Experimental Physiology

The effects of 5 weeks of moderate-intensity endurance training on pulmonary oxygen uptake kinetics ( on-kinetics) were studied in 15 healthy men (mean ± SD: age 22.7 ± 1.8 years, body weight 76.4 ± 8.9 kg and maximal 46.0 ± 3.7 ml kg–1 min–1). Training caused a significant acceleration (P = 0.003) of on-kinetics during moderate-intensity cycling (time constant of the ‘primary' component 30.0 ± 6.6 versus 22.8 ± 5.6 s before and after training, respectively) and a significant decrease (P = 0.04) in the amplitude of the primary component (837 ± 351 versus 801 ± 330 ml min–1). No changes in myosin heavy chain distribution, muscle fibre capillarization, level of peroxisome proliferator-activated receptor coactivator 1α and other markers of mitochondrial biogenesis (mitochondrial DNA copy number, cytochrome c and cytochrome oxidase subunit I contents) in the vastus lateralis were found after training. A significant downregulation in the content of the sarcoplasmic reticulum ATPase 2 (SERCA2; P = 0.03) and a tendency towards a decrease in SERCA1 (P = 0.055) was found after training. The decrease in SERCA1 was positively correlated (P = 0.05) with the training-induced decrease in the gain of the on-kinetics ( at steady state/power output). In the early stage of training, the acceleration in on-kinetics during moderate-intensity cycling can occur without enhanced mitochondrial biogenesis or changes in muscle myosin heavy chain distribution and in muscle fibre capillarization. The training-induced decrease of the O2 cost of cycling could be caused by the downregulation of SERCA pumps.

Increased maximal oxygen uptake ( ), mitochondrial capacity and energy coupling efficiency are reported after endurance training (ET) in adult subjects. Here we test whether leg exercise performance (power output of the legs, Pmax, at ) reflects these improvements with ET in the elderly. Fifteen male and female subjects were endurance trained for a 6 month programme, with 13 subjects (69.5 ± 1.2 years old, range 65–80 years old; n = 7 males; n = 6 females) completing the study. This training significantly improved Pmax (17%; P = 0.003), (5.4%; P = 0.021) and the increment in oxygen uptake ( ) above resting ( ; 9%; P < 0.02). In addition, evidence of improved energy coupling came from elevated leg power output per unit at the aerobic capacity [(Pmax/ ); P = 0.02] and during submaximal exercise in the ramp test as measured by delta efficiency (Pex/ ; P = 0.04). No change was found in blood lactate, muscle glycolysis or fibre type. The rise in Pmax paralleled the improvement in muscle oxidative phosphorylation capacity (ATPmax) in these subjects. In addition, the greater exercise energy coupling [(Pmax/ ) and delta efficiency] was accompanied by increased mitochondrial energy coupling as measured by elevated ATP production per unit mitochondrial content in these subjects. These results suggest that leg exercise performance benefits from elevations in energy coupling and oxidative phosphorylation capacity at both the whole-body and muscle levels that accompany endurance training in the elderly.

It is debatable whether differences in mitochondrial function exist across skeletal muscle types and whether mouse skeletal muscle mitochondrial function can serve as a valid model for human skeletal muscle mitochondrial function. The aims of this study were to compare and contrast three different mouse skeletal muscles and to identify the mouse muscle that most closely resembles human skeletal muscle respiratory capacity and control. Mouse quadriceps (QUADM), soleus (SOLM) and gastrocnemius (GASTM) skeletal muscles were obtained from 8- to 10-week-old healthy mice (n = 8), representing mixed, oxidative and glycolytic muscle, respectively. Skeletal muscle samples were also collected from young, active, healthy human subjects (n = 8) from the vastis lateralis (QUADH). High-resolution respirometry was used to examine mitochondrial function in all skeletal muscle samples, and mitochondrial content was quantified with citrate synthase activity. Mass-specific respiration was higher across all respiratory states in SOLM versus both GASTM and QUADH (P < 0.01). When controlling for mitochondrial content, however, SOLM respiration was lower than GASTM and QUADH (P < 0.05 and P < 0.01, respectively). When comparing respiratory capacity between mouse and human muscle, QUADM exhibited only one different respiratory state when compared with QUADH. These results demonstrate that qualitative differences in mitochondrial function exist between different mouse skeletal muscles types when respiratory capacity is normalized to mitochondrial content, and that skeletal muscle respiratory capacity in young, healthy QUADM does correspond well with that of young, healthy QUADH.

