nerve activity

SNS

sympathetic nervous system

SR

shear rate

SR_AUC

shear rate area under curve

SV

stroke volume

TPRi

total peripheral resistance index

     

Age protects from harmful effects produced by chronic intermittent hypoxia

Obstructive sleep apnoea (OSA) affects an estimated 3–7% of the adult population, the frequency doubling at ages >60–65 years. As it evolves, OSA becomes frequently associated with cardiovascular, metabolic and neuropsychiatric pathologies defining OSA syndrome (OSAS). Exposing experimental animals to chronic intermittent hypoxia (CIH) can be used as a model of the recurrent hypoxic and O₂ desaturation patterns observed in OSA patients. CIH is an important OSA event triggering associated pathologies; CIH induces carotid body (CB)‐driven exaggerated sympathetic tone and overproduction of reactive oxygen species, related to the pathogenic mechanisms of associated pathologies observed in OSAS. Aiming to discover why OSAS is clinically less conspicuous in aged patients, the present study compares CIH effects in young (3–4 months) and aged (22–24 months) rats. To define potential distinctive patterns of these pathogenic mechanisms, mean arterial blood pressure as the final CIH outcome was measured. In young rats, CIH augmented CB sensory responses to hypoxia, decreased hypoxic ventilation and augmented sympathetic activity (plasma catecholamine levels and renal artery content and synthesis rate). An increased brainstem integration of CB sensory input as a trigger of sympathetic activity is suggested. CIH also caused an oxidative status decreasing aconitase/fumarase ratio and superoxide dismutase activity. In aged animals, CIH minimally affected CB responses, ventilation and sympathetic‐related parameters leaving redox status unaltered. In young animals, CIH caused hypertension and in aged animals, whose baseline blood pressure was augmented, CIH did not augment it further. Plausible mechanisms of the differences and potential significance of these findings for the diagnosis and therapy of OSAS are discussed.

Abbreviations

24M

rats aged 22–24 months

24MCIH

rats aged 22–24 months exposed to chronic intermittent hypoxia

3M

rats aged 3–4 months

3MCIH

rats aged 3–4 months exposed to chronic intermittent hypoxia

A

adrenaline

AP

arterial pressure

CA

catecholamine

CB

carotid body

CI

confidence interval

CIH

chronic intermittent hypoxia

CRP

C‐reactive protein

CuZnSOD

cytoplasmic superoxide dismutase

DA

dopamine

E_GSH

glutathione redox potential

GPx

glutathione peroxidase

GSH

reduced glutathione

GSSG

oxidized glutathione

LPO

lipid peroxide

MnSOD

mitochondrial superoxide dismutase

MV

minute ventilation

NA

noradrenaline

NTS

nucleus of the tractus solitarius

OSA

obstructive sleep apnoea

OSAS

obstructive sleep apnoea syndrome

PaO₂

arterial oxygen pressure

RA

renal artery

ROS

reactive oxygen species

RVLM

rostroventrolateral medulla

SaO₂

arterial haemoglobin saturation

SOD

superoxide dismutase

SSA

5‐sulfosalicylic acid

TV

tidal volume

UA

upper airway

     

Limitations to oxygen transport and utilization during sprint exercise in humans: evidence for a functional reserve in muscle O2 diffusing capacity

Key points

• Severe acute hypoxia reduces sprint performance.
• Muscle V˙O2 during sprint exercise in normoxia is not limited by O₂ delivery, O₂ offloading from haemoglobin or structure‐dependent diffusion constraints in the skeletal muscle of young healthy men.
• A large functional reserve in muscle O₂ diffusing capacity exists and remains available at exhaustion during exercise in normoxia; this functional reserve is recruited during exercise in hypoxia.
• During whole‐body incremental exercise to exhaustion in severe hypoxia, leg V˙O2 is primarily dependent on convective O₂ delivery and less limited by diffusion constraints than previously thought.
• The kinetics of O₂ offloading from haemoglobin does not limit V˙O2 peak in hypoxia.
• Our results indicate that the limitation to V˙O2 during short sprints resides in mechanisms regulating mitochondrial respiration.

Abstract

To determine the contribution of convective and diffusive limitations to V˙O2 peak during exercise in humans, oxygen transport and haemodynamics were measured in 11 men (22 ± 2 years) during incremental (IE) and 30 s all‐out cycling sprints (Wingate test, WgT), in normoxia (Nx, P_IO2: 143 mmHg) and hypoxia (Hyp, P_IO2: 73 mmHg). Carboxyhaemoglobin (COHb) was increased to 6–7% before both WgTs to left‐shift the oxyhaemoglobin dissociation curve. Leg V˙O2 was measured by the Fick method and leg blood flow (BF) with thermodilution, and muscle O₂ diffusing capacity (D_MO2) was calculated. In the WgT mean power output, leg BF, leg O₂ delivery and leg V˙O2 were 7, 5, 28 and 23% lower in Hyp than Nx (P < 0.05); however, peak WgT D_MO2 was higher in Hyp (51.5 ± 9.7) than Nx (20.5 ± 3.0 ml min⁻¹ mmHg⁻¹, P < 0.05). Despite a similar P_aO2 (33.3 ± 2.4 and 34.1 ± 3.3 mmHg), mean capillary P_O2 (16.7 ± 1.2 and 17.1 ± 1.6 mmHg), and peak perfusion during IE and WgT in Hyp, D_MO2 and leg V˙O2 were 12 and 14% higher, respectively, during WgT than IE in Hyp (both P < 0.05). D_MO2 was insensitive to COHb (COHb: 0.7 vs. 7%, in IE Hyp and WgT Hyp). At exhaustion, the Y equilibration index was well above 1.0 in both conditions, reflecting greater convective than diffusive limitation to the O₂ transfer in both Nx and Hyp. In conclusion, muscle V˙O2 during sprint exercise is not limited by O₂ delivery, O₂ offloading from haemoglobin or structure‐dependent diffusion constraints in the skeletal muscle. These findings reveal a remarkable functional reserve in muscle O₂ diffusing capacity.

