Experimental Physiology

Secretin has been implicated in cardiovascular regulation through its specific receptors, as well as through β-adrenoceptors and nitric oxide, although data on its direct effect on coronary blood flow and cardiac function have remained scarce. The present study aimed to determine the primary in vivo effect of secretin on cardiac function and perfusion and the mechanisms related to the autonomic nervous system, secretin receptors and NO. In addition, in coronary endothelial cells the intracellular pathways involved in the effects of secretin on NO release were also examined. In 30 pigs, intracoronary secretin infusion at 2.97 pg for each millilitre per minute of coronary blood flow at constant heart rate and aortic blood pressure increased coronary blood flow, maximal rate of change of left ventricular pressure, segmental shortening, cardiac output and coronary NO release (P < 0.05). These responses were graded in a further five pigs. Moreover, while blockade of muscarinic cholinoreceptors (n = 5) and of α-adrenoceptors (n = 5) did not abolish the observed responses to secretin, blockade of β1-adrenoceptors (n = 5) prevented the effects of secretin on cardiac function. In addition, blockade of β2-adrenoceptors (n = 5) and NO synthase inhibition (n = 5) prevented the coronary response and the effect of secretin on NO release. All these effects were abolished by a secretin receptor inhibitor (n = 5). In coronary endothelial cells, the increased NO production caused by secretin was found to be related to cAMP/protein kinase A signalling activated as downstream effectors of stimulation of secretin receptors and β2-adrenoceptors. In conclusion, in anaesthetized pigs secretin primarily increased cardiac function and perfusion through the involvement of specific receptors, β-adrenoceptors and NO release.

Exercise hyperaemia is regulated by several factors, and one factor known to increase with exercise that evokes a powerful vasomotor action is extracellular ATP. The origin of ATP detected in plasma from exercising muscle of humans is, however, a matter of debate, and ATP has been suggested to arise from sympathetic nerves, blood sources (e.g. erythrocytes), endothelial cells and skeletal myocytes, among others. Therefore, we tested the hypothesis that acute augmentation of sympathetic nervous system activity (SNA) results in elevated plasma ATP draining skeletal muscle, and that SNA superimposition during exercise increases ATP more than exercise alone. We showed that increased SNA via –40 mmHg lower body negative pressure (LBNP) at rest did not increase plasma ATP (51 ± 8 nmol l–1 at rest versus 58 ± 7 nmol l–1 with LBNP), nor did it increase [ATP] above levels observed during rhythmic hand-grip exercise (79 ± 11 nmol l–1 with exercise alone versus 71 ± 8 nmol l–1 with LBNP). Next, we tested the hypothesis that active perfusion of skeletal muscle is essential to observe increased plasma ATP during exercise. We showed that complete obstruction of blood flow to contracting muscle abolished exercise-mediated increases in plasma ATP (from 90 ± 19 to 49 ± 12 nmol l–1), and that cessation of blood flow prior to exercise completely inhibited the typical rise in ATP (3 versus 61%, obstructed versus intact perfusion). The lack of change in ATP during occlusion occurred in the face of continued muscular work and elevated SNA, indicating that the rise of intravascular ATP did not result from these extravascular sources. Our collective observations indicated that the elevation in extracellular ATP observed in blood during exercise was unlikely to originate from sympathetic nerves or the contacting muscle itself, but rather was dependent on intact skeletal muscle perfusion. We conclude that an intravascular source for ATP is essential, which indicates an important role for blood sources (e.g. red blood cells) in augmenting and maintaining elevated plasma ATP during exercise.

Peroxisome proliferator-activated receptor  coactivator-1α (PGC-1α) is emerging as a novel factor that plays a critical role in integrating signalling pathways in the control of cellular and systemic metabolism. We investigated the role of vascular expression of PGC-1α and related factors, such as sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor  (PPAR) and adiponectin, during the atherosclerotic process. Endothelial function, vascular superoxide anion production and inflammatory mediators were also evaluated. This study was carried out in male New Zealand rabbits fed a diet containing 0.5% cholesterol and 14% coconut oil for 8 weeks. Animals developed mixed dyslipidaemia and atherosclerotic lesions, which were associated with endothelial dysfunction, aortic overproduction of superoxide anions and inflammation. Expression of PGC-1α, SIRT1, PPAR and adiponectin was reduced (P < 0.05) in aorta from atherosclerotic rabbits. Levels of PGC-1α were correlated negatively (P < 0.05) with total cholesterol levels, aortic superoxide anion production and tumour necrosis factor-α expression, and positively (P < 0.05) with maximal relaxation in response to acetylcholine. The observed results suggest that PGC-1α could be considered to be a link between the main atherosclerotic processes (endothelial dysfunction, oxidation and inflammation) and alterations of other factors involved in vascular wall integrity, such as SIRT1, PPAR and adiponectin.

