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1.  Ryanodine-induced vasoconstriction of the gerbil spiral modiolar artery depends on the Ca2+ sensitivity but not on Ca2+ sparks or BK channels 
BMC Physiology  2016;16:6.
In many vascular smooth muscle cells (SMCs), ryanodine receptor-mediated Ca2+ sparks activate large-conductance Ca2+-activated K+ (BK) channels leading to lowered SMC [Ca2+]i and vasodilation. Here we investigated whether Ca2+ sparks regulate SMC global [Ca2+]i and diameter in the spiral modiolar artery (SMA) by activating BK channels.
SMAs were isolated from adult female gerbils, loaded with the Ca2+-sensitive flourescent dye fluo-4 and pressurized using a concentric double-pipette system. Ca2+ signals and vascular diameter changes were recorded using a laser-scanning confocal imaging system. Effects of various pharmacological agents on Ca2+ signals and vascular diameter were analyzed.
Ca2+ sparks and waves were observed in pressurized SMAs. Inhibition of Ca2+ sparks with ryanodine increased global Ca2+ and constricted SMA at 40 cmH2O but inhibition of Ca2+ sparks with tetracaine or inhibition of BK channels with iberiotoxin at 40 cmH2O did not produce a similar effect. The ryanodine-induced vasoconstriction observed at 40 cmH2O was abolished at 60 cmH2O, consistent with a greater Ca2+-sensitivity of constriction at 40 cmH2O than at 60 cmH2O. When the Ca2+-sensitivity of the SMA was increased by prior application of 1 nM endothelin-1, ryanodine induced a robust vasoconstriction at 60 cmH2O.
The results suggest that Ca2+ sparks, while present, do not regulate vascular diameter in the SMA by activating BK channels and that the regulation of vascular diameter in the SMA is determined by the Ca2+-sensitivity of constriction.
PMCID: PMC5093982  PMID: 27806708
Ca2+ spark; Ca2+ sensitivity; Spiral modiolar artery; Ryanodine; Vascular diameter; BK channels; Tetracaine
2.  Trimester-specific thyroid hormone reference ranges in Sudanese women 
BMC Physiology  2016;16:5.
Trimester-specific reference ranges for T3, T4, and TSH need to be established in different communities. Neither Sudan nor other African countries have established trimester-specific reference ranges for TSH, free T3 (FT3), and free T4 (FT4) in healthy pregnant women. This study aimed to establish trimester-specific reference ranges for TSH, FT3, and FT4 in healthy pregnant Sudanese women.
We performed a longitudinal study, which included 63 women with singleton pregnancies who were followed since early pregnancy until the third trimester. The study was performed in Saad Abu-Alela Hospital, Khartoum, Sudan, during January to October 2014. An equal number of age- and parity-matched non-pregnant women were enrolled as a control group. Basic clinical and obstetrics data were gathered using questionnaires. TSH, FT3, and FT4 levels were measured. Median (5th–95th centile) values of TSH, FT3, and FT4 were 1.164 IU/ml (0.079–2.177 IU/ml), 4.639 nmol/l (3.843–6.562 nmol/l), and 16.86 pmol/l (13.02–31.48 pmol/l) in the first trimester. Median values of TSH, FT3, and FT4 were 1.364 IU/ml (0.540–2.521 IU/ml), 4.347 nmol/l (3.425–5.447 nmol/l), and 13.51 pmol/l (11.04–31.07 pmol/l) in the second trimester. These values were 1.445 IU/ml (0.588–2.460 IU/ml), 4.132 nmol/l (3.176–5.164 nmol/l), and 12.87 pmol/l (9.807–23.78 pmol/l) in the third trimester, respectively. TSH levels increased throughout the trimesters. FT3 and FT4 levels were significantly higher in the first trimester compared with the second and third trimesters. TSH, FT3, and FT4 levels were significantly lower in pregnant women compared with non-pregnant women (P < 0.001).
The present study is the first to establish trimester-specific reference ranges of TSH, FT3, and FT4 in Sudanese women with normal pregnancies. Our results suggest that pregnancy is likely to suppress TSH, T3, and T4 levels in healthy women.