Abbreviations

a‐vO₂diff

arteriovenous oxygen concentration difference

BF

blood flow

C_aO2

arterial content of oxygen

CO

carbon monoxide

COHb

carboxyhaemoglobin

D_LO2

lung O₂ diffusing capacity

D_MO2

muscle O₂ diffusing capacity

D_O2

O₂ diffusing capacity

ECG

electrocardiogram

F_IO2

inspired oxygen fraction

FV

femoral vein

HR_max

maximal heart rate

HR_peak

peak heart rate during Wingate

Hyp

hypoxia

LBF

leg blood flow

Nx

normoxia

S_O2

haemoglobin saturation with O₂

ODC

oxyhaemoglobin dissociation curve

P₅₀

partial oxygen pressure at 50% S_O2

P_aO2

arterial oxygen pressure

P_CO2

carbon dioxide pressure

P_O2

oxygen pressure

P_{O2 cap}

capillary O₂ pressure

P_{O2 mit}

mitochondrial O₂ pressure

P _{FV O2}

femoral vein P_O2

P_IO2

inspiratory O₂ pressure

V˙CO2

carbon dioxide production

V˙CO2 peak

peak carbon dioxide production

V˙ Epeak

peak pulmonary ventilation

V˙O2

oxygen consumption

V˙O2 max

maximal oxygen consumption

V˙O2 peak

peak oxygen uptake

W_peak‐i

instantaneous peak power output

W_mean‐10

mean power output during the first 10 s of the sprint exercise

W_mean‐30

mean power output during the whole sprint exercise

WgT

isokinetic 30 s Wingate test

     

Statin myalgia is not associated with reduced muscle strength,mass or protein turnover in older male volunteers,but is allied with a slowing of time to peak power output,insulin resistance and differential muscle mRNA expression

Key points

• Statins cause muscle‐specific side effects, most commonly muscle aches/weakness (myalgia), particularly in older people. Furthermore, evidence has linked statin use to increased risk of type 2 diabetes. However, the mechanisms involved are unknown.
• This is the first study to measure muscle protein turnover rates and insulin sensitivity in statin myalgic volunteers and age‐matched, non‐statin users under controlled fasting and fed conditions using gold standard methods.
• We demonstrate in older people that chronic statin myalgia is not associated with deficits in muscle strength and lean mass or the dysregulation of muscle protein turnover compared to non‐statin users. Furthermore, there were no between‐group differences in blood or muscle inflammatory markers.
• Statin users did, however, show blunting of muscle power output at the onset of dynamic exercise, increased abdominal adiposity, whole body and leg insulin resistance, and clear differential expression of muscle genes linked to mitochondrial dysfunction and apoptosis, which warrant further investigation.

Abstract

Statins are associated with muscle myalgia and myopathy, which probably reduce habitual physical activity. This is particularly relevant to older people who are less active, sarcopaenic and at increased risk of statin myalgia. We hypothesised that statin myalgia would be allied to impaired strength and work capacity in older people, and determined whether differences aligned with divergences in lean mass, protein turnover, insulin sensitivity and the molecular regulation of these processes. Knee extensor strength and work output during 30 maximal isokinetic contractions were assessed in healthy male volunteers, nine with no statin use (control 70.4 ± 0.7 years) and nine with statin myalgia (71.5 ± 0.9 years). Whole body and leg glucose disposal, muscle myofibrillar protein synthesis (MPS) and leg protein breakdown (LPB) were measured during fasting (≈5 mU l⁻¹ insulin) and fed (≈40 mU l⁻¹ insulin + hyperaminoacidaemia) euglyceamic clamps. Muscle biopsies were taken before and after each clamp. Lean mass, MPS, LPB and strength were not different but work output during the initial three isokinetic contractions was 19% lower (P < 0.05) in statin myalgic subjects due to a delay in time to reach peak power output. Statin myalgic subjects had reduced whole body (P = 0.05) and leg (P < 0.01) glucose disposal, greater abdominal adiposity (P < 0.05) and differential expression of 33 muscle mRNAs (5% false discovery rate (FDR)), six of which, linked to mitochondrial dysfunction and apoptosis, increased at 1% FDR. Statin myalgia was associated with impaired muscle function, increased abdominal adiposity, whole body and leg insulin resistance, and evidence of mitochondrial dysfunction and apoptosis.

