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1.  Alteration in circulating metabolites during and after heat stress in the conscious rat: potential biomarkers of exposure and organ-specific injury 
BMC Physiology  2014;14(1):14.
Heat illness is a debilitating and potentially life-threatening condition. Limited data are available to identify individuals with heat illness at greatest risk for organ damage. We recently described the transcriptomic and proteomic responses to heat injury and recovery in multiple organs in an in vivo model of conscious rats heated to a maximum core temperature of 41.8°C (Tc,Max). In this study, we examined changes in plasma metabolic networks at Tc,Max, 24, or 48 hours after the heat stress stimulus.
Circulating metabolites were identified by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry. Bioinformatics analysis of the metabolomic data corroborated proteomics and transcriptomics data in the tissue at the pathway level, supporting modulations in metabolic networks including cell death or catabolism (pyrimidine and purine degradation, acetylation, sulfation, redox alterations and glutathione metabolism, and the urea cycle/creatinine metabolism), energetics (stasis in glycolysis and tricarboxylic acid cycle, β-oxidation), cholesterol and nitric oxide metabolism, and bile acids. Hierarchical clustering identified 15 biochemicals that differentiated animals with histopathological evidence of cardiac injury at 48 hours from uninjured animals. The metabolic networks perturbed in the plasma corroborated the tissue proteomics and transcriptomics pathway data, supporting a model of irreversible cell death and decrements in energetics as key indicators of cardiac damage in response to heat stress.
Integrating plasma metabolomics with tissue proteomics and transcriptomics supports a diagnostic approach to assessing individual susceptibility to organ injury and predicting recovery after heat stress.
Electronic supplementary material
The online version of this article (doi:10.1186/s12899-014-0014-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4306243  PMID: 25623799
Heat stress; Metabolomics; Systems biology; Energetics; Metabolic networks
2.  CaMKII content affects contractile, but not mitochondrial, characteristics in regenerating skeletal muscle 
BMC Physiology  2014;14(1):7.
The multi-meric calcium/calmodulin-dependent protein kinase II (CaMKII) is the main CaMK in skeletal muscle and its expression increases with endurance training. CaMK family members are implicated in contraction-induced regulation of calcium handling, fast myosin type IIA expression and mitochondrial biogenesis. The objective of this study was to investigate the role of an increased CaMKII content for the expression of the contractile and mitochondrial phenotype in vivo. Towards this end we attempted to co-express alpha- and beta-CaMKII isoforms in skeletal muscle and characterised the effect on the contractile and mitochondrial phenotype.
Fast-twitch muscle m. gastrocnemius (GM) and slow-twitch muscle m. soleus (SOL) of the right leg of 3-month old rats were transfected via electro-transfer of injected expression plasmids for native α/β CaMKII. Effects were identified from the comparison to control-transfected muscles of the contralateral leg and non-transfected muscles. α/β CaMKII content in muscle fibres was 4-5-fold increased 7 days after transfection. The transfection rate was more pronounced in SOL than GM muscle (i.e. 12.6 vs. 3.5%). The overexpressed α/β CaMKII was functional as shown through increased threonine 287 phosphorylation of β-CaMKII after isometric exercise and down-regulated transcripts COXI, COXIV, SDHB after high-intensity exercise in situ. α/β CaMKII overexpression under normal cage activity accelerated excitation-contraction coupling and relaxation in SOL muscle in association with increased SERCA2, ANXV and fast myosin type IIA/X content but did not affect mitochondrial protein content. These effects were observed on a background of regenerating muscle fibres.
Elevated CaMKII content promotes a slow-to-fast type fibre shift in regenerating muscle but is not sufficient to stimulate mitochondrial biogenesis in the absence of an endurance stimulus.
PMCID: PMC4277655  PMID: 25515219
CaMKII; Skeletal muscle; Plasticity; Excitation; Contraction; Mitochondria
3.  Big data in wildlife research: remote web-based monitoring of hibernating black bears 
BMC Physiology  2014;14(1):13.
Numerous innovations for the management and collection of “big data” have arisen in the field of medicine, including implantable computers and sensors, wireless data transmission, and web-based repositories for collecting and organizing information. Recently, human clinical devices have been deployed in captive and free-ranging wildlife to aid in the characterization of both normal physiology and the interaction of animals with their environment, including reactions to humans. Although these devices have had a significant impact on the types and quantities of information that can be collected, their utility has been limited by internal memory capacities, the efforts required to extract and analyze information, and by the necessity to handle the animals in order to retrieve stored data.
We surgically implanted miniaturized cardiac monitors (1.2 cc, Reveal LINQ™, Medtronic Inc.), a newly developed human clinical system, into hibernating wild American black bears (N = 6). These devices include wireless capabilities, which enabled frequent transmissions of detailed physiological data from bears in their remote den sites to a web-based data storage and management system. Solar and battery powered telemetry stations transmitted detailed physiological data over the cellular network during the winter months. The system provided the transfer of large quantities of data in near-real time. Observations included changes in heart rhythms associated with birthing and caring for cubs, and in all bears, long periods without heart beats (up to 16 seconds) occurred during each respiratory cycle.
For the first time, detailed physiological data were successfully transferred from an animal in the wild to a web-based data collection and management system, overcoming previous limitations on the quantities of data that could be transferred. The system provides an opportunity to detect unusual events as they are occurring, enabling investigation of the animal and site shortly afterwards. Although the current study was limited to bears in winter dens, we anticipate that future systems will transmit data from implantable monitors to wearable transmitters, allowing for big data transfer on non-stationary animals.
