bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and functional characterization of a rainbow trout liver Oatp
Authors: Steiner Konstanze , Dietrich Daniel , Hagenbuch Bruno
Published: June 2021

Microcystins (MCs) are natural occurring toxins produced by cyanobacteria in aquatic ecosystems. MCs are known hepatotoxicants in fish and mammals, whereby toxicity in mammals has been demonstrated to result from Oatp‐mediated MC‐uptake into hepatocytes. To investigate whether MC‐uptake in fish liver is Oatp‐mediated, the goal of this study was to clone and functionally characterize a rainbow trout liver Oatp. We prepared a cDNA library from rainbow trout liver mRNA and isolated a full length Oatp. To functionally characterize this Oatp we transiently transfected HEK293 cells and determined uptake of radiolabelled Oatp substrates. The isolated cDNA consists of 2773 bp and has an open reading frame of 2115 bp, encoding a 705‐amino acid protein. Phylogentic analysis suggests that the isolated protein is Oatp1d1. Functional characterization revealed that Oatp1d1 can mediate the uptake of various Oatp substrates including bromosulfophthalein (BSP), D‐penicillamine(2,5)enkephalin (DPDPE), estradiol‐17β‐glucuronide (E17βG), estrone‐3‐sulfate, taurocholate, and methotrexate. In conclusion, we have isolated and characterized a multispecific Oatp1d1 from rainbow trout liver that may be instrumental in MC uptake.
Prolactin induces BCRP/ABCG2 expression in T‐47D breast cancer cells via activation of multiple signalling cascades
Authors: Wu Alex , Dalvi Pooja , Yang Mingdong , Lu Xiaoli , Harper Patricia A , Ito Shinya
Published: June 2021

The drug transporter Breast Cancer Resistance Protein (BCRP/ABCG2) has been shown to be upregulated in certain drug resistant breast cancer cells and in the normal mammary gland during lactation. Given the important role of the lactogenic hormone prolactin in breast cancer and lactation biology, we investigated the effect of prolactin on BCRP expression in T‐47D human breast cancer epithelial cells. Prolactin upregulated BCRP mRNA and protein expression in a dose‐dependent manner. Induction of BCRP mRNA by prolactin was blocked by the transcription inhibitor actinomycin D. To characterize the mechanism responsible for prolactin‐induced BCRP mRNA expression, we systematically inhibited members of the three major pathways activated in prolactin signalling: JAK2/STAT5, MAPK, and PI3K/AKT. Knockdown of JAK2 by siRNA attenuated the prolactin‐BCRP response. Knockdown or pharmacological inhibition of STAT5 by siRNA or small molecule inhibitor, respectively, blunted prolactin‐induced BCRP mRNA expression. Treating cells in the presence of the MAPK pathway inhibitors PD98059 (20μM) and U0126 (10μM), and the PI3K pathway inhibitors LY294002 (10μM) and Wortmannin (25nM), significantly attenuated the effect of prolactin on BCRP mRNA expression. Taken together, our results demonstrate overlapping roles for JAK2/STAT5, MAPK, and PI3K/AKT signalling in prolactin‐induced BCRP expression.

Funding provided by CIHR.
Evaluating In Situ (E)‐2,4‐Diene‐Valproic Acid in the Toxicity of Valproic Acid and (E)‐2‐Ene‐Valproic Acid in Sandwich‐Cultured Rat Hepatocytes
Authors: Surendradoss Jayakumar , Chang Thomas K. H. , Abbott Frank S.
Published: June 2021