Abbreviations

AA

amino acid

AV

arterialised venous

CHO

carbohydrate

CK

creatine kinase

FDR

false discovery rate

GATM

glycine amidinotransferase

GLM

general linear model

HMBS

hydroxymethylbilane synthase

MPS

muscle protein synthesis

LPB

leg protein breakdown

PCr

phosphocreatine

PDC

pyruvate dehydrogenase complex

PDK

pyruvate dehydrogenase kinase

SAM

significance analysis of microarrays

     

Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation

Key points

• Endothelial function in resistance vessels entails Ca²⁺ and electrical signalling to promote vasodilatation and increase tissue blood flow. Whether membrane potential (V _m) governs intracellular calcium concentration ([Ca²⁺]_i) of the endothelium remains controversial.
• [Ca²⁺]_i and V _m were evaluated simultaneously during intracellular current injection using intact endothelial tubes freshly isolated from mouse skeletal muscle resistance arteries.
• [Ca²⁺]_i did not change during hyperpolarization or depolarization under resting conditions. However in the presence of 100 nM ACh (∼EC₅₀), [Ca²⁺]_i increased during hyperpolarization and decreased during depolarization. These responses required extracellular Ca²⁺ and were attenuated by half with genetic ablation of TRPV4 channels.
• In native microvascular endothelium, half‐maximal stimulation of muscarinic receptors enables V _m to govern [Ca²⁺]_i by activating Ca²⁺‐permeable channels in the plasma membrane. This effect of V _m is absent at rest and can be masked during maximal receptor stimulation.

Abstract

In resistance arteries, coupling a rise of intracellular calcium concentration ([Ca²⁺]_i) to endothelial cell hyperpolarization underlies smooth muscle cell relaxation and vasodilatation, thereby increasing tissue blood flow and oxygen delivery. A controversy persists as to whether changes in membrane potential (V _m) alter endothelial cell [Ca²⁺]_i. We tested the hypothesis that V _m governs [Ca²⁺]_i in endothelium of resistance arteries by performing Fura‐2 photometry while recording and controlling V _m of intact endothelial tubes freshly isolated from superior epigastric arteries of C57BL/6 mice. Under resting conditions, [Ca²⁺]_i did not change when V _m shifted from baseline (∼−40 mV) via exposure to 10 μM NS309 (hyperpolarization to ∼−80 mV), via equilibration with 145 mm [K⁺]_o (depolarization to ∼−5 mV), or during intracellular current injection (±0.5 to 5 nA, 20 s pulses) while V _m changed linearly between ∼−80 mV and +10 mV. In contrast, during the plateau (i.e. Ca²⁺ influx) phase of the [Ca²⁺]_i response to approximately half‐maximal stimulation with 100 nm ACh (∼EC₅₀), [Ca²⁺]_i increased as V _m hyperpolarized below −40 mV and decreased as V _m depolarized above −40 mV. The magnitude of [Ca²⁺]_i reduction during depolarizing current injections correlated with the amplitude of the plateau [Ca²⁺]_i response to ACh. The effect of hyperpolarization on [Ca²⁺]_i was abolished following removal of extracellular Ca²⁺, was enhanced subtly by raising extracellular [Ca²⁺] from 2 mm to 10 mm and was reduced by half in endothelium of TRPV4^−/− mice. Thus, during submaximal activation of muscarinic receptors, V _m can modulate Ca²⁺ entry through the plasma membrane in accord with the electrochemical driving force.

Abbreviations

ACh

acetylcholine

BK_Ca

large‐conductance Ca²⁺‐activated K⁺ channel

[Ca²⁺]_i

intracellular Ca²⁺ concentration

[Ca²⁺]_o

extracellular Ca²⁺ concentration

EC

endothelial cell

EC₅₀

drug concentration giving half‐maximal response

E_K

Nernst equilibrium potential for K⁺

ER

endoplasmic reticulum

FCCP

carbonyl cyanide p‐trifluoromethoxyphenylhydrazone

GSK101

GSK1016790A

GSK219

GSK2193874

ID

internal diameter

[K⁺]_o

extracellular K⁺ concentration

NO

nitric oxide

OD

outer diameter

PSS

physiological salt solution

SEA

superior epigastric artery

SK_Ca/IK_Ca

small‐ and intermediate‐conductance Ca²⁺‐activated K⁺ channels

SMC

smooth muscle cell

TRP

transient receptor potential

TRPV4

transient receptor potential vanilloid type 4 channel

TRPV4^−/−

TRPV4 knockout

V_m

membrane potential

     

Pre‐emptive analgesia and its supraspinal mechanisms: enhanced descending inhibition and decreased descending facilitation by dexmedetomidine

Key points

• Despite the clinical importance of pre‐emptive analgesia, the mechanisms by which it attenuates pain associated with central sensitization are poorly understood.
• We find that fentanyl and the α2‐adrenoceptor agonist dexmedetomidine (Dex) differ significantly in their modulatory actions on noxious mechanical and noxious heat‐evoked nociception in vivo.
• Unlike fentanyl, Dex modified descending control of nociception by decreasing the threshold for descending inhibition and/or increasing the threshold for descending facilitation.
• Dex exhibited after‐actions on activities of thalamus in prolongation of noxious heat‐evoked paw withdrawal latency that persisted for at least 7 days.
• This study provides insight into the organization of thalamic modulation in pre‐emptive analgesia.