Bile duct ligation (BDL) causes congestive liver failure that initiates haemodynamic changes, including peripheral vasodilatation and generalized oedema. Peripheral vasodilatation is hypothesized to activate compensatory mechanisms, including increased drinking behaviour and neurohumoral activation. This study tested the hypothesis that changes in the expression of angiotensin II type 1 receptor (AT1R) mRNA and protein in the lamina terminalis are associated with BDL-induced hyposmolality in the rat. All rats received either BDL or sham-ligation surgery. The rats were housed in metabolic chambers for measurement of fluid and food intake and urine output. Expression of AT1R in the lamina terminalis was assessed by Western blot and quantitative real-time PCR (RT-qPCR). Average baseline water intake increased significantly in BDL rats compared with sham-operated rats, and upregulation of AT1R protein and AT1aR mRNA were observed in the subfornical organ of BDL rats. Separate groups of BDL and sham-ligated rats were instrumented with minipumps filled with either losartan (2.0 μg μl–1) or 0.9% saline for chronic intracerebroventricular or chronic subcutaneous infusion. Chronic intracerebroventricular losartan infusion attenuated the increased drinking behaviour and prevented the increased abundance of AT1R protein in the subfornical organ in BDL rats. Chronic subcutaneous infusion did not affect water intake or AT1R abundance in the subfornical organ. The data presented here indicate a possible role of increased central AT1R expression in the regulation of drinking behaviour during congestive cirrhosis.

Oxidative stress has been shown to play an important role in the development and progression of diabetic nephropathy, and the formation of reactive oxygen species (ROS) is a direct consequence of hyperglycaemia. We hypothesized that hyperglycaemia-induced ROS can activate the transforming growth factor-β1 (TGF-β1)–phosphoinositide 3-kinase (PI3K)–Akt–FoxO3a signalling pathway, negatively regulating expression of manganese superoxide dismutase (MnSOD), which promotes excessive ROS generation and accelerates the pathological process of diabetic nephropathy. In vitro, in rat mesangial cells, high glucose (30 mmol l–1), but not equimolar mannitol, stimulated ROS production, upregulated the levels of TGF-β1, increased the phosphorylated Akt/total Akt and phosphorylated FoxO3a/total FoxO3a protein ratios, altered the subcellular localization of FoxO3a and reduced the levels of MnSOD expression. These high-glucose-induced changes further promoted the generation of ROS. In vivo, in db/db mice treated with an inhibitor of TGF-β1 (SB431542) or PI3K (LY294002), the levels of phosphorylated Akt and phosphorylated FoxO3a in the kidney cortices were decreased, the level of MnSOD expression was increased and the level of the lipid peroxidation end-product, malondialdehyde, was reduced. We conclude that overproduction of ROS induced by a high glucose concentration decreases the expression of MnSOD via the PI3K–Akt–FoxO3a pathway and further aggravates oxidative stress in diabetic nephropathy.