PMCID: PMC4277652  PMID: 25496699
American black bear; Hibernation physiology; Heart rate; Implantable cardiac monitor; Wireless data transmission
4.  Identification of novel Kirrel3 gene splice variants in adult human skeletal muscle 
BMC Physiology  2014;14(1):11.
Multiple cell types including trophoblasts, osteoclasts and myoblasts require somatic cell fusion events as part of their physiological functions. In Drosophila Melanogaster the paralogus type 1 transmembrane receptors and members of the immunoglobulin superfamily Kin of Irre (Kirre) and roughest (Rst) regulate myoblast fusion during embryonic development. Present within the human genome are three homologs to Kirre termed Kin of Irre like (Kirrel) 1, 2 and 3. Currently it is unknown if Kirrel3 is expressed in adult human skeletal muscle.
We investigated (using PCR and Western blot) Kirrel3 in adult human skeletal muscle samples taken at rest and after mild exercise induced muscle damage. Kirrel3 mRNA expression was verified by sequencing and protein presence via blotting with 2 different anti-Kirrel3 protein antibodies. Evidence for three alternatively spliced Kirrel3 mRNA transcripts in adult human skeletal muscle was obtained. Kirrel3 mRNA in adult human skeletal muscle was detected at low or moderate levels, or not at all. This sporadic expression suggests that Kirrel3 is expressed in a pulsatile manner. Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.
The results presented here demonstrate for the first time that there are at least 3 splice variants of Kirrel3 expressed in adult human skeletal muscle, two of which have never previously been identified in human muscle. Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance. These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.
PMCID: PMC4269076  PMID: 25488023
Kirrel3; Human; Myogenesis; Biopsy; Drosophila
5.  Acetate transiently inhibits myocardial contraction by increasing mitochondrial calcium uptake 
BMC Physiology  2014;14(1):12.
There is a close relationship between cardiovascular disease and cardiac energy metabolism, and we have previously demonstrated that palmitate inhibits myocyte contraction by increasing Kv channel activity and decreasing the action potential duration. Glucose and long chain fatty acids are the major fuel sources supporting cardiac function; however, cardiac myocytes can utilize a variety of substrates for energy generation, and previous studies demonstrate the acetate is rapidly taken up and oxidized by the heart. In this study, we tested the effects of acetate on contractile function of isolated mouse ventricular myocytes.
Acute exposure of myocytes to 10 mM sodium acetate caused a marked, but transient, decrease in systolic sarcomere shortening (1.49 ± 0.20% vs. 5.58 ± 0.49% in control), accompanied by a significant increase in diastolic sarcomere length (1.81 ± 0.01 μm vs. 1.77 ± 0.01 μm in control), with a near linear dose response in the 1–10 mM range. Unlike palmitate, acetate caused no change in action potential duration; however, acetate markedly increased mitochondrial Ca2+ uptake. Moreover, pretreatment of cells with the mitochondrial Ca2+ uptake blocker, Ru-360 (10 μM), markedly suppressed the effect of acetate on contraction.
Lehninger and others have previously demonstrated that the anions of weak aliphatic acids such as acetate stimulate Ca2+ uptake in isolated mitochondria. Here we show that this effect of acetate appears to extend to isolated cardiac myocytes where it transiently modulates cell contraction.
PMCID: PMC4274725  PMID: 25488103
6.  White-nose syndrome initiates a cascade of physiologic disturbances in the hibernating bat host 
BMC Physiology  2014;14(1):10.
The physiological effects of white-nose syndrome (WNS) in hibernating bats and ultimate causes of mortality from infection with Pseudogymnoascus (formerly Geomyces) destructans are not fully understood. Increased frequency of arousal from torpor described among hibernating bats with late-stage WNS is thought to accelerate depletion of fat reserves, but the physiological mechanisms that lead to these alterations in hibernation behavior have not been elucidated. We used the doubly labeled water (DLW) method and clinical chemistry to evaluate energy use, body composition changes, and blood chemistry perturbations in hibernating little brown bats (Myotis lucifugus) experimentally infected with P. destructans to better understand the physiological processes that underlie mortality from WNS.
These data indicated that fat energy utilization, as demonstrated by changes in body composition, was two-fold higher for bats with WNS compared to negative controls. These differences were apparent in early stages of infection when torpor-arousal patterns were equivalent between infected and non-infected animals, suggesting that P. destructans has complex physiological impacts on its host prior to onset of clinical signs indicative of late-stage infections. Additionally, bats with mild to moderate skin lesions associated with early-stage WNS demonstrated a chronic respiratory acidosis characterized by significantly elevated dissolved carbon dioxide, acidemia, and elevated bicarbonate. Potassium concentrations were also significantly higher among infected bats, but sodium, chloride, and other hydration parameters were equivalent to controls.
Integrating these novel findings on the physiological changes that occur in early-stage WNS with those previously documented in late-stage infections, we propose a multi-stage disease progression model that mechanistically describes the pathologic and physiologic effects underlying mortality of WNS in hibernating bats. This model identifies testable hypotheses for better understanding this disease, knowledge that will be critical for defining effective disease mitigation strategies aimed at reducing morbidity and mortality that results from WNS.
Electronic supplementary material
The online version of this article (doi:10.1186/s12899-014-0010-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4278231  PMID: 25487871
White-nose syndrome; Bats; Doubly labeled water
7.  Differential role of STIM1 and STIM2 during transient inward (Tin) current generation and the maturation process in the Xenopus oocyte 
BMC Physiology  2014;14(1):9.
The Xenopus oocyte is a useful cell model to study Ca2+ homeostasis and cell cycle regulation, two highly interrelated processes. Here, we used antisense oligonucleotides to investigate the role in the oocyte of stromal interaction molecule (STIM) proteins that are fundamental elements of the store-operated calcium-entry (SOCE) phenomenon, as they are both sensors for Ca2+ concentration in the intracellular reservoirs as well as activators of the membrane channels that allow Ca2+ influx.