Abstract

We investigated and compared the antinociceptive effects of intraperitoneal administration of fentanyl (2–60 μg kg⁻¹) and dexmedetomidine (Dex, 1–10 μg kg⁻¹; a highly selective α2‐adrenoceptor agonist) in the regulation of nociception assessed by measuring noxious paw withdrawal reflexes in rats. Fentanyl elevated noxious mechanical paw withdrawal threshold and prolonged paw withdrawal heat latency within 1–1.5 h (P < 0.05). Dex failed to affect the mechanical paw withdrawal threshold, yet significantly prolonged the paw withdrawal heat latency in a bi‐phasic manner; a short transient 1–1.5 h period followed by a second, slowly developing increase in latency that persisted for at least 7 days (P < 0.05). Lesion of the dorsolateral funiculus (DLF) did not influence fentanyl‐induced antinociceptive effects, indicating peripheral and spinal antinociceptive mechanisms. By contrast, the Dex‐induced second, but not the first, phase of the prolonged paw withdrawal heat latency was significantly blocked by the lesion of either DLF or thalamic ventromedial (VM) nuclei, and was attenuated by intracerebral administration of either atipamezole (α2‐adrenoceptor antagonist) or WAY‐100635 (5‐HT_1A receptor antagonist) into the VM nuclei (P < 0.05). Upon intramuscular 5.8% saline‐induced muscle nociception, pre‐emptive injection of fentanyl enhanced mechanical hyperalgesia and blocked heat hypoalgesia, whereas Dex significantly prevented the occurrence of mechanical hyperalgesia and enhanced heat hypoalgesia. It is suggested that Dex, but not fentanyl, significantly enhances descending inhibition and/or decreases descending facilitation to modulate pain and nociception. The present study provides novel insight into thalamus‐mediated mechanisms in pre‐emptive analgesia.

Abbreviations

Dex

dexmedetomidine

GS

gastrocnemius

i.c.

intracerebal

DLF

dorsolateral funiculus

MD

mediodorsal

VM

ventromedial

     

Higher oesophageal temperature at rest and during exercise in humans with patent foramen ovale

Key points

• Patent foramen ovale (PFO) is present in ∼35% of the general population.
• The respiratory system participates in thermoregulation via evaporative and convective heat loss so blood flow that bypasses the respiratory system, e.g. through a PFO, may not participate in respiratory system cooling.
• We found that subjects with a PFO (PFO+) had a ∼0.4°C higher oesophageal temperature (T _oesoph) than subjects without a PFO (PFO−) during pre‐exercise and exercise.
• T _oesoph in PFO+ subjects was associated with the estimated size of the PFO whereby subjects with a large PFO had a greater T _oesoph than PFO− subjects and subjects with a small PFO.
• During high intensity exercise breathing cold and dry air, PFO+ subjects achieved a higher T _oesoph than PFO– subjects.
• Absence of respiratory system cooling of shunted blood partially explains the differences in T _oesoph between PFO+ and PFO– subjects; other differences in thermoregulatory responses that impact core temperature also likely exist.

Abstract

Respiratory system cooling occurs via convective and evaporative heat loss, so right‐to‐left shunted blood flow through a patent foramen ovale (PFO) would not be cooled. Accordingly, we hypothesized that PFO+ subjects would have a higher core temperature than PFO– subjects due, in part, to absence of respiratory system cooling of the shunted blood and that this effect would be dependent upon the estimated PFO size and inspired air temperature. Subjects were screened for the presence and size of a PFO using saline contrast echocardiography. Thirty well‐matched males (15 PFO−, 8 large PFO+, 7 small PFO+) completed cycle ergometer exercise trials on three separate days. During Trial 1, subjects completed a V˙O2 max test. For Trials 2 and 3, randomized, subjects completed four 2.5 min stages at 25, 50, 75 and 90% of the maximum workload achieved during Trial 1, breathing either ambient air (20.6 ± 1.0°C) or cold air (1.9 ± 3.5°C). PFO+ subjects had a higher oesophageal temperature (T _oesoph) (P < 0.05) than PFO− subjects on Trial 1. During exercise breathing cold and dry air, PFO+ subjects achieved a higher T _oesoph than PFO− subjects (P < 0.05). Subjects with a large PFO, but not those with a small PFO, had a higher T _oesoph than PFO− subjects (P < 0.05) during Trial 1 and increased T _oesoph breathing cold and dry air. These data suggest that the presence and size of a PFO are associated with T _oesoph in healthy humans but this is explained only partially by absence of respiratory system cooling of shunted blood.

Abbreviations

D_LCO

lung diffusion capacity for carbon monoxide

FEF_25–75

forced mid‐expiratory flow

FEV₁

forced expiratory volume in 1 s

FVC

forced vital capacity

HR

heart rate

PFO

patent foramen ovale

PFO+

subjects with a PFO

PFO−

subjects without a PFO

Q˙C

cardiac output

RER

respiratory exchange ratio

RPE_{leg discomfort}

rate of perceived exertion for legs

RPE _dyspnoea

rate of perceived exertion for lungs

S_aO2

arterial saturation of oxygen

S_pO2

peripheral arterial oxygen saturation

T_air

ambient air temperature

T_core

core temperature

T_oesoph

oesophageal temperature

T_exp

expired air temperature

T_insp

inspired air temperature

TLC

total lung capacity

V˙CO2

carbon dioxide elimination

V˙E

minute ventilation

V˙E/V˙O2

ventilatory equivalent of oxygen

V˙O2

oxygen uptake

V_t

tidal volume

     