Transcranial magnetic stimulation (TMS) can activate the corticobulbar system and briefly recruit upper airway dilator muscles, improving the inspiratory airflow dynamics of flow-limited respiratory cycles during sleep. The purpose of this investigation was to quantify the effects of TMS-induced twitches applied during sleep on flow-limited respiratory cycles in 14 obstructive sleep apnoea patients. Submental muscle motor threshold (SUBMT) and motor-evoked potential (SUBMEP) were examined during wakefulness and sleep. The TMS-induced twitches were applied during stable non-rapid eye movement (NREM) sleep, during non-consecutive flow-limited respiratory cycles at the beginning of inspiration, with intensities varying from sleep SUBMT up to maximal stimulation without arousal. Maximal inspiratory flow, inspiratory volume, shifts of electroencephalogram frequency and pulse rate variability were assessed. Cortical and/or autonomic arousal after TMS was observed in only 13.8% of all twitches applied. The SUBMT increased during NREM sleep (wakefulness, 24.8 ± 9.3%; and NREM sleep, 28.3 ± 9.5%; P = 0.003). Augmenting stimulator output from SUBMT to maximal stimulation before arousal enhanced SUBMEP peak-to-peak amplitude (from 0.09 ± 0.05 to 0.4 ± 0.3 mV; P = 0.005) with a concomitant rise in maximal inspiratory flow (from 376.2 ± 107.9 to 411.9 ± 109.3 ml s–1; P = 0.008) and inspiratory volume (from 594.8 ± 189.2 to 663.7 ± 203.1 ml; P = 0.001) in all but one patient. Corticobulbar excitability of submental muscles decreases during NREM sleep. Brief recruitment of submental muscles with TMS during sleep improves upper airway mechanics without arousing patients from sleep.

Calcium-activated potassium channels of small (KCa2, SK) and intermediate (KCa3.1, IK) conductance are involved in endothelium-dependent relaxation of pulmonary arteries. We hypothesized that the function and expression of KCa2 and KCa3.1 increase as a compensatory mechanism to counteract hypoxia-induced pulmonary hypertension in rats. For functional studies, pulmonary arteries were mounted in microvascular myographs for isometric tension recordings. The KCa channel expression was evaluated by immunoblotting and quantitative PCR. Although ACh induced similar relaxations, the ACh-induced relaxations were abolished by the combined inhibition of nitric oxide synthase (by l-nitro-arginine, l-NOARG), cyclo-oxygenase (by indomethacin) and soluble guanylate cyclase (by ODQ) in pulmonary arteries from hypoxic rats, whereas 20 ± 6% (n = 8) maximal relaxation in response to ACh persisted in arteries from normoxic rats. Inhibiting Na+,K+-ATPase with ouabain or blocking KCa2 and KCa3.1 channels reduced the persisting ACh-induced relaxation. In the presence of l-NOARG and indomethacin, a novel KCa2 and KCa3.1 channel activator, NS4591, induced concentration- and endothelium-dependent relaxations, which were markedly reduced in arteries from chronically hypoxic rats compared with arteries from normoxic rats. The mRNA levels of KCa2.3 and KCa3.1 were unaltered, whereas KCa2.3 protein expression was upregulated and KCa3.1 protein expression downregulated in pulmonary arteries from rats exposed to hypoxia. In conclusion, endothelium-dependent relaxation was conserved in pulmonary arteries from chronically hypoxic rats, while endothelium-derived hyperpolarization (EDH)-type relaxation was impaired in chronically hypoxic pulmonary small arteries despite upregulation of KCa2.3 channels. Since impaired EDH-type relaxation was accompanied by KCa3.1 channel protein downregulation, these findings suggest that KCa3.1 channels are important for the maintenance of EDH-type relaxation.