PMCID: PMC5087113  PMID: 27795209
Pregnancy; Reference range; Sudan; Trimester; Thyroid
3.  The gastric H,K-ATPase in stria vascularis contributes to pH regulation of cochlear endolymph but not to K secretion 
BMC Physiology  2016;17:1.
Disturbance of acid–base balance in the inner ear is known to be associated with hearing loss in a number of conditions including genetic mutations and pharmacologic interventions. Several previous physiologic and immunohistochemical observations lead to proposals of the involvement of acid–base transporters in stria vascularis.
We directly measured acid flux in vitro from the apical side of isolated stria vascularis from adult C57Bl/6 mice with a novel constant-perfusion pH-selective self-referencing probe. Acid efflux that depended on metabolism and ion transport was observed from the apical side of stria vascularis. The acid flux was decreased to about 40 % of control by removal of the metabolic substrate (glucose-free) and by inhibition of the sodium pump (ouabain). The flux was also decreased a) by inhibition of Na,H-exchangers by amiloride, dimethylamiloride (DMA), S3226 and Hoe694, b) by inhibition of Na,2Cl,K-cotransporter (NKCC1) by bumetanide, and c) by the likely inhibition of HCO3/anion exchange by DIDS. By contrast, the acid flux was increased by inhibition of gastric H,K-ATPase (SCH28080) but was not affected by an inhibitor of vH-ATPase (bafilomycin).  K flux from stria vascularis was reduced less than 5 % by SCH28080.
These observations suggest that stria vascularis may be an important site of control of cochlear acid–base balance and demonstrate a functional role of several acid–base transporters in stria vascularis, including basolateral H,K-ATPase and apical Na,H-exchange. Previous suggestions that H secretion is mediated by an apical vH-ATPase and that basolateral H,K-ATPase contributes importantly to K secretion in stria vascularis are not supported. These results advance our understanding of inner ear acid–base balance and provide a stronger basis to interpret the etiology of genetic and pharmacologic cochlear dysfunctions that are influenced by endolymphatic pH.
Electronic supplementary material
The online version of this article (doi:10.1186/s12899-016-0024-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4982335  PMID: 27515813
Acid–base balance; Endolymph; Inner ear; Hydrogen ion secretion; Potassium secretion; Stria vascularis; Ion-selective self-referencing electrode
4.  The novel in vitro reanimation of isolated human and large mammalian heart-lung blocs 
BMC Physiology  2016;16:4.
In vitro isolated heart preparations are valuable tools for the study of cardiac anatomy and physiology, as well as for preclinical device testing. Such preparations afford investigators a high level of hemodynamic control, independent of host or systemic interactions. Here we hypothesize that recovered human and swine heart-lung blocs can be reanimated using a clear perfusate and elicit viable cardiodynamic and pulmonic function. Further, this approach will facilitate multimodal imaging, which is particularly valuable for the study of both functional anatomy and device-tissue interactions. Five human and 18 swine heart-lung preparations were procured using techniques analogous to those for cardiac transplant. Specimens were then rewarmed and reperfused using modifications of a closed circuit, isolated, beating and ventilated heart-lung preparation. Positive pressure mechanical ventilation was also employed, and epicardial defibrillation was applied to elicit native cardiac sinus rhythm. Videoscopy, fluoroscopy, ultrasound, and infrared imaging were performed for anatomical and experimental study.
Systolic and diastolic left ventricular pressures observed for human and swine specimens were 68/2 ± 11/7 and 74/3 ± 17/5 mmHg, respectively, with associated native heart rates of 80 ± 7 and 96 ± 16 beats per minute. High-resolution imaging within functioning human pulmonary vasculature was obtained among other anatomies of interest. Note that one human specimen elicited poor cardiac performance post defibrillation.
We report the first dynamic videoscopic images of the pulmonary vasculature during viable cardiopulmonary function in isolated reanimated heart-lung blocs. This experimental approach provides unique in vitro opportunities for the study of novel medical therapeutics applied to large mammalian, including human, heart-lung specimens.