Blebbistatin (BS) is a recently discovered inhibitor of the myosin II isoform and has been adopted as the mechanical uncoupler of choice for optical mapping, because previous studies suggest that BS has no significant cardiac electrophysiological effects in a number of species. The aim of this study was to determine whether BS affects cardiac electrophysiology in isolated New Zealand White rabbit hearts. Langendorff-perfused hearts (n = 39) in constant-flow mode had left ventricular monophasic action potential duration (MAPD) measured at apical and basal regions during constant pacing (300 ms cycle length). Standard action potential duration restitution was obtained using the single extrastimulus method with measurement of the maximal restitution slope. Ventricular fibrillation threshold was measured as the minimal current inducing sustained ventricular fibrillation with burst pacing (30 stimuli, at 30 ms intervals). Optical action potentials were recorded using the voltage-sensitive dye di-4-ANEPPS. Measurements were taken at baseline and after 60 min perfusion with BS (5 μm). Blebbistatin significantly prolonged left ventricular apical (mean ± SEM; from 129.9 ± 2.9 to 170.7 ± 4.1 ms, P < 0.001, n = 8) and basal MAPD (from 135.0 ± 2.3 to 163.3 ± 5.6 ms, P < 0.001) and effective refractory period (from 141.3 ± 4.8 to 175.6 ± 3.7 ms, P < 0.001) whilst increasing the maximal slope of restitution (apex, from 0.79 ± 0.09 to 1.57 ± 0.16, P < 0.001; and base, from 0.71 ± 0.06 to 1.44 ± 0.24, P < 0.001) and ventricular fibrillation threshold (from 5.3 ± 1.1 to 17.0 ± 2.9 mA, P < 0.001). In other hearts, blebbistatin significantly prolonged optically recorded action potentials (from 136.5 ± 6.3 to 173.0 ± 7.9 ms, P < 0.05, n = 4). In control experiments, the increase of MAPD with blebbistatin was present whether the hearts were perfused in constant-pressure mode (n = 5) or in unloaded conditions (n = 5). These data show that blebbistatin significantly affects cardiac electrophysiology. Its use in optical mapping studies should be treated with caution.

Despite debate regarding its cardioprotective and pro-arrhythmic effects, the precise mechanisms of action of rosiglitazone on the heart are still unclear. We determined the mechanistic effects of rosiglitazone on cardiac function, arrhythmias and infarct size during cardiac ischaemia–reperfusion. Twenty-six rats were used. In each rat, either rosiglitazone or saline solution was administered intravenously prior to a 30 min left anterior descending coronary artery ligation and a 120 min reperfusion. Cardiac function, infarct size, myocardial levels of connexin43, Bax/Bcl-2, cytochrome c, caspase-3, caspase-8, Akt, tumour necrosis factor-α and interleukin-4 and cardiac mitochondrial function were determined. Isolated cardiomyocytes were used for studying intracellular calcium. Rosiglitazone did not alter cardiac function during the ischaemia–reperfusion periods, but increased the arrhythmia score and mortality rate, decreased the time to onset of ventricular fibrillation and prolonged the Ca2+ decay rate, in comparison to the saline-injected group (P < 0.05). However, the infarct size in the rosiglitazone-injected group was reduced (P < 0.05). Rosiglitazone decreased the levels of connexin43 phosphorylation, active caspase-8 and tumour necrosis factor-α, but increased the level of procaspase-3. However, levels of Bax/Bcl-2, cytochrome c, Akt and interleukin-4 and the cardiac mitochondrial function were not different between the two groups. Rosiglitazone simultaneously exerted both beneficial and adverse cardiac effects in the heart exposed to ischaemia–reperfusion. Although it decreased the infarct size via the extrinsic anti-apoptotic pathway and anti-inflammatory effects, rosiglitazone facilitated a fatal arrhythmia by decreasing connexin43 phosphorylation and prolonging the Ca2+ decay rate in ischaemia–reperfusion. The higher mortality rate in the rosiglitazone-injected group suggests that its undesirable effect was more pronounced than its benefit on infarct size reduction.

