Experimental Physiology

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Editorial Board

01 October 2014

Alterations in atrial perfusion during atrial fibrillation

01 October 2014

Left atrial (LA) perfusion during disease states has been a topic of much interest, because the clinical implications and detrimental effects of lack of blood flow to the atria are numerous. In the chronic setting, changes in perfusion may lead to LA ischaemia and structural remodelling, a factor implicated in the self-perpetuation of chronic atrial fibrillation (AF). The association between AF and altered LA perfusion has been studied, but a direct causal association between perfusion changes and AF has not been established. A comprehensive literature search of Medline, Embase and Google Scholar databases was conducted from 1960 to February 2014. We systematically analysed reference lists of physiological articles and reviews for other possibly relevant studies. The aim of this review is to provide a comprehensive discussion of the AF-mediated changes in LA perfusion and the potential mechanisms underlying the alterations in coronary flow to the LA in this setting. In addition, we discuss the clinical contexts in which changes in LA perfusion may be relevant. Finally, this article highlights the need for longitudinal studies of AF that would elucidate the changes in LA perfusion resulting from chronic AF and lead to advancements in effective treatments to prevent progression of this disease.

Calcium in the heart: from physiology to disease

01 October 2014

Contraction of the heart results from an increase of cytoplasmic Ca2+ concentration ([Ca2+]i), the so-called systolic Ca2+ transient. Most of this results from the release of Ca2+ from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). In turn, the amplitude of this Ca2+ transient determines the contractility of the heart. In this lecture, I consider the factors which govern the size and stability of this Ca2+ release. The amplitude of the Ca2+ transient is a steep function of SR Ca, resulting in a requirement for very precise beat-to-beat regulation of SR Ca content. This is achieved by a simple negative feedback mechanism, in which an increase of SR Ca content increases the size of the Ca2+ transient, resulting in a decrease of Ca2+ influx on the L-type Ca2+ current and an increase of efflux through Na+–Ca2+ exchange. Changing the activity of any of the Ca2+-cycling proteins will change the steady-state SR Ca content. This feedback mechanism has many consequences, including the fact that a change of RyR open probability has a only a temporary effect on the amplitude of the Ca2+ transient due to a compensating change of SR Ca content. The remainder of the article considers the link between intracellular Ca2+ waves and arrhythmias. This is done in the context of catecholaminergic polymorphic ventricular tachycardia, which is an inherited arrhythmia syndrome, in many cases due to a RyR mutation, where arrhythmias occur during exercise as a result of β-adrenergic stimulation. Calcium waves occur only when the SR Ca content exceeds a threshold level. Our data show that the threshold is reduced by the RyR mutation and that the adrenergic stimulation increases SR Ca content.

Inhale your blood volume!

01 October 2014

Respiratory modulation of muscle sympathetic nerve activity in obstructive sleep apnoea

01 October 2014
New Findings

  • What is the central question of this study?

    Muscle sympathetic nerve activity (MSNA) is increased in obstructive sleep apnoea (OSA), leading to hypertension. Is this due to an increase in respiratory–sympathetic coupling, as has been demonstrated in the spontaneously hypertensive rat?

  • What is the main finding and its importance?

    Using direct microelectrode recordings of MSNA in hypertensive OSA patients and normotensive control subjects, we show that the magnitude of respiratory modulation is not increased in OSA, arguing against an amplified respiratory–sympathetic coupling as the underlying cause of the neurogenic hypertension, although the temporal coupling of MSNA to respiration was stronger in OSA.