Endogenous STIM1 and STIM2 expression was demonstrated, and their synthesis was knocked down 48–72 h after injecting oocytes with specific antisense sequences. Selective elimination of their mRNA and protein expression was confirmed by PCR and Western blot analysis, and we then evaluated the effect of their absence on two endogenous responses: the opening of SOC channels elicited by G protein-coupled receptor (GPCR)-activated Ca2+ release, and the process of maturation stimulated by progesterone. Activation of SOC channels was monitored electrically by measuring the Tin response, a Ca2+-influx-dependent Cl− current, while maturation was assessed by germinal vesicle breakdown (GVBD) scoring and electrophysiology.
It was found that STIM2, but not STIM1, was essential in both responses, and Tin currents and GVBD were strongly reduced or eliminated in cells devoid of STIM2; STIM1 knockdown had no effect on the maturation process, but it reduced the Tin response by 15 to 70%. Thus, the endogenous SOCE response in Xenopus oocytes depended mainly on STIM2, and its expression was necessary for entry into meiosis induced by progesterone.
Electronic supplementary material
The online version of this article (doi:10.1186/s12899-014-0009-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4236480  PMID: 25399338
SOCE; STIM1; STIM2; Xenopus oocyte; Ca2+-entry; Maturation
8.  Longevity of Daphnia and the attenuation of stress responses by melatonin 
BMC Physiology  2014;14(1):8.
The widespread occurrence of melatonin in prokaryotes as well as eukaryotes indicates that this indoleamine is considerably old. This high evolutionary age has led to the development of diverse functions of melatonin in different organisms, such as the detoxification of reactive oxygen species and anti-stress effects. In insects, i.e. Drosophila, the addition of melatonin has also been shown to increase the life span of this arthropod, probably by reducing age-related increasing oxidative stress.
Although the presence of melatonin was recently found to exist in the ecological and toxicological model organism Daphnia, its function in this cladoceran has thus far not been addressed. Therefore, we challenged Daphnia with three different stressors in order to investigate potential stress-response attenuating effects of melatonin. i) Female and male daphnids were exposed to melatonin in a longevity experiment, ii) Daphnia were confronted with stress signals from the invertebrate predator Chaoborus sp., and iii) Daphnia were grown in high densities, i.e. under crowding-stress conditions.
In our experiments we were able to show that longevity of daphnids was not affected by melatonin. Therefore, age-related increasing oxidative stress was probably not compensated by added melatonin. However, melatonin significantly attenuated Daphnia’s response to acute predator stress, i.e. the formation of neckteeth which decrease the ability of the gape-limited predator Chaoborus sp. to handle their prey. In addition, melatonin decreased the extent of crowding-related production of resting eggs of Daphnia.
Our results confirm the effect of melatonin on inhibition of stress-signal responses of Daphnia. Until now, only a single study demonstrated melatonin effects on behavioral responses due to vertebrate kairomones, whereas we clearly show a more general effect of melatonin: i) on morphological predator defense induced by an invertebrate kairomone and ii) on life history characteristics transmitted by chemical cues from conspecifics. Therefore, we could generally confirm that melatonin plays a role in the attenuation of responses to different stressors in Daphnia.
PMCID: PMC4226899  PMID: 25373613
Daphnia; Chaoborus kairomone; Melatonin; Crowding; Longevity; Stress response
9.  MAPK-activated protein kinase 2-deficiency causes hyperacute tumor necrosis factor-induced inflammatory shock 
BMC Physiology  2014;14(1):5.
MAPK-activated protein kinase 2 (MK2) plays a pivotal role in the cell response to (inflammatory) stress. Among others, MK2 is known to be involved in the regulation of cytokine mRNA metabolism and regulation of actin cytoskeleton dynamics. Previously, MK2-deficient mice were shown to be highly resistant to LPS/d-Galactosamine-induced hepatitis. Additionally, research in various disease models has indicated the kinase as an interesting inhibitory drug target for various acute or chronic inflammatory diseases.
We show that in striking contrast to the known resistance of MK2-deficient mice to a challenge with LPS/D-Gal, a low dose of tumor necrosis factor (TNF) causes hyperacute mortality via an oxidative stress driven mechanism. We identified in vivo defects in the stress fiber response in endothelial cells, which could have resulted in reduced resistance of the endothelial barrier to deal with exposure to oxidative stress. In addition, MK2-deficient mice were found to be more sensitive to cecal ligation and puncture-induced sepsis.
The capacity of the endothelial barrier to deal with inflammatory and oxidative stress is imperative to allow a regulated immune response and maintain endothelial barrier integrity. Our results indicate that, considering the central role of TNF in pro-inflammatory signaling, therapeutic strategies examining pharmacological inhibition of MK2 should take potentially dangerous side effects at the level of endothelial barrier integrity into account.
PMCID: PMC4160542  PMID: 25185746
MK2; Inflammatory shock; Tumor necrosis factor; Reactive oxygen species; Actin cytoskeleton; Endothelial permeability; Cecal ligation and puncture
10.  Impact of maternal dietary fat supplementation during gestation upon skeletal muscle in neonatal pigs 
BMC Physiology  2014;14(1):6.
Maternal diet during pregnancy can modulate skeletal muscle development of the offspring. Previous studies in pigs have indicated that a fat supplemented diet during pregnancy can improve piglet outcome, however, this is in contrast to human studies suggesting adverse effects of saturated fats during pregnancy. This study aimed to investigate the impact of a fat supplemented (palm oil) “high fat” diet on skeletal muscle development in a porcine model. Histological and metabolic features of the biceps femoris muscle obtained from 7-day-old piglets born to sows assigned to either a commercial (C, n = 7) or to an isocaloric fat supplementation diet (“high fat” HF, n = 7) during pregnancy were assessed.