Formation of reactive metabolites like (E)‐2,4‐diene‐VPA is a proposed mechanism for the idiosyncratic hepatotoxicity of valproic acid (VPA). (E)‐2,4‐diene‐VPA is formed by cytochrome P450 (CYP)‐mediated desaturation of (E)‐2‐ene‐VPA or mitochondrial β‐oxidation of 4‐ene‐VPA, which itself is a CYP‐catalyzed metabolite of VPA. (E)‐2,4‐diene‐VPA is reactive and more hepatotoxic than VPA, but direct experimental evidence is needed to evaluate the effect of the in situ generated metabolite on VPA hepatotoxicity. We assessed the effect of modulating the in situ formation of (E)‐2,4‐diene‐VPA by pretreatment with phenobarbital (PB, a CYP inducer) and 1‐aminobenzotriazole (1‐ABT, a CYP inhibitor) on markers of oxidative stress, steatosis and necrosis in sandwich‐cultured rat hepatocytes treated with VPA or (E)‐2‐ene‐VPA. PB increased the metabolism of (E)‐2‐ene‐VPA to (E)‐2,4‐diene‐VPA, and this was accompanied by enhanced toxicity of (E)‐2‐ene‐VPA, whereas 1‐ABT attenuated the increase in the levels of the (E)‐2,4‐diene‐VPA metabolite and toxicity by PB. Neither PB nor 1‐ABT affected (E)‐2,4‐diene‐VPA formation and toxicity in VPA‐treated hepatocytes. In conclusion, in situ formed (E)‐2,4‐diene‐VPA contributes to the toxicity in sandwich‐cultured rat hepatocytes, if generated at sufficiently high levels. [Supported by CIHR and MSFHR]
TRPV4 channels regulate tumor angiogenesis through the modulation of Rho‐dependent tumor endothelial cell mechanosensitivity
Authors: Thodeti Charles K , Adapala Ravi K , Ghosh Kaustabh , Thoppil Roslin , Dudley Andrew C , Klagsbrun Michael , Chilian William , Ingber Donald
Published: June 2021

Tumor vessels are characterized by abnormal morphology and patterning that cause vascular hyperpermeability and inefficient delivery of anti‐cancer agents. We have previously shown that tumor vessel malformations may arise from aberrant Rho‐mediated mechanosensitivity of tumor‐derived capillary endothelial (CE) cells to mechanical cues, in particular blood flow‐induced cyclic strain. Here, we demonstrate that the expression and function of TRPV4, a mechanosenstive ion channel required for CE cell reorientation, is significantly lower in tumor CE cells compared to normal CE cells. Overexpression of TRPV4 increased GSK1016790A‐induced calcium influx in tumor CE cells and restored their shape sensitivity to substrate elasticity. Further, TRPV4 overexpression resulted in the suppression of the endogenously high Rho that, we previously showed, is implicated in aberrant mechanosensitivity of tumor CE cells. Importantly, overexpression of TRPV4 alone normalized the aberrant capillary formation and abnormal migration by tumor‐derived CE cells in vitro. Finally, tumor xenograft experiments revealed that, compared to tumors in wild type mice, those in TRPV4 KO mice exhibit a greater degree of vascular abnormality, as demonstrated by increased vessel diameter and reduced pericyte coverage, as well as enhanced growth. This work, therefore, demonstrates the critical role of TRPV4 channels in tumor endothelial cell mechanosensitivity and offers a novel therapeutic target for normalizing tumor vessel malformations and improving cancer drug delivery.
Exclusive interaction between α1D‐adrenergic receptor and pannexin 1 can mediate vasoconstriction
Authors: Billaud Marie , Lohman Alexander W , Chiu Yu-Hsin , Sandilos Joanna K , Bayliss Doug A , Isakson Brant E
Published: June 2021

We recently demonstrated that pannexin1 (Panx1) channels release ATP in vascular smooth muscle cells after phenylephrine (PE) stimulation (Billaud et al, Circ Res, 2011). However, the participation of Panx1 in other contractile pathways has not been investigated. Therefore, we stimulated thoracodorsal arteries with angiotensin II, endothelin‐1, serotonin, ATP, KCl, noradrenaline and PE. Only noradrenaline and PE responses were altered by pannexin inhibitors suggesting that these responses occurred through an association between the α1D‐adrenergic receptor (α1D‐AR) and Panx1. These results were verified using duolink imaging of the α1D‐AR/Panx1 interaction. We hypothesized that disruption of this complex would alter vasoconstriction and therefore designed peptides against the Panx1 intracellular loop (IL) and carboxyl tail (CT). Only the Panx1 IL peptide decreased constriction to PE. We further tested the effect of the peptides on the integrity of the Panx1/α1D‐AR complex using co‐immunoprecipitation and verified that the peptides had no effect on Panx1 hexamerization and trafficking. We demonstrated this in vitro by co‐transfection of Panx1 and α1D‐AR plasmids in HEK cells. Electrophysiology confirmed PE‐induced Panx1 activation which was altered by Panx1 IL peptide. Our results show that Panx1 has an exclusive interaction with α1D‐AR in the regulation of vascular tone.
Effect of desmin knockout on skeletal muscle extracellular matrix organization
Authors: Gillies Allison , Lieber Richard L.
Published: June 2021