  • Obstructive sleep apnoea (OSA) is associated with elevated muscle sympathetic nerve activity (MSNA) during normoxic daytime wakefulness, leading to hypertension. We tested the hypothesis that respiratory–sympathetic coupling, postulated to be the underlying cause of neurogenic hypertension, is increased in OSA. Muscle sympathetic nerve activity, blood pressure, ECG and respiration were recorded in 21 normotensive control subjects and 21 newly diagnosed patients with OSA before and after 6 and 12 months of treatment with continuous positive airway pressure. Muscle sympathetic nerve activity was recorded via tungsten microelectrodes inserted percutaneously into the peroneal nerve. Cardiac and respiratory modulation of MSNA was quantified from the cross-correlation histograms constructed between the sympathetic spikes and either ECG or respiration. Muscle sympathetic nerve activity was significantly elevated in newly diagnosed OSA patients compared with control subjects (53 ± 2 versus 28 ± 2 bursts min–1). There was a significant fall in MSNA after 6 months of continuous positive airway pressure (37 ± 2 bursts min–1), with no further change after 12 months (37 ± 2 bursts min–1). There were no significant differences in the magnitude of respiratory modulation of MSNA between the OSA patients and control subjects (40 ± 3.1 versus 39 ± 3.4%). However, when considering the normalized temporal profile there were changes in the respiratory patterning of MSNA in OSA, with more activity occurring in postinspiration and less in inspiration and expiration. This was largely reversed following long-term continuous positive airway pressure.

    Neuronal activation by mucosal biopsy supernatants from irritable bowel syndrome patients is linked to visceral sensitivity

    01 October 2014

    Based on the discomfort/pain threshold during rectal distension, irritable bowel syndrome (IBS) patients may be subtyped as normo- or hypersensitive. We previously showed that mucosal biopsy supernatants from IBS patients activated enteric and visceral afferent neurons. We tested the hypothesis that visceral sensitivity is linked to the degree of neuronal activation. Normo- and hypersensitive IBS patients were distinguished by their discomfort/pain threshold to rectal balloon distension with a barostat. Using potentiometric and Ca2+ dye imaging, we recorded the response of guinea-pig enteric submucous and mouse dorsal root ganglion (DRG) neurons, respectively, to mucosal biopsy supernatants from normosensitive (n = 12 tested in enteric neurons, n = 9 tested in DRG) and hypersensitive IBS patients (n = 9, tested in both types of neurons). In addition, we analysed the association between neuronal activation and individual discomfort/pain pressure thresholds. The IBS supernatants evoked Ca2+ transients in DRG neurons and spike discharge in submucous neurons. Submucous and DRG neurons showed significantly stronger responses to supernatants from hypersensitive IBS patients as reflected by higher spike frequency or stronger [Ca2+]i transients in a larger proportion of neurons. The neuroindex as a product of spike frequency or [Ca2+]i transients and proportion of responding neurons correlated significantly with the individual discomfort/pain thresholds of the IBS patients. Supernatants from hypersensitive IBS patients caused stronger activation of enteric and DRG neurons. The level of activation correlated with the individual discomfort/pain threshold pressure values. These findings support our hypothesis that visceral sensitivity is linked to activation of peripheral neurons by biopsy supernatants.

    Increased cardiac output contributes to the development of chronic intermittent hypoxia-induced hypertension

    01 October 2014

    Chronic intermittent hypoxia (CIH) in animal models has been shown to result in hypertension and elevation of sympathetic nervous system activity. Sympathetically mediated vasoconstriction is believed to be the primary mechanism underpinning CIH-induced hypertension; however, the potential contribution of the heart is largely overlooked. We sought to determine the contribution of cardiac output (CO) and lumbar sympathetic control of the hindlimb circulation to CIH-induced hypertension. Male Wistar rats (n = 64) were exposed to 2 weeks of CIH [cycles of 90 s hypoxia (5% O2 nadir) and 210 s normoxia] or normoxia for 8 h day–1. Under urethane anaesthesia, CIH-treated animals developed hypertension (81.4 ± 2.2 versus 91.6 ± 2.4 mmHg; P < 0.001), tachycardia (397 ± 8 versus 445 ± 7 beats min–1; P < 0.001) and an increased haematocrit (42.4 ± 0.4 versus 45.0 ± 0.4%; P < 0.001). Echocardiography revealed that CIH exposure increased the CO [19.3 ± 1.7 versus 25.8 ± 2.6 ml min–1 (100 g)–1; P = 0.027] with no change in total peripheral resistance (4.93 ± 0.49 versus 4.17 ± 0.34 mmHg ml–1 min–1; P = 0.123). Sympathetic ganglionic blockade revealed that sympathetic control over blood pressure was not different (–27.7 ± 1.6 versus –32.3 ± 2.9 mmHg; P = 0.095), and no chronic vasoconstriction was found in the hindlimb circulation of CIH-treated animals (39.4 ± 2.5 versus 38.0 ± 2.4 μl min–1 mmHg–1; P = 0.336). Lumbar sympathetic control over the hindlimb circulation was unchanged in CIH-treated animals (P = 0.761), although hindlimb arterial sympathetic density was increased (P = 0.012) and vascular sensitivity to phenylephrine was blunted (P = 0.049). We conclude that increased CO is sufficient to explain the development of CIH-induced hypertension, which may be an early adaptive response to raise O2 flow. We propose that sustained elevated cardiac work may ultimately lead to heart failure.