Offspring exposed to a maternal HF diet demonstrated enhanced muscular development, reflected by an increase in fractional growth rate, rise in myofibre cross-sectional area, increased storage of glycogen and reduction in lipid staining of myofibres. Although both groups had similar intramuscular protein and triglyceride concentrations, the offspring born to HF mothers had a higher proportion of arachidonic acid (C20:4n6) and a reduction in α-linolenic acid (C18:3n3) compared to C group offspring. The HF group muscle also exhibited a higher ratio of C20:3n6 to C20:4n6 and total n-6 to n-3 in conjunction with up-regulation of genes associated with free fatty acid uptake and biogenesis.
In conclusion, a HF gestational diet accelerates the maturation of offspring biceps femoris muscle, reflected in increased glycolytic metabolism and fibre cross sectional area, differences accompanied with a potential resetting of myofibre nutrient uptake.
PMCID: PMC4243771  PMID: 25189710
Nutrition; Muscle; Fetal development; Growth
11.  Sympathoactivation and rho-kinase-dependent baroreflex function in experimental renovascular hypertension with reduced kidney mass 
BMC Physiology  2014;14:4.
Dysregulation of the autonomic nervous system is frequent in subjects with cardiovascular disease. The contribution of different forms of renovascular hypertension and the mechanisms contributing to autonomic dysfunction in hypertension are incompletely understood. Here, murine models of renovascular hypertension with preserved (2-kidneys-1 clip, 2K1C) and reduced (1-kidney-1 clip, 1K1C) kidney mass were studied with regard to autonomic nervous system regulation (sympathetic tone: power-spectral analysis of systolic blood pressure; parasympathetic tone: power-spectral analysis of heart rate) and baroreflex sensitivity of heart rate by spontaneous, concomitant changes of systolic blood pressure and pulse interval. Involvement of the renin-angiotensin system and the rho-kinase pathway were determined by application of inhibitors.
C57BL6N mice (6 to 11) with reduced kidney mass (1K1C) or with preserved kidney mass (2K1C) developed a similar degree of hypertension. In comparison to control mice, both models presented with a significantly increased sympathetic tone and lower baroreflex sensitivity of heart rate. However, only 2K1C animals had a lower parasympathetic tone, whereas urinary norepinephrine excretion was reduced in the 1K1C model. Rho kinase inhibition given to a subset of 1K1C and 2K1C animals improved baroreflex sensitivity of heart rate selectively in the 1K1C model. Rho kinase inhibition had no additional effects on autonomic nervous system in either model of renovascular hypertension and did not change the blood pressure. Blockade of AT1 receptors (in 2K1C animals) normalized the sympathetic tone, decreased resting heart rate, improved baroreflex sensitivity of heart rate and parasympathetic tone.
Regardless of residual renal mass, blood pressure and sympathetic tone are increased, whereas baroreflex sensitivity is depressed in murine models of renovascular hypertension. Reduced norepinephrine excretion and/or degradation might contribute to sympathoactivation in renovascular hypertension with reduced renal mass (1K1C). Overall, the study helps to direct research to optimize medical therapy of hypertension.
PMCID: PMC4074138  PMID: 24946879
Arterial hypertension; Sympathetic nervous system; Baroreflex; Irbesartan
12.  Reductions in expression of growth regulating genes in skeletal muscle with age in wild type and myostatin null mice 
BMC Physiology  2014;14:3.
Genes that decline in expression with age and are thought to coordinate growth cessation have been identified in various organs, but their expression in skeletal muscle is unknown. Therefore, our objective was to determine expression of these genes (Ezh2, Gpc3, Mdk, Mest, Mycn, Peg3, and Plagl1) in skeletal muscle from birth to maturity. We hypothesized that expression of these genes would decline with age in skeletal muscle but differ between sexes and between wild type and myostatin null mice.
Female and male wild type and myostatin null mice (C57BL/6J background) were sacrificed by carbon dioxide asphyxiation followed by decapitation at d -7, 0, 21, 42, and 70 days of age. Whole bodies at d -7, all muscles from both hind limbs at d 0, and bicep femoris muscle from d 21, 42 and 70 were collected. Gene expression was determined by quantitative real-time PCR. In general, expression of these growth-regulating genes was reduced at d 21 compared with day 0 and d -7. Expression of Gpc3, Mest, and Peg3 was further reduced at d 42 and 70 compared with d 21, however the expression of Mycn increased from d 21 to d 42 and 70. Myostatin null mice, as expected, were heavier with increased biceps femoris weight at d 70. However, with respect to sex and genotype, there were few differences in expression. Expression of Ezh2 was increased at d 70 and expression of Mdk was increased at d 21 in myostatin null mice compared with wild type, but no other genotype effects were present. Expression of Mdk was increased in females compared to males at d 70, but no other sex effects were present.
Overall, these data suggest the downregulation of these growth-regulating genes with age might play a role in the coordinated cessation of muscle growth similar to organ growth but likely have a limited role in the differences between sexes or genotypes.
PMCID: PMC3986603  PMID: 24678801
Ezh2; Gpc3; Mdk; Mest; Mycn; Peg3; Plagl1; Muscle growth; Myostatin
13.  Cardiac responses to elevated seawater temperature in Atlantic salmon 
BMC Physiology  2014;14:2.