Caveolae regulate vasoconstriction of conduit arteries to angiotensin II in hindlimb unweighted rats

Key points

• Exposure to microgravity induces postflight orthostatic intolerance on re‐exposure to 1 G gravity which is second to the structural and functional remodelling of arteries.
• We found the maximal developed force (E _max) of angiotensin II‐elicited vasoconstriction was decreased in abdominal aorta, unchanged in thoracic aorta and increased in carotid artery by simulated weightlessness. However, the sensitivity of the response (EC₅₀) was decreased in all of the arteries as was the desensitization of angiotensin II type I receptor (AT₁) upon angiotensin II stimulation.
• We demonstrate that caveolae on vascular smooth muscle cells play a key role in the adaptation of EC₅₀ and AT₁ desensitization, but not E _max of the response to simulated weightlessness.
• This study gives insight into the mechanism underlying the arterial functional remodelling during weightlessness. Further, the findings might stimulate new ideas for research into countermeasures to postflight orthostatic intolerance upon astronauts returning to the earth.

Abstract

Weightlessness induces the functional remodelling of arteries, but the changes to angiotensin II (Ang II)‐elicited vasoconstriction and the underlying mechanism have never been reported. Caveolae are invaginations of the cell membrane crucial for the contraction of vascular smooth muscle cells, so we investigated the adaptation of Ang II‐elicited vasoconstriction to simulated weightlessness and the role of caveolae in it. The 4 week hindlimb unweighted (HU) rat was used to simulate the effects of weightlessness. Ang II‐elicited vasoconstriction was measured by isometric force recording. The morphology of caveolae was examined by transmission electron microscope. The binding of the angiotensin II type 1 receptor (AT₁) and caveolin‐1 (cav‐1) was examined by coimmunoprecipitation and Western blot. We found that the maximal developing force (E _max) of Ang II‐elicited vasoconstriction was decreased in abdominal aorta by 30.6%, unchanged in thoracic aorta and increased in carotid artery by 17.9% after HU, while EC₅₀ of the response was increased in all three arteries (P < 0.05). AT₁ desensitization upon activation was significantly reduced by HU in all three arteries, as was the number of caveolae (P < 0.05). Furthermore, Ang II promoted the binding of AT₁ and cav‐1 significantly in control but not HU arteries. Both the number of caveolae and the binding of AT₁ and cav‐1 in HU arteries were restored by cholesterol pretreatment which also reinstated the change in EC₅₀ as well as the level of AT₁ desensitization. These results indicate that modified caveolae in vascular smooth muscle cells could interfere with the binding of AT₁ and cav‐1 mediating the adaptation of Ang II‐elicited vasoconstriction to HU.

Abbreviations

AA

abdominal aorta

Ang II

angiotensin II

AT₁

angiotensin II type 1 receptor

CA

carotid artery

cav‐1

caveolin‐1

co‐IP

coimmunoprecipitation

CON

control

E_max

maximal effect

HU

hindlimb unweighted

TA

thoracic aorta

TEM

transmission electron microscope

VSMCs

vascular smooth muscle cells

     

Early redox imbalance is associated with liver dysfunction at weaning in overfed rats

Key points

• Childhood obesity is associated with precocious oxidative stress, which can contribute to future diseases.
• Early overfed rats have increased oxidative stress in liver and plasma at weaning.
• The model of litter size reduction causes adipocyte hypertrophy and lower PPARγ at weaning, suggesting a decrease in adipocyte proliferation.

Abstract

Neonatal overfeeding induced by litter size reduction leads to further obesity and other metabolic disorders, such as liver oxidative stress and microsteatosis at adulthood. We hypothesized that overfeeding causes an early redox imbalance at weaning, which could programme the animals to future liver dysfunction. Thus, we studied lipogenesis, adipogenesis, catecholamine status and oxidative balance in weaned overfed pups. To induce early overfeeding, litters were adjusted to three pups at the 3rd day of lactation (SL group). The control group contained 10 pups per litter until weaning (NL group). Peripheral autonomic nerve function was determined in vivo at 21 days old. Thereafter, pups were killed for further analysis. Differences were considered significant when P < 0.05. The SL pups presented with a higher visceral adipocyte area, higher content of lipogenic enzymes (ACC, FAS) and with a lower content of adipogenic factors (CEBP, PPARγ) in visceral adipose tissue (VAT). Although autonomic nerve activity and adrenal catecholamine production were not significantly altered, catecholamine receptor (β3ADR) content was lower in VAT. The SL pups also presented with higher triglyceride, PPARγ, PPARα and PGC1α contents in liver. In plasma and liver, the SL pups showed an oxidative imbalance, with higher lipid peroxidation and protein oxidation. The SL group presented with a higher serum alanine aminotransferase (ALT). The early increase in lipogenesis in adipose tissue and liver in weaned overfed rats suggests that the higher oxidative stress and lower catecholamine content in VAT are associated with the early development of liver dysfunction and adipocyte hypertrophy.