Catecholamines modulate the production of inflammatory mediators by macrophages in an autocrine/paracrine manner. They also tune β2-adrenoceptor expression. Glucocorticoids influence catecholamine metabolism and adrenoceptor expression in many cell types. We hypothesized that adrenal hormones affect the production of tumour necrosis factor-α (TNF-α) and NO by macrophages by altering the modulatory influence of catecholamines. To prove the hypothesis, peritoneal exudate macrophages from propranolol-treated non-operated and adrenalectomized rats and from corticosterone-supplemented adrenalectomized rats were examined for lipopolysaccharide-stimulated NO and TNF-α production in vitro and for expression of β2-adrenoceptors and major catecholamine-metabolizing enzymes. Glucocorticoid deprivation increased NO production by macrophages, whereas 4 days of propranolol treatment was ineffective in this respect. However, propranolol treatment, via β2-adrenoceptor blockade, increased production of TNF-α by macrophages in both non-operated and adrenalectomized rats (showing dramatically enhanced TNF-α production due to a lack of circulating glucocorticoids) for the same value. The expression of β2-adrenoceptor was increased in peritoneal macrophages that were freshly isolated from non-operated, propranolol-treated and adrenalectomized rats (due to adrenal catecholamine deficiency). Propranolol did not affect macrophage β2-adrenoceptor expression in adrenalectomized rats. Given that propranolol increased the density of macrophage tyrosine hydroxylase expression only in non-operated rats and affected the mRNA expression of monoamine oxidase-A in neither non-operated nor adrenalectomized animals, a significant influence of propranolol on peritoneal exudate cell noradrenaline content was found only in non-operated rats. A lack of circulating adrenal hormones also affected noradrenaline metabolism and content in peritoneal exudate cells including macrophages. Collectively, despite differences in the abundance of macrophage catecholamine–β2-adrenoceptor system components and in the TNF-α response to lipopolysaccharide between adrenalectomized and non-operated rats, propranolol increased TNF-α production by the same amount in macrophages from these two groups of animals.

The G protein-coupled estrogen receptor (GPER) has been identified in several brain regions, including cholinergic neurons of the nucleus ambiguus, which are critical for parasympathetic cardiac regulation. Using calcium imaging and electrophysiological techniques, microinjection into the nucleus ambiguus and blood pressure measurement, we examined the in vitro and in vivo effects of GPER activation in nucleus ambiguus neurons. A GPER selective agonist, G-1, produced a sustained increase in cytosolic Ca2+ concentration in a concentration-dependent manner in retrogradely labelled cardiac vagal neurons of nucleus ambiguus. The increase in cytosolic Ca2+ produced by G-1 was abolished by pretreatment with G36, a GPER antagonist. G-1 depolarized cultured cardiac vagal neurons of the nucleus ambiguus. The excitatory effect of G-1 was also identified by whole-cell visual patch-clamp recordings in nucleus ambiguus neurons, in medullary slices. To validate the physiological relevance of our in vitro studies, we carried out in vivo experiments. Microinjection of G-1 into the nucleus ambiguus elicited a decrease in heart rate; the effect was blocked by prior microinjection of G36. Systemic injection of G-1, in addition to a previously reported decrease in blood pressure, also reduced the heart rate. The G-1-induced bradycardia was prevented by systemic injection of atropine, a muscarinic antagonist, or by bilateral microinjection of G36 into the nucleus ambiguus. Our results indicate that GPER-mediated bradycardia occurs via activation of cardiac parasympathetic neurons of the nucleus ambiguus and support the involvement of the GPER in the modulation of cardiac vagal tone.

Hypoxia changes the regional distribution of cerebral blood flow and stimulates the ventilatory chemoreflex, thereby reducing CO2 tension. We examined the effects of both hypoxia and isocapnic hypoxia on acute changes in internal carotid (ICA) and vertebral artery (VA) blood flow. Ten healthy male subjects underwent the following two randomly assigned respiratory interventions after a resting baseline period with room air: (i) hypoxia; and (ii) isocapnic hypoxia with a controlled gas mixture (12% O2; inspiratory mmHg). In the isocapnic hypoxia intervention, subjects were instructed to maintain the rate and depth of breathing to maintain the level of end-tidal partial pressure of CO2 ( ) during the resting baseline period. The ICA and VA blood flow (velocity x cross-sectional area) were measured using Doppler ultrasonography. The was decreased (–6.3 ± 0.9%, P < 0.001) during hypoxia by hyperventilation (minute ventilation +12.9 ± 2.2%, P < 0.001), while was unchanged during isocapnic hypoxia. The ICA blood flow was unchanged (P = 0.429), while VA blood flow increased (+10.3 ± 3.1%, P = 0.010) during hypoxia. In contrast, isocapnic hypoxia increased both ICA (+14.5 ± 1.4%, P < 0.001) and VA blood flows (+10.9 ± 2.4%, P < 0.001). Thus, hypoxic vasodilatation outweighed hypocapnic vasoconstriction in the VA, but not in the ICA. These findings suggest that acute hypoxia elicits an increase in posterior cerebral blood flow, possibly to maintain essential homeostatic functions of the brainstem.