The genetic deletion of desmin, an intermediate filament in skeletal muscle, leads to lower muscle stress production, muscle stiffness, and fibrosis. The extracellular matrix (ECM) of desmin knockout muscle was studied to determine how its structure and organization change in fibrotic muscle. The abundance and organization of perimysial collagen was quantified by stereological analysis of transmission electron micrographs, and perimysial collagen structure was observed by scanning electron microscopy and confocal imaging of fiber bundles stained for type I collagen. The organization of perimysial collagen termination on muscle fibers was determined by serial block face imaging (3View, Gatan, Inc.). An endomysial mesh was identified, as well as perimysium arranged in large bundles of collagen fibrils. These perimysial “cables” were ~1μm in diameter and tortuous at resting length, but straightened in response to muscle elongation. The apparent load bearing differences of endomysium and perimysium seem to be based on structure, and this structural specialization has not previously been appreciated. Increased stiffness observed in desmin‐null muscle may be partially due to altered ECM organization or material properties in addition to a generalized increase in ECM quantity. Supported by Allergan, Inc. and NIH grants R24 HD05083 and R01 AR057393.
Developmental Changes in Airway Smooth Muscle Relaxation Mediated by NO and PGE2
Authors: Jakupaj Muharrem , Temaj Gazmend , Cenaj Asllan , Sopi Ramadan B
Published: June 2021

In this study we investigated changes in relaxation of airways during development and we sought to determine to what extent NO and PGE2 contribute to this response during postnatal maturation. Airway segments were excised from trachea of rats at four different ages (1, 2, and 4 wk and 3 mo). In precontracted tracheal smooth muscle (TSM), electrical field stimulation (EFS) was applied to induce relaxation, in absence or presence of NO synthase (NOS) inhibitor (L‐NAME) or cyclooxygenase inhibitor (indomethacin) or combined. EFS‐induced relaxation of TSM significantly decreased with advancing postnatal age (overall, P < 0.001). The maximal relaxant responses were as follows: at 1wk, 70.2 ± 4.6%; at 2 wk, 55.7 ± 5.2%; at 4 wk, 37.4 ± 4.2% and 3 mo, 22.3 ± 3.7%. Pretreatment with L‐NAME or indomethacin or both, significantly reduced the EFS‐induced relaxation (P < 0.001) compared with controls. Although both inhibitors reduced relaxation, there was a significant difference (P < 0.05) between inhibitors in the extent of relaxation reduce. While at 1 and 2 wk, the relaxation was predominantly reduced by indomethacin, at 3 mo. it was predominated by L‐NAME, whereas there was no difference at 4 wk. We conclude that relaxation of ASM decreases with age and the relaxation mediatory mechanisms alternate during maturation, predominating at by PGE2 at first weeks and replaced by NO at later stages.

Supported by Ministry of Health.
Hyperoxia Increases Elastic Modulus of Alveolar Epithelial Cells Through Rho Kinase
Authors: Wilhelm Kristina R. , Roan Esra , Waters Christopher M.
Published: June 2021