Abbreviations

ACC

acetyl‐CoA carboxylase

β2ADR

β2 adrenergic receptor

β3ADR

β3 adrenergic receptor

ALT

alanine aminotransferase

AST

aspartate aminotransferase

CAT

catalase

C/EBPs

CCAAT‐enhancer‐binding proteins

FAS

fatty acid synthase

GPx

glutathione peroxidase

MDA

malondialdehyde

NL

normal litter

PGC1α

peroxisome proliferator‐activated receptor‐gamma coactivator 1 α

PPARγ

nuclear receptor peroxisome proliferator‐activated receptor γ

ROS

reactive oxygen species

SL

small litter

SOD

superoxide dismutase

TG

triglyceride

TH

tyrosine hydroxylase

VAT

visceral adipose tissue

     

In vivo physiological recording from the lateral line of juvenile zebrafish

AbstractHair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post‐fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l⁻¹ was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l⁻¹ did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS‐222, which reduces the size of basolateral membrane K⁺ currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More generally, this method would allow functional studies involving live imaging and electrophysiology from juvenile and adult zebrafish.

Abbreviations

dpf

days post‐fertilization

MET

mechanoelectrical transducer

OHC

outer hair cell

PLLg

posterior lateral line ganglion

wpf

weeks post fertilization

     

Shear stress induces a longitudinal Ca2+ wave via autocrine activation of P2Y1 purinergic signalling in rat atrial myocytes

AbstractAtrial myocytes are exposed to shear stress during the cardiac cycle and haemodynamic disturbance. In response, they generate a longitudinally propagating global Ca²⁺ wave. Here, we investigated the cellular mechanisms underlying the shear stress‐mediated Ca²⁺ wave, using two‐dimensional confocal Ca²⁺ imaging combined with a pressurized microflow system in single rat atrial myocytes. Shear stress of ∼16 dyn cm⁻² for 8 s induced ∼1.2 aperiodic longitudinal Ca²⁺ waves (∼79 μm s⁻¹) with a delay of 0.2−3 s. Pharmacological blockade of ryanodine receptors (RyRs) or inositol 1,4,5‐trisphosphate receptors (IP₃Rs) abolished shear stress‐induced Ca²⁺ wave generation. Furthermore, in atrial myocytes from type 2 IP₃R (IP₃R2) knock‐out mice, shear stress failed to induce longitudinal Ca²⁺ waves. The phospholipase C (PLC) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, but not its inactive analogue {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343, abolished the shear‐induced longitudinal Ca²⁺ wave. However, pretreating atrial cells with blockers for stretch‐activated channels, Na⁺−Ca²⁺ exchanger, transient receptor potential melastatin subfamily 4, or nicotinamide adenine dinucleotide phosphate oxidase did not suppress wave generation under shear stress. The P2 purinoceptor inhibitor suramin, and the potent P2Y₁ receptor antagonist MRS 2179, both suppressed the Ca²⁺ wave, whereas the P2X receptor antagonist, iso‐PPADS, did not alter it. Suppression of gap junction hemichannels permeable to ATP or extracellular application of ATP‐metabolizing apyrase inhibited the wave. Removal of external Ca²⁺ to enhance hemichannel opening facilitated the wave generation. Our data suggest that longitudinally propagating, regenerative Ca²⁺ release through RyRs is triggered by P2Y₁–PLC–IP₃R2 signalling that is activated by gap junction hemichannel‐mediated ATP release in atrial myocytes under shear stress.

Abbreviations

9‐AC

9‐anthracenecarboxylic acid

2‐APB

2‐aminoethoxydiphenyl borate

CICR

Ca²⁺‐induced Ca²⁺ release

DPI

diphenyleneiodonium

FDHM

full duration at half‐maximum

IP₃R2

type 2 inositol 1,4,5‐trisphosphate receptor

KO

knock‐out

NCX

Na⁺−Ca²⁺ exchanger

NOX

nicotinamide adenine dinucleotide phosphate oxidase

PLC

phospholipase C

ROI

region‐of‐interest

RyR

ryanodine receptor

SAC

stretch‐activated channel

SR

sarcoplasmic reticulum

T_p

time‐to‐peak

TRPM4

transient receptor potential melastatin subfamily 4

V_p

propagation velocity

WT

wild‐type

     

The exercise‐induced biochemical milieu enhances collagen content and tensile strength of engineered ligaments

Key points

• Exercise acutely increases the concentrations of metabolites and hormones such as growth hormone (GH) and, to a lesser extent, insulin‐like growth factor 1 (IGF‐1); however, the biological function of this response is unclear.
• Pharmacological administration of these hormones stimulates collagen synthesis in muscle and tendon; however, whether the post‐exercise biochemical milieu has a similar action is unknown.
• Treating engineered ligaments with serum obtained from young healthy men after exercise resulted in more collagen and improved tensile strength over those treated with serum from resting men.
• Further, we show that the increase in collagen induced by post‐exercise serum (i) is not reproduced by treatment with recombinant GH or IGF‐1, and (ii) is associated with the activation of PI3 kinase/mTORC1 and ERK1/2 signalling.