    Vasopressin regulation of epithelial colonic proliferation and permeability is mediated by pericryptal platelet-derived growth factor A

    01 October 2014

    Arginine vasopressin (AVP) has trophic effects on the rat distal colon, increasing the growth of pericryptal myofibroblasts and reducing the colonic crypt wall permeability. This study aimed to reproduce in vitro the effects of AVP observed in vivo using cultures of human CCD-18Co myofibroblasts and T84 colonic epithelial cells. Proliferation of myofibroblasts was quantified by bromodeoxyuridine incorporation; the expression of platelet-derived growth factor A (PDGFA), platelet-derived growth factor B, epidermal growth factor, transforming growth factor-β and vascular endothelial growth factor was measured by PCR and the expression of epithelial junction proteins by Western blot. Arginine vasopressin stimulated myofibroblast proliferation and the expression of PDGFA without affecting the expression of platelet-derived growth factor B, epidermal growth factor, transforming growth factor-β or vascular endothelial growth factor. These effects were prevented when AVP receptor inhibitors were present in the medium. Pre-incubation of CCD-18Co cells with anti-PDGF antibody or with an inhibitor of the PDGF receptor abolished the effects of AVP. When colonocytes were incubated with medium obtained from myofibroblasts incubated with AVP, both cell proliferation and the expression of epithelial junction proteins increased; however, direct incubation of colonocytes with AVP did not modify these variables. These results demonstrate that AVP stimulates myofibroblast proliferation and induces PDGFA secretion, implying that PDGFA mediates local myofibroblast proliferation by an autocrine feedback loop and regulates epithelial proliferation and permeability by a paracrine mechanism.

    Characterization of butyrate transport across the luminal membranes of equine large intestine

    01 October 2014

    The diet of the horse, pasture forage (grass), is fermented by the equine colonic microbiota to short-chain fatty acids, notably acetate, propionate and butyrate. Short-chain fatty acids provide a major source of energy for the horse and contribute to many vital physiological processes. We aimed to determine both the mechanism of butyrate uptake across the luminal membrane of equine colon and the nature of the protein involved. To this end, we isolated equine colonic luminal membrane vesicles. The abundance and activity of cysteine-sensitive alkaline phosphatase and villin, intestinal luminal membrane markers, were significantly enriched in membrane vesicles compared with the original homogenates. In contrast, the abundance of GLUT2 protein and the activity of Na+–K+-ATPase, known markers of the intestinal basolateral membrane, were hardly detectable. We demonstrated, by immunohistochemistry, that monocarboxylate transporter 1 (MCT1) protein is expressed on the luminal membrane of equine colonocytes. We showed that butyrate transport into luminal membrane vesicles is energized by a pH gradient (out < in) and is not Na+ dependent. Moreover, butyrate uptake is time and concentration dependent, with a Michaelis–Menten constant of 5.6 ± 0.45 mm and maximal velocity of 614 ± 55 pmol s–1 (mg protein)–1. Butyrate transport is significantly inhibited by p-chloromercuribenzoate, phloretin and α-cyano-4-hydroxycinnamic acid, all potent inhibitors of MCT1. Moreover, acetate and propionate, as well as the monocarboxylates pyruvate and lactate, also inhibit butyrate uptake. Data presented here support the conclusion that transport of butyrate across the equine colonic luminal membrane is predominantly accomplished by MCT1.