Patients with acute lung injury are administered high concentrations of oxygen during mechanical ventilation, and both hyperoxia and mechanical ventilation can independently cause injury. However, the precise mechanisms that lead to epithelial injury are not well understood. We hypothesized that alveolar epithelial cells may be more susceptible to injury caused by mechanical ventilation because hyperoxia causes cells to be more stiff due to increased f‐action formation via activation of the GTPase RhoA and its downstream effecter Rho kinase (ROCK). We used fluorescence microscopy to examine cytoskeletal structures in murine lung alveolar epithelial cells. We measured cell elasticity (E) using atomic force microscopy in the indenter mode. Hyperoxia caused increased f‐actin stress fibers formation, and cells became stiffer (higher E). Treatment with an inhibitor of ROCK significantly decreased E and prevented the cytoskeletal changes. Pre‐exposure of cells to hyperoxia promoted detachment when cells were subsequently stretched using the Flexercell device, but the ROCK inhibitor prevented this effect. Loss of cell adhesion would result in decreased barrier function. In summary, hyperoxia caused cytoskeletal remodeling that resulted in increased cell stiffness and promoted cell detachment during stretch. These effects were ameliorated by inhibition of ROCK. Supported by NIH grant HL094366 (CMW).
cGMP induces the degradation of internalized NKCC2 in thick ascending limbs: role of the proteasome
Authors: Ares Gustavo , Ortiz Pablo
Published: June 2021

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter NKCC2. cGMP decreases NKCC2 activity by decreasing the amount of NKCC2 in the apical membrane. Furthermore, we found that internalized NKCC2 is degraded slowly (0.33%/min) but it is not known whether NKCC2 degradation is regulated. We hypothesized that internalized NKCC2 is degraded by the ubiquitin‐proteasome system in a process stimulated by cGMP. TAL surface proteins were biotinylated, allowed to internalize and biotin remaining in surface proteins stripped away. Internalized NKCC2 was measured by western blot. cGMP enhanced the rate of disappearance of internalized NKCC2 by 83 % and this was blocked by a proteasomal (MG132) but not lysosomal (leupeptin) inhibitor (Control: 0.29 ± 0.04%/min; cGMP: 0.53 ± 0.10%/min; cGMP + MG132: 0.10 ± 0.10%/min; cGMP + Leupeptin: 0.44 ± 0.06 %/min; p < 0.05). Ubiquitination of membrane proteins is involved in their degradation. We found that NKCC2 inmunoprecipitated with ubiquitin. Proteasome inhibition induced the accumulation of ubiquitin‐NKCC2 and this was enhanced by cGMP (MG132: 59 ± 14%, MG132+cGMP: 111 ± 25%; p<0.05). We concluded that internalized NKCC2 is degraded via the proteasome pathway in a process stimulated by cGMP. cGMP induced degradation of internalized NKCC2 may contribute to decreased NKCC2 trafficking to the apical membrane of TALs.
The influence of FAs complexity and architectures of cytoskeleton on morphology during cell initial spreading
Authors: Lin Wei-Yin , Chen Ting-Jung , Wu Chia-Ching , Su Fong-Chin
Published: June 2021

The cytoskeleton controls intracellular force equilibrium and force transmission from attachment substrate to focal adhesions (FAs). During epithelial‐mesenchymal transition (EMT), essential process for numerous developmental processes, cell morphology has dramatically change by alter force balance between extracellular matrix (ECM) and cytoskeletons. The force and tension pattern change in cytoskeleton in relation to different morphology is still unknown. Our objective is to simulate the dynamics of intracellular force distribution and total stored energy during cell spreading by using the cytoskeleton tensegrity model with the pre‐tensed actin filaments and the pre‐compressed microtubules. The Madin‐Darby Canine Kidney (MDCK) cell, bovine aortic endothelium cell (BAEC), and myoblastoma cell (C2C12) and NIH 3T3 fibroblast (NIH 3T3) represents each step of EMT. The coordination of FA was used for stimulation. Our results indicated that most of all MDCK and C2C12 is circle shape as compared to BAEC and NIH 3T3 which is polygon. FAs in C2C12 but not BAEC and MDCK fuse together during cell spreading. The total energy in stress fiber and microtubules will increase and decrease with spreading area respectively. Cells have different FA distribution and stress fiber arrangement present different cell morphology due to energy distribution. This study was supported by NSC in Taiwan (NSC 98‐2627‐B‐006‐010).
Downregulation of the (pro)renin receptor by insulin is potentiated by high glucose in mouse renal collecting duct cells
Authors: Gonzalez Alexis A , Rajo Marco , Kassan Modar , Matrougui Khalid , Prieto Minolfa C
Published: June 2021