Dopamine is commonly used for blood pressure support in the neonate, but has limited empirical evidence to support its use. We tested the hypothesis that after near-terminal asphyxia in utero, dopamine infusions would prevent secondary hypotension. Fetal sheep (122–129 days of gestation; term is 147 days) received umbilical cord occlusion for 15 min or sham occlusion (n = 5). If the mean arterial blood pressure fell below 90% of baseline within 6 h after occlusion, fetuses were randomized to either dopamine infusion starting at 4 μg kg–1 min–1 and titrated according to mean arterial blood pressure up to a maximum of 40 μg kg–1 min–1 (n = 5) or to the same volume of normal saline (n = 5). Dopamine infusion, initiated at a median of 180 min after occlusion (range 96–280 min), was associated with a marked but transient increase in mean arterial blood pressure and fall in femoral blood flow compared with saline. Terminal hypotension developed later in four of the five fetuses that received maximal dopamine infusions than in five of five receiving saline infusion [517 (range 240–715) versus 106 min (range 23–497) after the start of infusions, P < 0.05]. In conclusion, dopamine infusion delayed but did not prevent terminal hypotension after severe asphyxia.

Physical inactivity and exercise training result in opposite adaptations of vascular structure. However, the molecular mechanisms behind these adaptations are not completely understood. We used a unique study design to examine both vascular characteristics of the superficial femoral artery (using ultrasound) and gene expression levels (from a muscle biopsy) in human models for physical deconditioning and exercise training. Initially, we compared able-bodied control subjects (n = 6) with spinal cord-injured individuals (n = 8) to assess the effects of long-term deconditioning. Subsequently, able-bodied control subjects underwent short-term lower limb deconditioning using 3 weeks of unilateral limb suspension. Spinal cord-injured individuals were examined before and after 6 weeks of functional electrical stimulation exercise training. Baseline femoral artery diameter and hyperaemic flow were lower after short- and long-term deconditioning and higher after exercise training, whilst intima–media thickness/lumen ratio was increased with short- and long-term deconditioning and decreased with exercise training. Regarding gene expression levels of vasculature-related genes, we found that groups of genes including the vascular endothelial growth factor pathway, transforming growth factor β1 and extracellular matrix proteins were strongly associated with vascular adaptations in humans. This approach resulted in the identification of important genes that may be involved in vascular adaptations after physical deconditioning and exercise.

Thyroid hormone exerts broad effects on the adult heart, but little is known regarding the role of thyroid hormone in the regulation of cardiac growth early in development and in response to pathophysiological conditions. To address this issue, we determined the effects of fetal thyroidectomy on cardiac growth and growth-related gene expression in control and pulmonary-artery-banded fetal sheep. Fetal thyroidectomy (THX) and/or placement of a restrictive pulmonary artery band (PAB) were performed at 126 ± 1 days of gestation (term, 145 days). Four groups of animals [n = 5–6 in each group; (i) control; (ii) fetal THX; (iii) fetal PAB; and (iv) fetal PAB + THX] were monitored for 1 week prior to being killed. Fetal heart rate was significantly lower in the two THX groups compared with the non-THX groups, while mean arterial blood pressure was similar among groups. Combined left and right ventricle free wall + septum weight, expressed per kilogram of fetal weight, was significantly increased in PAB (6.27 ± 0.85 g kg–1) compared with control animals (4.72 ± 0.12 g kg–1). Thyroidectomy significantly attenuated the increase in cardiac mass associated with PAB (4.94 ± 0.13 g kg–1), while THX alone had no detectable effect on heart mass (4.95 ± 0.27 g kg–1). The percentage of binucleated cardiomyocytes was significantly decreased in THX and PAB +THX groups (~16%) compared with the non-THX groups (~27%). No differences in levels of activated Akt, extracellular signal-regulated kinase or c-Jun N-terminal kinase were detected among the groups. Markers of cellular proliferation but not apoptosis or expression of growth-related genes were lower in the THX and THX+ PAB groups relative to thyroid-intact animals. These findings suggest that in the late-gestation fetal heart, thyroid hormone has important cellular growth functions in both physiological and pathophysiological states. Specifically, thyroid hormone is required for adaptive fetal cardiac growth in response to pressure overload.