    Endoplasmic reticulum stress-mediated autophagy protects against lipopolysaccharide-induced apoptosis in HL-1 cardiomyocytes

    01 October 2014

    Apoptosis of cardiomyocytes limits the contractile efficiency of the heart during sepsis. Prosurvival autophagy has been proposed as a novel mechanism to maintain normal heart function. Here, we demonstrated that autophagy was activated in lipopolysaccharide (LPS)-treated HL-1 cells, and it counteracted the LPS-induced apoptosis. We investigated further the mechanism by which LPS triggered autophagy in HL-1 cells. We discovered that endoplasmic reticulum (ER) stress played an important role in LPS-triggered autophagy. The ER activated a survival pathway through the ER-localized transmembrane protein PERK, which was essential for LPS-induced autophagy. Lipopolysaccharide increased expression of GRP78, phosphorylated PERK and phosphorylated eukaryotic initiation factor 2α. Similar results were observed after administration of tunicamycin, a well-known ER stressor. Most importantly, we found that 4-phenylbutyrate, an inhibitor of ER stress, suppressed LPS-activated autophagy in the presence of LPS in HL-1 cells. The same results were observed after small interfering RNA-mediated silencing of PERK protein. We also noticed that LPS-induced apoptosis appeared early, at 4 h. Our findings revealed that PERK, one arm of ER stress, facilitated survival of LPS-treated HL-1 cells by promoting autophagy, and could serve as a potential therapeutic strategy to alleviate septic myocardial dysfunction.

    Mechanomyogram amplitude correlates with human gastrocnemius medialis muscle and tendon stiffness both before and after acute passive stretching

    01 October 2014

    The study aimed to assess the level of correlation between muscle–tendon unit (MTU) stiffness and mechanomyogram (MMG) signal amplitude of the human gastrocnemius medialis muscle, both before and after acute passive stretching. The passive torque (Tpass), electrically evoked peak torque (pT) and myotendinous junction displacement were determined at different angles of dorsiflexion (0, 10 and 20 deg), while maximum voluntary isometric torque (Tmax) was assessed only at 0 deg. Measurements were repeated after a bout of passive stretching. From the MMG signal, the root mean square (RMS) and peak to peak (p-p) were calculated. The MTU, muscle and tendon stiffness were determined by ultrasound and Tpass measurements. Before stretching, correlations between MMG RMS and MTU, muscle and tendon stiffness were found (R2 = 0.22–0.46). After stretching, Tpass, Tmax, pT and MTU, muscle and tendon stiffness decreased by 25 ± 7, 16 ± 2, 9 ± 2, 22 ± 7, 23 ± 8 and 28 ± 5%, respectively (P < 0.05). During voluntary and electrically evoked contractions, MMG p-p decreased by 9 ± 2 and 5 ± 1%, while MMG RMS increased by 48 ± 7 and 50 ± 8%, respectively (P < 0.05). Correlations between MMG RMS and MTU, muscle and tendon stiffness were still present after stretching (R2 = 0.44–0.60). In conclusion, correlations between MMG RMS and stiffness exist both before and after stretching, suggesting that a slacker MTU leads to larger muscle fibre oscillations. However, care must be taken in using MMG amplitude as an indirect index to estimate stiffness owing to the relatively small R2 values of the investigated correlations.

    Amitriptyline is efficacious in ameliorating muscle inflammation and depressive symptoms in the mdx mouse model of Duchenne muscular dystrophy

    01 October 2014

    Mutations in the structural protein dystrophin underlie muscular dystrophies characterized by progressive deterioration of muscle function. Dystrophin-deficient mdx mice are considered a model for Duchenne muscular dystrophy (DMD). Individuals with DMD are also susceptible to mood disorders, such as depression and anxiety. Therefore, the study objectives were to investigate the effects of the tricyclic antidepressant amitriptyline on mood, learning, central cytokine expression and skeletal muscle inflammation in mdx mice. Amitriptyline-induced effects (10 mg kg–1 daily s.c. injections, 25 days) on the behaviour of mdx mice were investigated using the open field arena and tail suspension tests. The effects of chronic amitriptyline treatment on inflammatory markers were studied in the muscle and plasma of mdx mice, and mood-associated monoamine and cytokine concentrations were measured in the amygdala, hippocampus, prefrontal cortex, striatum, hypothalamus and midbrain. The mdx mice exhibited increased levels of anxiety and depressive-like behaviour compared with wild-type mice. Amitriptyline treatment had anxiolytic and antidepressant effects in mdx mice associated with elevations in serotonin levels in the amygdala and hippocampus. Inflammation in mdx skeletal muscle tissue was also reduced following amitriptyline treatment as indicated by decreased immune cell infiltration of muscle and lower levels of the pro-inflammatory cytokines tumour necrosis factor-α and interleukin-6 in the forelimb flexors. Interleukin-6 mRNA expression was remarkably reduced in the amygdala of mdx mice by chronic amitriptyline treatment. Positive effects of amitriptyline on mood, in addition to its anti-inflammatory effects in skeletal muscle, may make it an attractive therapeutic option for individuals with DMD.