Electronic supplementary material
The online version of this article (doi:10.1186/s12899-016-0023-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4893289  PMID: 27259478
Cardiac anatomy; Cardiac physiology; Heart-lung bloc; Reanimation
5.  Renal effects of angiotensin II in the newborn period: role of type 1 and type 2 receptors 
BMC Physiology  2016;16:3.
Evidence suggests a critical role for the renin-angiotensin system in regulating renal function during postnatal development. However, the physiological relevance of a highly elevated renin-angiotensin system early in life is not well understood, nor which angiotensin receptors might be involved. This study was designed to investigate the roles of angiotensin receptors type 1 (AT1R) and type 2 (AT2R) in regulating glomerular and tubular function during postnatal development.
The renal effects of the selective antagonist to AT1R, ZD 7155 and to AT2R, PD 1233319 were evaluated in two groups of conscious chronically instrumented lambs aged ~ one week (N = 8) and ~ six weeks (N = 10). Two experiments were carried out in each animal and consisted of the assessment of renal variables including glomerular and tubular function, for 30 min before (Control) and 60 min after infusion of ZD 7155 and PD 123319, respectively. Statistical significance was determined using parametric testing (Student t-test, analysis of variance ANOVA) as appropriate.
ZD 7155 infusion was associated with a significant decrease in glomerular filtration rate and filtration fraction at one but not six weeks; urinary flow rate decreased significantly in older animals, whereas sodium excretion and free water clearance were not altered. There was an age-dependent effect on potassium handling along the nephron, potassium excretion decreasing after ZD 7155 infusion in younger but not in older lambs. PD 123319 had no significant effects on glomerular filtration rate and tubular function in either age group.
These results provide evidence to support an important role for AT1Rs in mediating the renal effects of angiotensin II during postnatal maturation in conscious developing animals. In contrast to a role for AT2Rs later in life, there appears to be no role for AT2Rs in influencing the renal effects of Angiotensin II in the postnatal period.
Electronic supplementary material
The online version of this article (doi:10.1186/s12899-016-0022-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4835895  PMID: 27090941
Newborn; Kidney; Angiotensin receptor type 1; Angiotensin receptor type 2; Angiotensin II; Electrolytes; Development
6.  Subcellular dissemination of prothymosin alpha at normal physiology: immunohistochemical vis-a-vis western blotting perspective 
BMC Physiology  2016;16:2.
The cell type, cell status and specific localization of Prothymosin α (PTMA) within cells seemingly determine its function. PTMA undergoes 2 types of protease proteolytic modifications that are useful in elucidating its interactions with other molecules; a factor that typifies its roles. Preferably a nuclear protein, PTMA has been shown to function in the cytoplasm and extracellularly with much evidence leaning on pathognomonic status. As such, determination of its cellular distribution under normal physiological context while utilizing varied techniques is key to illuminating prospective validation of its distinct functions in different tissues. Differential distribution insights at normal physiology would also portent better basis for further clarification of its interactions and proteolytic modifications under pathological conditions like numerous cancer, ischemic stroke and immunomodulation. We therefore raised an antibody against the C terminal of PTMA to use in tandem with available antibody against the N terminal in a murine model to explicate the differences in its distribution in brain cell types and major peripheral organs through western blotting and immunohistochemical approaches.
The newly generated antibody was applied against the N-terminal antibody to distinguish truncated versions of PTMA or deduce possible masking of the protein by other interacting molecules. Western blot analysis indicated presence of a truncated form of the protein only in the thymus, while immunohistochemical analysis showed that in brain hippocampus the full-length PTMA was stained prominently in the nucleus whereas in the stomach full-length PTMA staining was not observed in the nucleus but in the cytoplasm.
Truncated PTMA could not be detected by western blotting when both antibodies were applied in all tissues examined except the thymus. However, immunohistochemistry revealed differential staining by these antibodies suggesting possible masking of epitopes by interacting molecules. The differential localization patterns observed in the context of nucleic versus cytoplasmic presence as well as punctate versus diffuse pattern in tissues and cell types, warrant further investigations as to the forms of PTMA interacting partners.
Electronic supplementary material
The online version of this article (doi:10.1186/s12899-016-0021-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4774093  PMID: 26932824
Prothymosin α; Anti-C terminal antibody; Anti- N terminal antibody; Immunohistochemistry; Western blotting and localization
7.  The normal electrocardiograms in the conscious newborn lambs in neonatal period and its progression 
BMC Physiology  2016;16:1.