Upregulation of the prorenin receptor (PRR) in glomeruli leads to diabetic nephropathy and high glucose increases PRR expression in mesangial cells in vitro. Angiotensin II (Ang II) upregulates PRR in the collecting duct (CD), which is the main source of prorenin in diabetes. However, there is no evidence indicating that the expression of the PRR in the CD is altered during this condition. To test the hypothesis that PRR expression in the CD is augmented by high glucose and that this effect is prevented by insulin, we examined the PRR mRNA levels in renal inner medullay tissues of db ‐/‐ knockout mice and wild type mice. PRR transcript levels were increased in db ‐/‐ knockout mice (129 ± 8 vs. 100 ± 6 %; P<0.05), which exhibited high blood glucose levels (431 ± 3 vs. 110 ± 11 mMol/L). Treatment with insulin (0.1U/implant × 30 days) prevented this upregulation yet in the presence of chronic Ang II infusion (68 ± 6 % vs. 100 ± 6 %; P<0.05). In M‐1 cells, a mouse collecting duct cell line grown in normal glucose levels (5 mM), PRR mRNA levels were downregulated by 100 nM insulin for 8 hours (68 ± 4 % vs. 100 ± 5 % P<0.05), and even further with concurrent treatment of 30 mM glucose (45 ± 8 % vs. 100 ± 5 %; P<0.001). No significant effects were found by high glucose alone. The downregulation of PRR expression in the CD by insulin even in the presence of high glucose suggest that insulin may protect the kidney from tubulointerstitial damage in diabetic subjects.
Vacuolar H+‐ATPase Regulation by AMPK in the Kidney Proximal Tubule
Authors: Al-bataineh Mohammad , Pastor-Soler Nuria
Published: June 2021

The vacuolar H+‐ATPase (V‐ATPase) mediates transport of H+ across membranes. The V‐ATPase is expressed at the apical membrane of kidney proximal tubule cells. PKA regulates V‐ATPase subcellular localization in intercalated cells and it phosphorylates the A subunit at Ser‐175. Moreover, activation of the metabolic sensor AMP‐activated protein kinase (AMPK) prevents PKA‐mediated V‐ATPase apical accumulation in A‐type intercalated cells. We examined the role of AMPK in the regulation of V‐ATPase in proximal tubule S3 segment of rat kidney tissue slices ex vivo and in an S3 proximal tubule cell line. Incubation of kidney slices (75 min) in Ringer buffer alone caused V‐ATPase apical accumulation in S3 segments, while the AMPK activator AICAR induced a decrease in V‐ATPase apical accumulation. We also detected ubiquitinated V‐ATPase A subunit in S3 cells. Our results suggest that in S3 cells the V‐ATPase is regulated by metabolic signaling via AMPK (supported by NIDDK)
Modulation Of Renal Glucose Transport By Sweet Taste Sensing At The Proximal Tubule Brush Border Membrane
Authors: Chichger Havovi , Marks Joanne , Srai Kaila , Debnam Edward S , Unwin Robert J
Published: June 2021

Taste sensing by the T1R family of G protein‐coupled receptors is important for nutrient recognition in response to energy needs. In the intestinal epithelium, expression of the sweet taste heterodimer T1R2/3 is elevated by luminal glucose or artificial sweetener. This in turn promotes glucose uptake across the brush border membrane (BBM) via increased GLUT2 and SGLT1 protein levels. Renal glucose reabsorption occurs in the proximal tubule (PT) and is mediated by SGLT1, SGLT2 and GLUT2, however whether renal taste receptors exist and influence renal glucose transport is unknown.