    Discharge characteristics of motor units during long-duration contractions

    01 October 2014

    The purpose of the study was to determine how long humans could sustain the discharge of single motor units during a voluntary contraction. The discharge of motor units in first dorsal interosseus of subjects (27.8 ± 8.1 years old) was recorded for as long as possible. The task was terminated when the isolated motor unit stopped discharging action potentials, despite the ability of the individual to sustain the abduction force. Twenty-three single motor units were recorded. Task duration was 21.4 ± 17.8 min. When analysed across discharge duration, mean discharge rate (10.6 ± 1.8 pulses s–1) and mean abduction force (5.5 ± 2.8% maximum) did not change significantly (discharge rate, P = 0.119; and abduction force, P = 0.235). In contrast, the coefficient of variation for interspike interval during the initial 30 s of the task was 22.2 ± 6.0% and increased to 31.9 ± 7.0% during the final 30 s (P < 0.001). All motor units were recruited again after 60 s of rest. Although subjects were able to sustain a relatively constant discharge rate, the cessation of discharge was preceded by a gradual increase in discharge variability. The findings also showed that the maximal duration of human motor unit discharge exceeds that previously reported for the discharge elicited in motor neurons by intracellular current injection in vitro.

    Infusion of ATP increases leg oxygen delivery but not oxygen uptake in the initial phase of intense knee-extensor exercise in humans

    01 October 2014

    The present study examined whether an increase in leg blood flow and oxygen delivery at the onset of intense exercise would speed the rate of rise in leg oxygen uptake. Nine healthy men (25 ± 1 years old, mean ± SEM) performed one-leg knee-extensor exercise (62 ± 3 W, 86 ± 3% of incremental test peak power) for 4 min during a control setting (CON) and with infusion of ATP into the femoral artery in order to increase blood flow before and during exercise. In the presence of ATP, femoral arterial blood flow and O2 delivery were higher (P < 0.001) at the onset of exercise and throughout exercise (femoral arterial blood flow after 10 s, 5.1 ± 0.5 versus 2.7 ± 0.3 l min–1; after 45 s, 6.0 ± 0.5 versus 4.1 ± 0.4 l min–1; after 90 s, 6.6 ± 0.6 versus 4.5 ± 0.4 l min–1; and after 240 s, 7.0 ± 0.6 versus 5.1 ± 0.3 l min–1 in ATP and CON conditions, respectively). Leg oxygen uptake was not different in ATP and CON conditions during the first 20 s of exercise but was lower (P < 0.05) in the ATP compared with CON conditions after 30 s and until the end of exercise (30 s, 436 ± 42 versus 549 ± 45 ml min–1; and 240 s, 705 ± 31 versus 814 ± 59 ml min–1 in ATP and CON, respectively). Lactate release was lower after 60, 120 and 180 s of exercise with ATP infusion. These results suggest that O2 delivery is not limiting the rise in skeletal muscle oxygen uptake in the initial phase of intense exercise.

    Erythropoietin administration alone or in combination with endurance training affects neither skeletal muscle morphology nor angiogenesis in healthy young men