Atlantic salmon aquaculture operations in the Northern hemisphere experience large seasonal fluctuations in seawater temperature. With summer temperatures often peaking around 18-20°C there is growing concern about the effects on fish health and performance. Since the heart has a major role in the physiological plasticity and acclimation to different thermal conditions in fish, we wanted to investigate how three and eight weeks exposure of adult Atlantic salmon to 19°C, previously shown to significantly reduce growth performance, affected expression of relevant genes and proteins in cardiac tissues under experimental conditions.
Transcriptional responses in cardiac tissues after three and eight weeks exposure to 19°C (compared to thermal preference, 14°C) were analyzed with cDNA microarrays and validated by expression analysis of selected genes and proteins using real-time qPCR and immunofluorescence microscopy. Up-regulation of heat shock proteins and cell signaling genes may indicate involvement of the unfolded protein response in long-term acclimation to elevated temperature. Increased immunofluorescence staining of inducible nitric oxide synthase in spongy and compact myocardium as well as increased staining of vascular endothelial growth factor in epicardium could reflect induced vascularization and vasodilation, possibly related to increased oxygen demand. Increased staining of collagen I in the compact myocardium of 19°C fish may be indicative of a remodeling of connective tissue with long-term warm acclimation. Finally, higher abundance of transcripts for genes involved in innate cellular immunity and lower abundance of transcripts for humoral immune components implied altered immune competence in response to elevated temperature.
Long-term exposure of Atlantic salmon to 19°C resulted in cardiac gene and protein expression changes indicating that the unfolded protein response, vascularization, remodeling of connective tissue and altered innate immune responses were part of the cardiac acclimation or response to elevated temperature.
PMCID: PMC3944800  PMID: 24581386
Temperature; Thermal acclimation; Cardiac tissue; Gene expression; Microarray; Immunofluorescence microscopy; iNOS; VEGF; Collagen I; Immune response
14.  Kcnq1-5 (Kv7.1-5) potassium channel expression in the adult zebrafish 
BMC Physiology  2014;14:1.
KCNQx genes encode slowly activating-inactivating K+ channels, are linked to physiological signal transduction pathways, and mutations in them underlie diseases such as long QT syndrome (KCNQ1), epilepsy in adults (KCNQ2/3), benign familial neonatal convulsions in children (KCNQ3), and hearing loss or tinnitus in humans (KCNQ4, but not KCNQ5). Identification of kcnqx potassium channel transcripts in zebrafish (Danio rerio) remains to be fully characterized although some genes have been mapped to the genome. Using zebrafish genome resources as the source of putative kcnq sequences, we investigated the expression of kcnq1-5 in heart, brain and ear tissues.
Overall expression of the kcnqx channel transcripts is similar to that found in mammals. We found that kcnq1 expression was highest in the heart, and also present in the ear and brain. kcnq2 was lowest in the heart, while kcnq3 was highly expressed in the brain, heart and ear. kcnq5 expression was highest in the ear. We analyzed zebrafish genomic clones containing putative kcnq4 sequences to identify transcripts and protein for this highly conserved member of the Kcnq channel family. The zebrafish appears to have two kcnq4 genes that produce distinct mRNA species in brain, ear, and heart tissues.
We conclude that the zebrafish is an attractive model for the study of the KCNQ (Kv7) superfamily of genes, and are important to processes involved in neuronal excitability, cardiac anomalies, epileptic seizures, and hearing loss or tinnitus.
PMCID: PMC4016485  PMID: 24555524
Zebrafish (Danio rerio); kcnq1-5; RNA transcripts; Kcnq protein; Zebrafish genome; qRTPCR; Tinnitus
16.  Inference of mechanical states of intestinal motor activity using hidden Markov models 
BMC Physiology  2013;13:14.
Contractions and relaxations of the muscle layers within the digestive tract alter the external diameter and the internal pressures. These changes in diameter and pressure move digesting food and waste products. Defining these complex relationships is a fundamental step for neurogastroenterologists to be able define normal and abnormal gut motility.
Utilising an in vitro technique that allows for the simultaneous recording of intraluminal pressure (manometry) and gut diameter (video) in an isolated section of rabbit colon, we developed a technique to help define the mechanical states of the muscle at any point in space and time during actual peristaltic movements. This was achieved by directly relating the changes in pressure to the changes in diameter along the length of the gut studied. For each individual measure of pressure or diameter, 3 dynamic state components were identified; increasing or decreasing changes or a stable period. Two additional static state components, fully contracted and fully distended, were defined for the diameter. Then qualitative mechanical states of the muscle activity were defined as combinations of these state components. A hidden Markov model was used to correlate adjacent-in-time samples, and the Viterbi algorithm was used to infer the most likely sequence of mechanical states based on the observed data. From this a spatiotemporal map of the mechanical states was produced, showing the regions of active contractions, active relaxations, or passive states along the length of the gut throughout the entire recording period.
The identification of mechanical muscles states based on gut diameter and intraluminal pressure was possible by modelling muscle activation with a hidden Markov model.
PMCID: PMC3909344  PMID: 24330642
Manometry; Peristalsis; Time-series analysis; Muscle mechanics
17.  The endotoxin/toll-like receptor-4 axis mediates gut microvascular dysfunction associated with post-prandial lipidemia 
BMC Physiology  2013;13:12.
Postprandial lipidemia is important in the development of coronary artery disease (CAD). Consumption of a meal high in monounsaturated fat was correlated with acute impairment of endothelial function. However, the mechanisms underlying impaired endothelial function in the postprandial state have not yet been elucidated. The effects of polyunsaturated fat (corn oil) and monounsaturated fat (olive oil) on vascular dysfunction in intestinal postcapillary venules and arterioles were examined in wild-type (WT) mice, mice genetically deficient in TLR4 (TLR4-/-) and mice pre-treated with antibiotics by intravital microscopy which was performed 1.0, 1.5, 2.0, 2.5 hours after oil administration. After intravital microscopy, samples of jejunum were therefore collected to test TLR4, pNF-kB p65 and SIRT1 protein expression by western blotting.