Abstract

Exercise stimulates a dramatic change in the concentration of circulating hormones, such as growth hormone (GH), but the biological functions of this response are unclear. Pharmacological GH administration stimulates collagen synthesis; however, whether the post‐exercise systemic milieu has a similar action is unknown. We aimed to determine whether the collagen content and tensile strength of tissue‐engineered ligaments is enhanced by serum obtained post‐exercise. Primary cells from a human anterior cruciate ligament (ACL) were used to engineer ligament constructs in vitro. Blood obtained from 12 healthy young men 15 min after resistance exercise contained GH concentrations that were ∼7‐fold greater than resting serum (P < 0.001), whereas IGF‐1 was not elevated at this time point (P = 0.21 vs. rest). Ligament constructs were treated for 7 days with medium supplemented with serum obtained at rest (RestTx) or 15 min post‐exercise (ExTx), before tensile testing and collagen content analysis. Compared with RestTx, ExTx enhanced collagen content (+19%; 181 ± 33 vs. 215 ± 40 μg per construct P = 0.001) and ligament mechanical properties – maximal tensile load (+17%, P = 0.03 vs. RestTx) and ultimate tensile strength (+10%, P = 0.15 vs. RestTx). In a separate set of engineered ligaments, recombinant IGF‐1, but not GH, enhanced collagen content and mechanics. Bioassays in 2D culture revealed that acute treatment with post‐exercise serum activated mTORC1 and ERK1/2. In conclusion, the post‐exercise biochemical milieu, but not recombinant GH, enhances collagen content and tensile strength of engineered ligaments, in association with mTORC1 and ERK1/2 activation.

Abbreviations

ACL

anterior cruciate ligament

ERK

extracellular signal‐regulated kinase

GH

growth hormone

IGF‐1

insulin‐like growth factor 1

mTORC1

mechanistic/mammalian target of rapamycin complex 1

TGF‐β1

transforming growth factor‐β1

     

Perivascular tissue inhibits rho‐kinase‐dependent smooth muscle Ca2+ sensitivity and endothelium‐dependent H2S signalling in rat coronary arteries

Key points

• Local regulation of vascular resistance adjusts coronary blood flow to metabolic demand, although the mechanisms involved are not comprehensively understood
• We show that heart tissue surrounding rat coronary arteries releases diffusible factors that regulate vasoconstriction and relaxation
• Perivascular tissue reduces rho‐kinase‐dependent smooth muscle Ca²⁺ sensitivity and constriction of coronary arteries to serotonin, the thromboxane analogue U46619 and the α₁‐adrenergic agonist phenylephrine
• Endothelium‐dependent relaxation of coronary arteries in response to cholinergic stimulation is inhibited by perivascular tissue as a result of reduced endothelial Ca²⁺ responses and attenuated H₂S‐dependent signalling
• These results establish cellular mechanisms by which perivascular heart tissue can modify local vascular tone and coronary blood flow

Abstract

Interactions between perivascular tissue (PVT) and the vascular wall modify artery tone and contribute to local blood flow regulation. Using isometric myography, fluorescence microscopy, membrane potential recordings and phosphospecific immunoblotting, we investigated the cellular mechanisms by which PVT affects constriction and relaxation of rat coronary septal arteries. PVT inhibited vasoconstriction to thromboxane, serotonin and α₁‐adrenergic stimulation but not to depolarization with elevated extracellular [K⁺]. When PVT was wrapped around isolated arteries or placed at the bottom of the myograph chamber, a smaller yet significant inhibition of vasoconstriction was observed. Resting membrane potential, depolarization to serotonin or thromboxane stimulation, and resting and serotonin‐stimulated vascular smooth muscle [Ca²⁺]‐levels were unaffected by PVT. Serotonin‐induced vasoconstriction was almost abolished by rho‐kinase inhibitor Y‐27632 and modestly reduced by protein kinase C inhibitor bisindolylmaleimide X. PVT reduced phosphorylation of myosin phosphatase targeting subunit (MYPT) at Thr850 by ∼40% in serotonin‐stimulated arteries but had no effect on MYPT‐phosphorylation in arteries depolarized with elevated extracellular [K⁺]. The net anti‐contractile effect of PVT was accentuated after endothelial denudation. PVT also impaired vasorelaxation and endothelial Ca²⁺ responses to cholinergic stimulation. Methacholine‐induced vasorelaxation was mediated by NO and H₂S, and particularly the H₂S‐dependent (dl‐propargylglycine‐ and XE991‐sensitive) component was attenuated by PVT. Vasorelaxation to NO‐ and H₂S‐donors was maintained in arteries with PVT. In conclusion, cardiomyocyte‐rich PVT surrounding coronary arteries releases diffusible factors that reduce rho‐kinase‐dependent smooth muscle Ca²⁺ sensitivity and endothelial Ca²⁺ responses. These mechanisms inhibit agonist‐induced vasoconstriction and endothelium‐dependent vasorelaxation and suggest new signalling pathways for metabolic regulation of blood flow.

Abbreviations

8‐SPT

8‐(p‐sulphophenyl)theophylline

ACh

acetylcholine

AUC

area under the curve

Bis‐10

bisindolylmaleimide X

CSE

cystathionine γ‐lyase

EC

endothelial cell

K‐PSS

physiological saline solution with elevated [K⁺]

l‐NAME

N‐nitro‐l‐arginine methyl ester

MYPT

myosin phosphatase targeting subunit

PKC

protein kinase C

PPG

dl‐propargylglycine

PSS

physiological saline solution

PVT

perivascular tissue

SNAP

S‐nitroso‐N‐acetyl‐d,l‐penicillamine

SNP

sodium nitroprusside

VSMC

vascular smooth muscle cell

     

Potassium inhibits nitric oxide and adenosine arteriolar vasodilatation via KIR and Na+/K+ATPase: implications for redundancy in active hyperaemia