Veterinary cardiology, especially electrocardiography, has shown major advancements for all animal species. Consequently, the number of ovine species used as experimental animals has increased to date. Few studies have been published on ovine systematic electrocardiography, particularly with respect to lamb physiology and neonatology. This study aimed to standardize the values of normal waves, complexes, and intervals of the electrocardiogram (ECG) in clinically Bergamasca healthy neonatal lambs, used as experimental animals. Serial computerized electrocardiography was performed in 10 male and 12 female neonates on the 1st, 7th, 14th, 21st, 28th, and 35th days of age. The following parameters were analyzed: heart rate and rhythm, duration and amplitude of waves, duration of intervals, and heart electrical axis.
During the first 35 days of life, (1) the sinusal heart rhythm was predominant, (2) there was a progressive decrease in the heart rate and R and T wave amplitude, and (3) a progressive increase in the PR, QT, and RR intervals. Finally, we confirmed that various components of neonatal evolution were more discernible in the augmented unipolar leads (aVF), which we recommend should be preferentially used in future studies. No significant statistical alterations were observed between males and females in relation to the analyzed parameters.
The information assimilated in this study is anticipated to enhance the diagnosis of multiple congenital heart defects in Bergamasca lambs and could be implemented in studies that use ovine species as experimental models.
PMCID: PMC4719313  PMID: 26786197
Electrocardiogram; Newborn; Electrophysiology; Ovine; Cardiac development
8.  An investigation of the efficacy and mechanism of contrast-enhanced X-ray Computed Tomography utilizing iodine for large specimens through experimental and simulation approaches 
BMC Physiology  2015;15:5.
Iodine-based solutions have long been known to be effective in aiding the differentiation among soft tissues in both fundamental anatomical research and for clinical diagnoses. Recently the combination of this particular contrasting agent with micro-computed tomography (micro-CT) has resulted in an array of high-quality image data, in which anatomical structures not visible in conventional CT can be identified and quantified. However, there has been only limited data available to inform detailed protocols for staining large specimens. Further, modeling of the staining mechanism has focused on simple diffusion processes.
A low concentration of iodine-based buffered formalin solution with a long staining period was used to visualize soft-tissue structures in a large goose head. The staining effect was analyzed by serially measuring the micro-CT profiles across coronal sections throughout the staining period. Regular replacement of the staining solution combined with a longer staining period significantly improved contrast within tissues. A simplified one-dimensional Diffusion-Sorption model with a three-zone domain was used to simulate the diffusion process by calculating the concentration profile of iodine across the adductor region, which fits well with the experiment data. Observations of changes in the concentration of the staining agent and simulation results suggest that the sorption of iodine by tissues significantly affects the effective diffusion coefficient for the contrasting agent.
The Diffusion-Sorption model better explains previously reported difficulties in staining large samples comprised of tissues with high partition coefficients (Kd). Differences in partition coefficient (Kd), bulk density (ρb), and porosity (θ) could further explain the observed variation in staining rate and maximal staining effect among different tissues. Recommended protocols for staining large specimens are detailed.
PMCID: PMC4687389  PMID: 26691327
I2KI; Formalin; Staining; Diffusion; Sorption; CT-scan; Soft-tissue contrast; Aves
9.  Hypoxia mediated pulmonary edema: potential influence of oxidative stress, sympathetic activation and cerebral blood flow 
BMC Physiology  2015;15:4.
Neurogenic pulmonary edema (NPE) is a non-cardiogenic form of pulmonary edema that can occur consequent to central neurologic insults including stroke, traumatic brain injury, and seizure. NPE is a public health concern due to high morbidity and mortality, yet the mechanism(s) are unknown. We hypothesized that NPE, evoked by cerebral hypoxia in the presence of systemic normoxia, would be accompanied by sympathetic activation, oxidative stress, and compensatory antioxidant mechanisms.