Skeletal muscle is a highly dynamic tissue that responds to endogenous and external stimuli, including alterations in mechanical loading and growth factors. In particular, the antigravity soleus muscle experiences significant muscle atrophy during disuse and extensive muscle damage upon reloading. Given that insulin-like growth factor-1 (IGF-1) has been implicated as a central regulator of muscle repair and modulation of muscle size, we examined the effect of virally mediated overexpression of IGF-1 on the soleus muscle following hindlimb cast immobilization and upon reloading. Recombinant IGF-1 cDNA virus was injected into one of the posterior hindlimbs of the mice, while the contralateral limb was injected with saline (control). At 20 weeks of age, both hindlimbs were immobilized for 2 weeks to induce muscle atrophy in the soleus and ankle plantarflexor muscle group. Subsequently, the mice were allowed to reambulate, and muscle damage and recovery were monitored over a period of 2–21 days. The primary finding of this study was that IGF-1 overexpression attenuated reloading-induced muscle damage in the soleus muscle, and accelerated muscle regeneration and force recovery. Muscle T2 assessed by magnetic resonance imaging, a non-specific marker of muscle damage, was significantly lower in IGF-1-injected compared with contralateral soleus muscles at 2 and 5 days reambulation (P < 0.05). The reduced prevalence of muscle damage in IGF-1-injected soleus muscles was confirmed on histology, with a lower fractional area of abnormal muscle tissue in IGF-1-injected muscles at 2 days reambulation (33.2 ± 3.3 versus 54.1 ± 3.6%, P < 0.05). Evidence of the effect of IGF-1 on muscle regeneration included timely increases in the number of central nuclei (21% at 5 days reambulation), paired-box transcription factor 7 (36% at 5 days), embryonic myosin (37% at 10 days) and elevated MyoD mRNA (7-fold at 2 days) in IGF-1-injected limbs (P < 0.05). These findings demonstrate a potential role of IGF-1 in protecting unloaded skeletal muscles from damage and accelerating muscle repair and regeneration.

Understanding body weight regulation is essential to fight obesity. Mouse studies, using different types of diets, showed conflicting results in terms of body weight persistence after changing from an ad libitum high-fat diet to an ad libitum low-fat diet. In this study, we questioned specifically whether the energy content of the diet has a lasting effect on energy balance and body weight, using multiple switches and two purified diets with a different fat-to-sugar ratio, but otherwise identical ingredients. Young-adult obesity-prone male C57BL/6J mice were fed single or double switches of semi-purified diets with either 10 energy % (en%) fat (LF) or 40en% fat (HF), with starch replaced by fat, while protein content remained equal. After none, one or two dietary changes, energy metabolism was assessed at 5, 14 and 19 weeks. We observed no systematic continuous compensation in diet and energy intake when returning to LF after HF consumption. Body weight, white adipose tissue mass and histology, serum metabolic parameters, energy expenditure and substrate usage all significantly reflected the current diet intake, independent of dietary changes. This contrasts with studies that used diets with different ingredients and showed persistent effects of dietary history on body weight, suggesting diet-dependent metabolic set points. We conclude that body weight and metabolic parameters ‘settle', based on current energetic input and output. This study also highlights the importance of considering the choice of diet in physiological and metabolic intervention studies.

During exercise, the cardiovascular response is rapidly and appropriately matched to the intensity of the physical activity. The autonomic nervous system plays an important role in achieving this closely matched circulatory response by an increase in the sympathetic nerve activity to the heart, blood vessels and adrenal medulla and a decrease in the parasympathetic nerve activity to the heart. Early insights into the mechanisms that controlled these cardiovascular changes during exercise were reported in the 19th century. At that time, two mechanisms were hypothesized to be responsible for these changes. In one mechanism, a signal arising in a central area of the brain causes a parallel activation of skeletal muscle contraction and of autonomic nervous system changes (now termed ‘central command'). In the other mechanism, a signal arising in the contracting skeletal muscle causes a reflex activation of the autonomic nervous system changes (now termed ‘exercise pressor reflex'). Some important investigators involved in early studies include Johan Johansson, August Krogh, Johannes Lindhard and Horace Smirk. Also, Florence Buchanan and Louis Fridericia should be recognized for their contributions. In more recent years, the important involvement of a third mechanism, the arterial baroreflex, has been elucidated. Since those early insights, experiments in both animals and humans have added important findings that strongly support these early hypotheses.

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.