AbstractRedundancy, in active hyperaemia, where one vasodilator can compensate for another if the first is missing, would require that one vasodilator inhibits the effects of another; therefore, if the first vasodilator is inhibited, its inhibitory influence on the second vasodilator is removed and the second vasodilator exerts a greater vasodilatory effect. We aimed to determine whether vasodilators relevant to skeletal muscle contraction [potassium chloride (KCl), adenosine (ADO) and nitric oxide] inhibit one another and, in addition, to investigate the mechanisms for this interaction. We used the hamster cremaster muscle and intravital microscopy to directly visualize 2A arterioles when exposed to a range of concentrations of one vasodilator [10⁻⁸ to 10⁻⁵  m S‐nitroso‐N‐acetyl penicillamine (SNAP), 10⁻⁸ to 10⁻⁵  m ADO, 10 and 20 mm KCl] in the absence and then in the presence of a second vasodilator (10⁻⁷  m ADO, 10⁻⁷  m SNAP, 10 mm KCl). We found that KCl significantly attenuated SNAP‐induced vasodilatations by ∼65.8% and vasodilatations induced by 10⁻⁸ to 10⁻⁶  m ADO by ∼72.8%. Furthermore, we observed that inhibition of KCl vasodilatation, by antagonizing either Na⁺/K⁺ ATPase using ouabain or inward rectifying potassium channels using barium chloride, could restore the SNAP‐induced vasodilatation by up to ∼53.9% and 30.6%, respectively, and also restore the ADO‐induced vasodilatations by up to ∼107% and 76.7%, respectively. Our data show that vasodilators relevant to muscle contraction can interact in a way that alters the effectiveness of other vasodilators. These data suggest that active hyperaemia may be the result of complex interactions between multiple vasodilators via a redundant control paradigm.

Abbreviations

ADO

adenosine

BaCl

barium chloride

K_IR

inward rectifying potassium channels

NO

nitric oxide

Oua

ouabain

PKA

protein kinase A

PSS

physiological salt solution

SNAP

S‐nitroso‐N‐acetyl penicillamine

VSM

vascular smooth muscle

     

Estimating reflex responses in large populations of motor units by decomposition of the high‐density surface electromyogram

Key points

• Reflex responses of single motor units have been used for the study of spinal circuitries but the methods employed are invasive and limited to the assessment of a relatively small number of motor units.
• We propose a new approach to investigate reflexes on individual motor units based on high‐density surface electromyography (HDsEMG) decomposition.
• The decomposition of HDsEMG has been previously validated in voluntary isometric contractions but never during reflex activities.
• The use of HDsEMG decomposition for reflex studies at the individual motor unit level, during constant force contractions, with excitatory and inhibitory stimuli, was validated here by the comparison of results with concurrently recorded intramuscular EMG signals.
• The validation results showed that HDsEMG decomposition allows an accurate quantification of reflex responses for a large number of individual motor units non‐invasively, for both excitatory and inhibitory stimuli.

Abstract

We propose and validate a non‐invasive method that enables accurate detection of the discharge times of a relatively large number of motor units during excitatory and inhibitory reflex stimulations. High‐density surface electromyography (HDsEMG) and intramuscular EMG (iEMG) were recorded from the tibialis anterior muscle during ankle dorsiflexions performed at 5%, 10% and 20% of the maximum voluntary contraction (MVC) force, in nine healthy subjects. The tibial nerve (inhibitory reflex) and the peroneal nerve (excitatory reflex) were stimulated with constant current stimuli. In total, 416 motor units were identified from the automatic decomposition of the HDsEMG. The iEMG was decomposed using a state‐of‐the‐art decomposition tool and provided 84 motor units (average of two recording sites). The reflex responses of the detected motor units were analysed using the peri‐stimulus time histogram (PSTH) and the peri‐stimulus frequencygram (PSF). The reflex responses of the common motor units identified concurrently from the HDsEMG and the iEMG signals showed an average disagreement (the difference between number of observed spikes in each bin relative to the mean) of 8.2 ± 2.2% (5% MVC), 6.8 ± 1.0% (10% MVC) and 7.5 ± 2.2% (20% MVC), for reflex inhibition, and 6.5 ± 4.1%, 12.0 ± 1.8% and 13.9 ± 2.4%, for reflex excitation. There was no significant difference between the characteristics of the reflex responses, such as latency, amplitude and duration, for the motor units identified by both techniques. Finally, reflex responses could be identified at higher force (4 of the 9 subjects performed contraction up to 50% MVC) using HDsEMG but not iEMG, because of the difficulty in decomposing the iEMG at high forces. In conclusion, single motor unit reflex responses can be estimated accurately and non‐invasively in relatively large populations of motor units using HDsEMG. This non‐invasive approach may enable a more thorough investigation of the synaptic input distribution on active motor units at various force levels.

Abbreviations

CoV_ISI

coefficient of variation of the inter‐spike interval

CPN

common peroneal nerve

CUSUM

cumulative sum

HDsEMG

high‐density surface electromyography

iEMG

intramuscular EMG

MU

motor unit

MVC

maximum voluntary contraction

PNR

pulse‐to‐noise ratio

PSF

peri‐stimulus frequencygram

PSTH

peri‐stimulus time histogram

RA

rate of agreement

SIR

signal‐to‐interference ratio

TA muscle

tibialis anterior muscle

TN

tibial nerve