Thirteen Walker hounds were assigned to cerebral hypoxia (SaO2 ~ 55 %) with systemic normoxia (SaO2 ~ 90 %) (CH; n = 6), cerebral and systemic (global) hypoxia (SaO2 ~ 60 %) (GH; n = 4), or cerebral and systemic normoxia (SaO2 ~ 90 %) (CON; n = 3). Femoral venous (CH and CON) perfusate was delivered via cardiopulmonary bypass to the brain and GH was induced by FiO2 = 10 % to maintain the SaO2 at ~60 %. Lung wet to lung dry weight ratios (LWW/LDW) were assessed as an index of pulmonary edema in addition to hemodynamic measurements. Plasma catecholamines were measured as markers of sympathetic nervous system (SNS) activity. Total glutathione, protein carbonyls, and malondialdehyde were assessed as indicators of oxidative stress. Brain and lung compensatory antioxidants were measured with immunoblotting.
Compared to CON, LWW/LDW and pulmonary artery pressure were greater in CH and GH. Expression of hemeoxygenase-1 in brain was higher in CH compared to GH and CON, despite no group differences in oxidative damage in any tissue. Catecholamines tended to be higher in CH and GH.
Cerebral hypoxia, with systemic normoxia, is not systematically associated with an increase in oxidative stress and compensatory antioxidant enzymes in lung, suggesting oxidative stress did not contribute to NPE in lung. However, increased SNS activity may play a role in the induction of NPE during hypoxia.
PMCID: PMC4599206  PMID: 26449218
Cerebral hypoxia; Nrf2; HO-1; Catecholamines; Cerebral autoregulation
10.  Effects of postnatal growth restriction and subsequent catch-up growth on neurodevelopment and glucose homeostasis in rats 
BMC Physiology  2015;15:3.
There is increasing evidence that poor growth of preterm infants is a risk factor for poor long-term development, while the effects of early postnatal growth restriction are not well known. We utilized a rat model to examine the consequences of different patterns of postnatal growth and hypothesized that early growth failure leads to impaired development and insulin resistance. Rat pups were separated at birth into normal (N, n = 10) or restricted intake (R, n = 16) litters. At d11, R pups were re-randomized into litters of 6 (R-6), 10 (R-10) or 16 (R-16) pups/dam. N pups remained in litters of 10 pups/dam (N-10). Memory and learning were examined through T-maze test. Insulin sensitivity was measured by i.p. insulin tolerance test and glucose tolerance test.
By d10, N pups weighed 20 % more than R pups (p < 0.001). By d15, the R-6 group caught up to the N-10 group in weight, the R-10 group showed partial catch-up growth and the R-16 group showed no catch-up growth. All R groups showed poorer scores in developmental testing when compared with the N-10 group during T-Maze test (p < 0.05). Although R-16 were more insulin sensitive than R-6 and R-10, all R groups were more glucose tolerant than N-10.
In rats, differences in postnatal growth restriction leads to changes in development and in insulin sensitivity. These results may contribute to better elucidating the causes of poor developmental outcomes in human preterm infants.
PMCID: PMC4455975  PMID: 26040642
Growth restriction; Catch-up growth; Development; Insulin sensitivity
11.  Antenatal maternal low protein diet: ACE-2 in the mouse lung and sexually dimorphic programming of hypertension 
BMC Physiology  2015;15:2.
Elevated blood pressure is an important global health problem, and in-utero under-nutrition may be an important factor in the pathogenesis of hypertension. In the present study, we tested the hypothesis that antenatal maternal low protein diet (MLPD) leads to sexually dimorphic developmental programming of the components of the pulmonary renin-angiotensin system. This may be important in the antenatal MLPD-associated development of hypertension. In pregnant mice, we administered normal (control) and isocaloric 50 % protein restricted diet, commencing one week before mating and continuing until delivery of the pups. From the 18th to 24th week postnatal, we measured blood pressure in the offspring by use of a non-invasive tail-cuff method. In the same mice, we examined the mRNA and protein expression of the key components of the pulmonary renin-angiotensin system. Also, we examined microRNA complementary to angiotensin converting enzymes (ACE) 2 in the offspring lungs. Our results demonstrate that as a consequence of antenatal MLPD: 1) pup birthweight was significantly reduced in both sexes. 2) female offspring developed hypertension, but males did not. 3) In female offspring, ACE-2 protein expression was significantly reduced without any change in the mRNA levels. 4) miRNA 429, which has a binding site on ACE-2 - 3’ UTR was significantly upregulated in the female antenatal MLPD offspring. 5) In males, ACE-2 mRNA and protein expression were unaltered. We conclude that in the mouse, antenatal MLPD-induced reduction of ACE-2 in the female offspring lung may be an important mechanisms in sexually dimorphic programming of hypertension.