Our findings showed that feeding mono-unsaturated olive oil or polyunsaturated corn oil promoted leukocyte and platelet trafficking in the gut microvasculature, and impaired endothelium-dependent arteriolar vasodilator responses during postprandial lipidemia. The expression of TLR4, pNF-kB p65 was significantly increased in mice gavaged with olive oil at 2 h and was significantly reduced in mice gavaged for 7 days with antibiotics and in TLR4 knockout (TLR4-/-) mice. At the same time, SIRT1 protein expression is diminished by feeding olive oil for 2 h, a phenomenon that is attenuated in mice pre-treated with antibiotics and in TLR4-/- mice. Corn oil treated mice exhibited a pattern of response similar to olive oil.
Dietary oils may be negative regulators of SIRT1 which activate the innate immune response through the endotoxin/TLR4 axis. Our findings establish a link between innate immunity (i.e. the endotoxin/TLR4 axis) and epigenetic controls mediated by SIRT1 in the genesis of diet associated vascular stress.
PMCID: PMC3833857  PMID: 24219792
Microcirculation; Post-prandial lipidemia; TLR4; SIRT1
18.  Overtraining is associated with DNA damage in blood and skeletal muscle cells of Swiss mice 
BMC Physiology  2013;13:11.
The alkaline version of the single-cell gel (comet) assay is a useful method for quantifying DNA damage. Although some studies on chronic and acute effects of exercise on DNA damage measured by the comet assay have been performed, it is unknown if an aerobic training protocol with intensity, volume, and load clearly defined will improve performance without leading to peripheral blood cell DNA damage. In addition, the effects of overtraining on DNA damage are unknown. Therefore, this study aimed to examine the effects of aerobic training and overtraining on DNA damage in peripheral blood and skeletal muscle cells in Swiss mice. To examine possible changes in these parameters with oxidative stress, we measured reduced glutathione (GSH) levels in total blood, and GSH levels and lipid peroxidation in muscle samples.
Performance evaluations (i.e., incremental load and exhaustive tests) showed significant intra and inter-group differences. The overtrained (OTR) group showed a significant increase in the percentage of DNA in the tail compared with the control (C) and trained (TR) groups. GSH levels were significantly lower in the OTR group than in the C and TR groups. The OTR group had significantly higher lipid peroxidation levels compared with the C and TR groups.
Aerobic and anaerobic performance parameters can be improved in training at maximal lactate steady state during 8 weeks without leading to DNA damage in peripheral blood and skeletal muscle cells or to oxidative stress in skeletal muscle cells. However, overtraining induced by downhill running training sessions is associated with DNA damage in peripheral blood and skeletal muscle cells, and with oxidative stress in skeletal muscle cells and total blood.
PMCID: PMC3852772  PMID: 24099482
DNA damage; Aerobic training; Overtraining; Oxidative stress
19.  Estrogen-related receptor β deficiency alters body composition and response to restraint stress 
BMC Physiology  2013;13:10.
Estrogen-related receptors (ERRs) are orphan nuclear hormone receptors expressed in metabolically active tissues and modulate numerous homeostatic processes. ERRs do not bind the ligand estrogen, but they are able to bind the estrogen response element (ERE) embedded within the ERR response elements (ERREs) to regulate transcription of genes. Previous work has demonstrated that adult mice lacking Errβ have altered metabolism and meal patterns. To further understand the biological role of Errβ, we characterized the stress response of mice deficient for one or both alleles of Errβ.
Sox2-Cre:Errβ mice lack Errβ expression in all tissues of the developing embryo. Sox2-Cre:Errβ+/lox heterozygotes were obese, had increased Npy and Agrp gene expression in the arcuate nucleus of the hypothalamus, and secreted more corticosterone in response to stress. In contrast, Sox2-Cre:Errβlox/lox homozygotes were lean and, despite increased Npy and Agrp gene expression, did not secrete more corticosterone in response to stress. Sox2-Cre:Errβ+/lox and Sox2-Cre:Errβlox/lox mice treated with the Errβ and Errγ agonist DY131 demonstrated increased corticotropin-releasing hormone (Crh) expression in the paraventricular nucleus of the hypothalamus, although corticosterone levels were not affected. Nes-Cre:Errβlox/lox mice, which selectively lack Errβ expression in the nervous system, also demonstrated elevated stress response during an acoustic startle response test and decreased expression of both Crh and corticotropin-releasing hormone receptor 2 (Crhr2).
Loss of Errβ affects body composition, neuropeptide levels, stress hormones, and centrally-modulated startle responses of mice. These results indicate that Errβ alters the function of the hypothalamic-pituitary-adrenocortical axis and indicates a role for Errβ in regulating stress response.
PMCID: PMC3850731  PMID: 24053666
20.  Dynamically regulated miRNA-mRNA networks revealed by exercise 
BMC Physiology  2013;13:9.
MiRNAs are essential mediators of many biological processes. The aim of this study was to investigate the dynamics of miRNA-mRNA regulatory networks during exercise and the subsequent recovery period.
Here we monitored the transcriptome changes using microarray analysis of the whole blood of eight highly trained athletes before and after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. We combined expression profiling and bioinformatics and analysed metabolic pathways enriched with differentially expressed mRNAs and mRNAs which are known to be validated targets of differentially expressed miRNAs. Finally we revealed four dynamically regulated networks comprising differentially expressed miRNAs and their known target mRNAs with anti-correlated expression profiles over time. The data suggest that hsa-miR-21-5p regulated TGFBR3, PDGFD and PPM1L mRNAs. Hsa-miR-24-2-5p was likely to be responsible for MYC and KCNJ2 genes and hsa-miR-27a-5p for ST3GAL6. The targets of hsa-miR-181a-5p included ROPN1L and SLC37A3. All these mRNAs are involved in processes highly relevant to exercise response, including immune function, apoptosis, membrane traffic of proteins and transcription regulation.