Using RT‐PCR, we identified T1R2 and T1R3 mRNA in the rat kidney, while western blotting and immunohistochemistry revealed that the proteins are expressed at the PT BBM. Infusion of the artificial sweetener saccharin, for 2 hrs, caused a significant increase in BBM protein levels of T1R2 and T1R3. Concomitantly, we observed an increase in the Vmax for SGLT‐mediated glucose transport across the PT BBM (saccharin: 2.33 ± 0.44 vs saline: 0.95 ± 0.09 nmol/mg protein/4 sec, p<0.01) and a 77% increase in the expression of SGLT1, with no change in SGLT2 protein levels.

Our findings demonstrate the presence of sweet taste receptors at the PT BBM and indicate that these receptors influence renal glucose handling. Furthermore, we postulate that T1R2/3 presents a novel target to modulate renal glucose reabsorption in obesity and diabetes.
Epithelial sodium channel (ENaC) plasma membrane turnover is modified in channels containing δ subunits
Authors: de la Rosa Diego Alvarez , Wesch Diana Luise , Miranda Pablo , Giraldez Teresa
Published: June 2021

Epithelial Na+ channel (ENaC) formed by α, β and γ subunits plays a key role in Na+ reabsorption across tight epithelia. Strict control of ENaC activity is essential for extracellular volume homeostasis. ENaC endocytosis is controlled by Nedd4‐2 binding to PPxY motifs in the c‐termini of β and γ subunits and subsequent ubiquitination of channel subunits. A fourth ENaC subunit, named δ, can interact with β and γ to form channels that are also regulated by ubiquitination. In this study we compared the trafficking of αβγ and δβγ channels in Xenopus oocytes. The use of brefeldin A to block the insertion of newly synthesized channels to the membrane showed a higher endocytosis rate for αβγ than for δβγ. FRAP experiments were then used to study channel membrane insertion. The results indicate that the insertion rate of αβγ is higher than that of δβγ. Our data suggests that δ modifies ENaC turnover in the membrane and thus plays a dominant role in the regulation of channel trafficking. This work was funded by the Spanish Ministry of Science (grants CSD2008‐00005, BFU2010‐16265 and FIS‐PS09/00406).
Convergence of acid‐base regulation in embryonic cephalopod and fish
Authors: Tseng Yung-Che , Hu Marian Y. , Lin Li-Yih , Liu Sian-Tai , Melzner Frank , Hwang Pung-Pung
Published: June 2021

The present study provides a first finding of the acid‐base regulating machinery (ion‐transporters relevant for acid‐base regulation) in cephalopod, and series of studies showed that they exhibit specialized ion regulatory cells (ionocytes) on their skin and yolk epithelium. A feature that was so far only reported for fish. In addition, several ion regulatory genes were identified in cephalopod to be involved in the compensation of CO2 induced acid‐base disturbances, including Na+/H+‐exchanger (NHE3), ammonium transporters (Rhcg) and vacuolar H+‐ATPase (VHA) by being significantly up regulated in response to elevated sea water pCO2. Here we show for the first time that cephalopod embryos exhibit epidermal ionocytes and that the skin is a mayor site for proton excretion. Similar to fish, ionocytes located on the skin and yolk of cephalopod embryos are characterized by high concentrations of mitochondria. These similar responses towards elevated water pCO2 and sensitivity thresholds during life history may be explained by convergent acid‐base regulatory features of cephalopods and fish.
Rhcg1 and NHE3b are involved in ammonium‐dependent sodium uptake by zebrafish larvae acclimated to low‐sodium water
Authors: Shih Tin-Han , Horng Jiun-Lin , Liu Sian-Tai , Hwang Pung-Pung , Lin Li-Yih
Published: June 2021