Electronic supplementary material
The online version of this article (doi:10.1186/s12899-015-0016-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4430899  PMID: 25971747
Angiotensin converting enzyme; microRNA; Epigenetics; DOHaD; Fetal programming; Developmental origins; Barker hypothesis
12.  Exercise performed around MLSS decreases systolic blood pressure and increases aerobic fitness in hypertensive rats 
BMC Physiology  2015;15:1.
Exercise is a non-pharmacologic agent widely used for hypertension control, where low intensity is often associated with blood pressure reduction. Maximal lactate steady state (MLSS) was recently identified in spontaneously hypertensive rats (SHRs) as an important step in establishing secure intensities for prescribing exercise for hypertensive phenotypes. Here we verified the effects of training around MLSS, 20% below MLSS, and 15% above MLSS on aerobic fitness and blood pressure status of SHR. Eighteen-week-old SHRs (n = 5, ~ 172.4 ± 8.1 mm Hg systolic blood pressure) were trained on a treadmill for 4 weeks for 30 min/day, 5 days/week at a velocity of 20 m.min−1. After training, a novel MLSS and incremental test was performed to evaluate the animals’ aerobic fitness. Furthermore, ~ 22-week-old SHRs (n = 12, ~169.8 ± 13.8 mm Hg systolic blood pressure) were divided into non-exercised (CG, n = 4), low intensity (LIG, n = 4) and high intensity (HIG, n = 4) groups, where rats were trained at 16 m.min−1 and 23 m.min−1 respectively for 30 min/day, 5 days/week for 4 weeks.
Exercise performed at MLSS enhanced aerobic fitness, leading to a novel MLSS, identified around 30 m.min−1. Low and high intensity training reduced systolic blood pressure and only high intensity training led to improved aerobic fitness (28.1%, p < 0.01).
Therefore, our data indicate a decrease in blood pressure due to low and high exercise intensity, and an increase in aerobic fitness provided by high-intensity exercise in SHRs.
PMCID: PMC4367833  PMID: 25888441
Blood pressure; Hypertension; SHR; Maximal lactate steady state; Treadmill running exercise; Incremental test; Vmax
13.  Alteration in circulating metabolites during and after heat stress in the conscious rat: potential biomarkers of exposure and organ-specific injury 
BMC Physiology  2014;14:14.
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
14.  CaMKII content affects contractile, but not mitochondrial, characteristics in regenerating skeletal muscle 
BMC Physiology  2014;14:7.
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
15.  Big data in wildlife research: remote web-based monitoring of hibernating black bears 
BMC Physiology  2014;14:13.
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
16.  Identification of novel Kirrel3 gene splice variants in adult human skeletal muscle 
BMC Physiology  2014;14:11.
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
17.  Acetate transiently inhibits myocardial contraction by increasing mitochondrial calcium uptake 
BMC Physiology  2014;14:12.
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
18.  White-nose syndrome initiates a cascade of physiologic disturbances in the hibernating bat host 
BMC Physiology  2014;14:10.
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
19.  Differential role of STIM1 and STIM2 during transient inward (Tin) current generation and the maturation process in the Xenopus oocyte 
BMC Physiology  2014;14:9.
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
20.  Longevity of Daphnia and the attenuation of stress responses by melatonin 
BMC Physiology  2014;14:8.
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
21.  MAPK-activated protein kinase 2-deficiency causes hyperacute tumor necrosis factor-induced inflammatory shock 
BMC Physiology  2014;14:5.
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
22.  Impact of maternal dietary fat supplementation during gestation upon skeletal muscle in neonatal pigs 
BMC Physiology  2014;14:6.
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
23.  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
24.  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
25.  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

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