We have identified metabolic pathways involved in response to exercise and revealed four miRNA-mRNA networks dynamically regulated following exercise. This work is the first study to monitor miRNAs and mRNAs in parallel into the recovery period. The results provide a novel insight into the regulatory role of miRNAs in stress adaptation.
PMCID: PMC3681679  PMID: 24219008
Exercise; Regulation; miRNA-mRNA networks
21.  Significance of KATP channels, L-type Ca2+ channels and CYP450-4A enzymes in oxygen sensing in mouse cremaster muscle arterioles In vivo 
BMC Physiology  2013;13:8.
ATP-sensitive K+ channels (KATP channels), NO, prostaglandins, 20-HETE and L-type Ca2+ channels have all been suggested to be involved in oxygen sensing in skeletal muscle arterioles, but the role of the individual mechanisms remain controversial. We aimed to establish the importance of these mechanisms for oxygen sensing in arterioles in an in vivo model of metabolically active skeletal muscle. For this purpose we utilized the exteriorized cremaster muscle of anesthetized mice, in which the cremaster muscle was exposed to controlled perturbation of tissue PO2.
Change from “high” oxygen tension (PO2 = 153.4 ± 3.4 mmHg) to “low” oxygen tension (PO2 = 13.8 ± 1.3 mmHg) dilated cremaster muscle arterioles from 11.0 ± 0.4 μm to 32.9 ± 0.9 μm (n = 28, P < 0.05). Glibenclamide (KATP channel blocker) caused maximal vasoconstriction, and abolished the dilation to low oxygen, whereas the KATP channel opener cromakalim caused maximal dilation and prevented the constriction to high oxygen. When adding cromakalim on top of glibenclamide or vice versa, the reactivity to oxygen was gradually restored. Inhibition of L-type Ca2+ channels using 3 μM nifedipine did not fully block basal tone in the arterioles, but rendered them unresponsive to changes in PO2. Inhibition of the CYP450-4A enzyme using DDMS blocked vasoconstriction to an increase in PO2, but had no effect on dilation to low PO2.
We conclude that: 1) L-type Ca2+ channels are central to oxygen sensing, 2) KATP channels are permissive for the arteriolar response to oxygen, but are not directly involved in the oxygen sensing mechanism and 3) CYP450-4A mediated 20-HETE production is involved in vasoconstriction to high PO2.
PMCID: PMC3663688  PMID: 23663730
Hypoxic vasodilation; Hyperoxic vasoconstriction; Oxygen sensing; ATP-sensitive K+ channels; 20-HETE; L-type Ca2+ channels; Prostaglandin; NO-synthase; Skeletal muscle; Arterioles
22.  Liver-derived endocrine IGF-I is not critical for activation of skeletal muscle protein synthesis following oral feeding 
BMC Physiology  2013;13:7.
Insulin-like growth factor-1 (IGF-1) is produced in various tissues to stimulate protein synthesis under different conditions. It is however, difficult to distinguish effects by locally produced IGF-1 compared to liver-derived IGF-1 appearing in the circulation. In the present study the role of liver-derived endocrine IGF-I for activation of skeletal muscle protein synthesis following feeding was evaluated.
Transgenic female mice with selective knockout of the IGF-I gene in hepatocytes were freely fed, starved overnight and subsequently refed for 3 hours and compared to wild types (wt). Liver IGF-I knockout mice had 70% reduced plasma IGF-I. Starvation decreased and refeeding increased muscle protein synthesis (p < 0.01), similarly in both IGF-I knockouts and wt mice. Phosphorylation of p70s6k and mTOR increased and 4EBP1 bound to eIF4E decreased in both IGF-I knockouts and wt mice after refeeding (p < 0.05). Muscle transcripts of IGF-I decreased and IGF-I receptor increased (p < 0.01) in wild types during starvation but similar alterations did not reach significance in knockouts (p>0.05). mTOR mRNA increased in knockouts only during starvation. Plasma glucose decreased during starvation in all groups in parallel to insulin, while plasma IGF-I and GH did not change significantly among the groups during starvation-refeeding. Plasma amino acids declined and increased during starvation-refeeding in wild type mice (p < 0.05), but less so in IGF-I (−/−) knockouts (p < 0.08).
This study demonstrates that re-synthesis of muscle proteins following starvation is not critically dependent on endocrine liver-derived IGF-I.
PMCID: PMC3659091  PMID: 23657003
IGF-I; Muscle protein synthesis; Cell signaling; Amino acid
23.  cAMP-stimulated Cl- secretion is increased by glucocorticoids and inhibited by bumetanide in semicircular canal duct epithelium 
BMC Physiology  2013;13:6.
The vestibular system controls the ion composition of its luminal fluid through several epithelial cell transport mechanisms under hormonal regulation. The semicircular canal duct (SCCD) epithelium has been shown to secrete Cl- under β2-adrenergic stimulation. In the current study, we sought to determine the ion transporters involved in Cl- secretion and whether secretion is regulated by PKA and glucocorticoids.