    01 October 2014

    The aim was to investigate the ability of an erythropoiesis-stimulating agent (ESA), alone or in combination with endurance training, to induce changes in human skeletal muscle fibre and vascular morphology. In a comparative study, 36 healthy untrained men were randomly dispersed into the following four groups: sedentary–placebo (SP, n = 9); sedentary–ESA (SE, n = 9); training–placebo (TP, n = 10); or training–ESA (TE, n = 8). The ESA or placebo was injected once weekly. Training consisted of progressive bicycling three times per week for 10 weeks. Before and after the intervention period, muscle biopsies and magnetic resonance images were collected from the thigh muscles, blood was collected, body composition measured and endurance exercise performance evaluated. The ESA treatment (SE and TE) led to elevated haematocrit, and both ESA treatment and training (SE, TP and TE) increased maximal O2 uptake. With regard to skeletal muscle morphology, TP alone exhibited increases in whole-muscle cross-sectional area and fibre diameter of all fibre types. Also exclusively for TP was an increase in type IIa fibres and a corresponding decrease in type IIx fibres. Furthermore, an overall training effect (TP and TE) was statistically demonstrated in whole-muscle cross-sectional area, muscle fibre diameter and type IIa and type IIx fibre distribution. With regard to muscle vascular morphology, TP and TE both promoted a rise in capillary to muscle fibre ratio, with no differences between the two groups. There were no effects of ESA treatment on any of the muscle morphological parameters. Despite the haematopoietic effects of ESA, we provide novel evidence that endurance training rather than ESA treatment induces adaptational changes in angiogenesis and muscle morphology.

    Respiratory pump contributes to increased physiological reserve for compensation during simulated haemorrhage

    01 October 2014

    Intrathoracic pressure regulation (IPR) represents a therapy for increasing systemic circulation through the creation of negative intrathoracic pressure. We hypothesized that using this ‘respiratory pump’ effect would slow the diminution of the physiological reserve to compensate during progressive reductions in central blood volume. The compensatory reserve index (CRI) algorithm was used to measure the proportion (from 100 to 0%) of reserve capacity that remained to compensate for central volume loss before the onset of cardiovascular decompensation. Continuous analog recordings of arterial waveforms were extracted from data files of seven healthy volunteers. Subjects had previously participated in experiments designed to induce haemodynamic decompensation (presyncope) by progressive reduction in central blood volume using graded lower-body negative pressure. The lower-body negative pressure protocol was completed while breathing spontaneously through a standard medical face mask without (placebo) and with a resistance (approximately –7 cmH2O; active IPR) applied during inspiration. At the onset of presyncope in the placebo conditions, CRI was smaller than the CRI observed at the same time point in the active IPR conditions. The CRI at the onset of presyncope during active IPR (0.08 ± 0.01) was similar to the CRI at presyncope with placebo. Kaplan–Meier and log rank tests indicated that CRI survival curves were shifted to the right by active IPR. Optimizing the respiratory pump contributed a small but significant effect of increasing tolerance to progressive reductions in central blood volume by extending the compensatory reserve.

    Double disruption of {alpha}2A- and {alpha}2C-adrenoceptors induces endothelial dysfunction in mouse small arteries: role of nitric oxide synthase uncoupling

    01 October 2014

    Knockout mice lacking both α2A- and α2C-adrenergic receptors (α2A/α2C-ARKO) provide a model for understanding the mechanisms underlying the deleterious effects of sympathetic hyperactivity on the cardiovascular system. Thus, in the present study we investigated the vascular reactivity of large and small arteries of α2A/α2C-ARKO mice. Aorta and mesenteric small arteries (MSAs) from 7-month-old male α2A/α2C-ARKO mice and congenic C57BL6/J mice (wild-type, WT) were studied. In the aorta, noradrenaline- and serotonin-induced contraction was similar between groups, but in MSAs there was an increase in agonist-induced contraction in α2A/α2C-ARKO compared with WT. The l-NAME effect was reduced in MSAs of α2A/α2C-ARKO mice compared with WT mice, as was basal NO evaluated by a 4,5-diaminofluorescein diacetate probe. Increased total endothelial nitric oxide synthase (eNOS) protein expression was observed in MSAs from α2A/α2C-ARKO mice, while the dimer/monomer ratio of eNOS was decreased. Mesenteric small arteries from α2A/α2C-ARKO mice showed an increase in ethidium bromide-positive nuclei, indicating oxidative stress, which was attenuated by incubation with l-NAME. The sympathetic hyperactivity present in α2A/α2C-ARKO mice alters vascular reactivity only in certain types of arteries. Moreover, after chronic sympathetic hyperactivity, uncoupling eNOS may be a significant source of superoxide anion and reduced NO bioavailability in small vessels, increasing the contractile tone.