The formation of the cryopyrin inflammasome in the heart induces an intense inflammatory response during acute myocardial infarction (AMI), which mediates further damage and promotes adverse cardiac remodelling. Active interleukin-1β (IL-1β) is a key product of the inflammasome, being cleaved by active caspase-1. The aim of this study was to dissect the role of IL-1β from that of the inflammasome by using a neutralizing monoclonal antibody directed against IL-1β and measuring the intensity of the inflammatory response, the activity of caspase-1 in the inflammasome, cardiomyocyte apoptosis and cardiac remodelling in a mouse model of non-reperfused AMI. A mouse monoclonal IgG2a antibody directed against IL-1β (IL-1β-AB; 10 mg kg–1) was given i.p. immediately after surgery and repeated 1 week later. Cardiac tissue was analysed at 72 h after surgery in a subgroup of mice for inflammasome aggregates and caspase-1 activity (inflammasome) and for DNA fragmentation and caspase-3 activity (apoptosis). All sham-operated mice were alive at 10 weeks, whereas 40% of the control-antibody-treated mice and 30% of the IL-1β-AB-treated mice died during the 4 weeks after surgery. When compared with vehicle, treatment with the IL-1β-AB did not affect inflammasome formation or caspase-1 activation in the heart tissue at 72 h after AMI nor circulating plasma IL-6 levels, but did inhibit cardiomyocyte apoptosis, limit left ventricular enlargement by 40% (P < 0.01) and improve systolic dysfunction by 17% (P < 0.01) after AMI. These findings suggest that IL-1β mediates the deleterious effects on the heart during the sterile inflammatory response.

Insulin resistance, which characterizes type 2 diabetes, is associated with reduced translocation of glucose transporter 4 (GLUT4) to the plasma membrane following insulin stimulation, and diabetic patients with insulin resistance show a higher incidence of ischaemia, arrhythmias and sudden cardiac death. The aim of this study was to examine whether GLUT4 deficiency leads to more severe alterations in cardiac electrical activity during cardiac stress due to hypoxia. To fulfil this aim, we compared cardiac electrical activity from cardiac-selective GLUT4-ablated (G4H–/–) mouse hearts and corresponding control (CTL) littermates. A custom-made cylindrical ‘cage' electrode array measured potentials (Ves) from the epicardium of isolated, perfused mouse hearts. The normalized average of the maximal downstroke of Ves (|dVes/dtmin|na), which we previously introduced as an index of electrical activity in normal, ischaemic and hypoxic hearts, was used to assess the effects of GLUT4 deficiency on electrical activity. The |dVes/dtmin|na of G4H–/– and CTL hearts decreased by 75 and 47%, respectively (P < 0.05), 30 min after the onset of hypoxia. Administration of insulin attenuated decreases in values of |dVes/dtmin|na in G4H–/– hearts as well as in CTL hearts, during hypoxia. In general, however, G4H–/– hearts showed a severe alteration of the propagation sequence and a prolonged total activation time. Results of this study demonstrate that reduced glucose availability associated with insulin resistance and a reduction in GLUT4-mediated glucose transport impairs electrical activity during hypoxia, and may contribute to cardiac vulnerability to arrhythmias in diabetic patients.