Short circuit current (Isc) from rat SCCD epithelia demonstrated stimulation by forskolin (EC50: 0.8 μM), 8-Br-cAMP (EC50: 180 μM), 8-pCPT-cAMP (100 μM), IBMX (250 μM), and RO-20-1724 (100 μM). The PKA activator N6-BNZ-cAMP (0.1, 0.3 & 1 mM) also stimulated Isc. Partial inhibition of stimulated Isc individually by bumetanide (10 & 50 μM), and [(dihydroindenyl)oxy]alkanoic acid (DIOA, 100 μM) were additive and complete. Stimulated Isc was also partially inhibited by CFTRinh-172 (5 & 30 μM), flufenamic acid (5 μM) and diphenylamine-2,2′-dicarboxylic acid (DPC; 1 mM). Native canals of CFTR+/− mice showed a stimulation of Isc from isoproterenol and forskolin+IBMX but not in the presence of both bumetanide and DIOA, while canals from CFTR−/− mice had no responses. Nonetheless, CFTR−/− mice showed no difference from CFTR+/− mice in their ability to balance (rota-rod). Stimulated Isc was greater after chronic incubation (24 hr) with the glucocorticoids dexamethasone (0.1 & 0.3 μM), prednisolone (0.3, 1 & 3 μM), hydrocortisone (0.01, 0.1 & 1 μM), and corticosterone (0.1 & 1 μM) and mineralocorticoid aldosterone (1 μM). Steroid action was blocked by mifepristone but not by spironolactone, indicating all the steroids activated the glucocorticoid, but not mineralocorticoid, receptor. Expression of transcripts for CFTR; for KCC1, KCC3a, KCC3b and KCC4, but not KCC2; for NKCC1 but not NKCC2 and for WNK1 but only very low WNK4 was determined.
These results are consistent with a model of Cl- secretion whereby Cl- is taken up across the basolateral membrane by a Na+-K+-2Cl- cotransporter (NKCC) and potentially another transporter, is secreted across the apical membrane via a Cl- channel, likely CFTR, and demonstrate the regulation of Cl- secretion by protein kinase A and glucocorticoids.
PMCID: PMC3622586  PMID: 23537040
Chloride secretion; Rat; Knockout mouse; Primary culture; Epithelium; Inner ear; Bumetanide; DIOA; Glucocorticoid; NKCC; KCC
24.  Quinine controls body weight gain without affecting food intake in male C57BL6 mice 
BMC Physiology  2013;13:5.
Quinine is a natural molecule commonly used as a flavouring agent in tonic water. Diet supplementation with quinine leads to decreased body weight and food intake in rats. Quinine is an in vitro inhibitor of Trpm5, a cation channel expressed in taste bud cells, the gastrointestinal tract and pancreas. The objective of this work is to determine the effect of diet supplementation with quinine on body weight and body composition in male mice, to investigate its mechanism of action, and whether the effect is mediated through Trpm5.
Compared with mice consuming AIN, a regular balanced diet, mice consuming AIN diet supplemented with 0.1% quinine gained less weight (2.89 ± 0.30 g vs 5.39 ± 0.50 g) and less fat mass (2.22 ± 0.26 g vs 4.33 ± 0.43 g) after 13 weeks of diet, and had lower blood glucose and plasma triglycerides. There was no difference in food intake between the mice consuming quinine supplemented diet and those consuming control diet. Trpm5 knockout mice gained less fat mass than wild-type mice. There was a trend for a diet-genotype interaction for body weight and body weight gain, with the effect of quinine less pronounced in the Trpm5 KO than in the WT background. Faecal weight, energy and lipid contents were higher in quinine fed mice compared to regular AIN fed mice and in Trpm5 KO mice compared to wild type mice.
Quinine contributes to weight control in male C57BL6 mice without affecting food intake. A partial contribution of Trpm5 to quinine dependent body weight control is suggested.
PMCID: PMC3575391  PMID: 23394313
Obesity; Food intake; Fat; Body composition; Gastrointestinal tract
25.  Importance of uncharged polar residues and proline in the proximal two-thirds (Pro107–Ser128) of the highly conserved region of mouse ileal Na+-dependent bile acid transporter, Slc10a2, in transport activity and cellular expression 
BMC Physiology  2013;13:4.
SLC10A2-mediated reabsorption of bile acids at the distal end of the ileum is the first step in enterohepatic circulation. Because bile acids act not only as detergents but also as signaling molecules in lipid metabolism and energy production, SLC10A2 is important as the key transporter for understanding the in vivo kinetics of bile acids. SLC10A family members and the homologous genes of various species share a highly conserved region corresponding to Gly104–Pro142 of SLC10A2. The functional importance of this region has not been fully elucidated.
To elucidate the functional importance of this region, we previously performed mutational analysis of the uncharged polar residues and proline in the distal one-third (Thr130–Pro142) of the highly conserved region in mouse Slc10a2. In this study, proline and uncharged polar residues in the remaining two-thirds of this region in mouse Slc10a2 were subjected to mutational analysis, and taurocholic acid uptake and cell surface localization were examined. Cell surface localization of Slc10a2 is necessary for bile acid absorption. Mutants in which Asp or Leu were substituted for Pro107 (P107N or P107L) were abundantly expressed, but their cell surface localization was impaired. The S126A mutant was completely impaired in cellular expression. The T110A and S128A mutants exhibited remarkably enhanced membrane expression. The S112A mutant was properly expressed at the cell surface but transport activity was completely lost. Replacement of Tyr117 with various amino acids resulted in reduced transport activity. The degree of reduction roughly depended on the van der Waals volume of the side chains.
The functional importance of proline and uncharged polar residues in the highly conserved region of mouse Slc10a2 was determined. This information will contribute to the design of bile acid-conjugated prodrugs for efficient drug delivery or SLC10A2 inhibitors for hypercholesterolemia treatment.
PMCID: PMC3570448  PMID: 23374508
Bile acid; Enterohepatic circulation; Ileal sodium-dependent bile acid transporter

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