To investigate whether Na+ uptake by zebrafish is dependent on NH4 + excretion, a scanning ion‐selective electrode technique (SIET) was applied to measure Na+ and NH4 + gradients at the yolk‐sac surface of zebrafish larvae. Low‐Na+ acclimation induced an inward Na+ gradient (uptake), and a combination of low‐Na+ and high‐ NH4 + induced a larger inward Na+ gradient. When measuring the ionic gradients, raising the external NH4 + level (5 mM) simultaneously blocked NH4 + excretion and Na+ uptake; in contrast, raising the external Na+ level (10 mM) simultaneously enhanced Na+ uptake and NH4 + excretion. The addition of MOPS buffer (5 mM) which is known to block NH4 + excretion also suppressed Na+ uptake. These results showed that Na+ uptake and NH4 + excretion by larval skin are associated when ambient Na+ level is low. Knockdown of Rhcg1 translation with morpholino‐oligonucleotides decreased both NH4 + excretion and Na+ uptake by the skin and Na+ content of whole larvae. Knockdown of nhe3b translation or inhibitor (EIPA) treatment also decreased both the NH4 + excretion and Na+ uptake. This study provides loss‐of‐function evidence for the involvement of Rhcg1 and NHE3b in the ammonium‐dependent Na+ uptake mechanism in zebrafish larvae subjected to low‐Na+ water.
Modulation of the onset of air‐breathing of the Siamese Fighting Fish and the Blue Gourami
Authors: Sanchez Jose Fernando Mendez , Burggren Warren
Published: June 2021

Onset of air‐breathing in larval amphibious fishes represents a distinct developmental landmark and thus an ideal process to investigate heterokairy (intraspecific plasticity in the timing of the developmental events). We have determined the effect of hypoxia on air breathing onset and physiological and morphological characters in larvae of the Blue Gourami (Trichogaster trichopterus) and the Siamese Fighting Fish (Betta splendens). Larvae were exposed intermittently (12/12 h daily) to 21, 18, and 15% O2 from 1 to 40 days post‐fertilization (dpf). Survival, onset of air breathing, wet mass, mass‐specific oxygen consumption (VO2), and critical PO2 (Pcrit) were measured every 5 dpf. Hypoxia advanced by 4 days, and delayed by 5 days, the onset of air breathing in Betta and Trichogaster, respectively. Hypoxia had no effect on larval wet mass in either species, but larval mass at the onset of air‐breathing was 28–81% higher than identically aged larvae not yet breathing air. Hypoxic exposure increased VO2 by 50–100% at each day throughout larval development in Betta, but had no effect on larval Trichogaster. Hypoxia increased Pcrit in Betta by 1.5 mmHg, but reduced Pcrit by 1.8 mmHg in Trichogaster. We attribute these interspecific differences to their ecophysiological differences: Betta is a facultative air breather after 36 dpf while in Trichogaster air breathing is obligatory past 32 dpf.
Variation in cuticular hydrocarbons with age, feeding, and insemination status of Anopheles gambiae
Authors: Artis Monica , Huestis Diana , Lehmann Tovi
Published: June 2021

The primary vector of malaria in sub‐Saharan Africa is the African malaria mosquito, Anopheles gambiae. Mosquito age is the most important indicator of a female's potential to transmit disease. Little is known about various aspects of this vectorˈs natural biology, such as mating/insemination rate, feeding habits and age structure. We evaluated the effect of age and related factors on composition of cuticular hydrocarbons. We predicted that the CHC amount and composition would change with age, namely that the total amount of CHCs would increase with age. We also predicted that a change in CHC amount and composition due to feeding status of females and mating status of male and females would be observed. Gas chromatography was used to detect amounts of CHCs in male and female mosquitoes in different treatments (mated vs. virgin, un‐fed vs. blood‐fed, and across age classes). Composition of CHCs with respect to the above factors will also be analyzed and presented. Our data showed that CHCs can be a predictive factor in determining female mosquitoe age. These laboratory results thus provide an essential benchmark to understanding variation in the field
Immune responses to parasitic infections in mourning doves (Zenaida macroura)
Authors: Juan Tiffany K , Jarrett Catherine L , Sweazea Karen L
Published: June 2021

Wild mourning doves (Zenaida macroura) captured during the summer of 2011 in Tempe, AZ were found to have an increased prevalence of parasitic worms (trematodes) within the mesenteric veins. In humans, trematode infections begin as mild cercarial dermatitis and, if left untreated, lead to significant inflammation and organ damage. It is not widely known what effects these parasites have on birds. We hypothesized that doves carrying trematodes would exhibit weight loss and have increased expression of heat shock proteins (HSP) 60 and 70 as indicators of immune system activation. Effects of the parasites on body condition were assessed by weighing the infected birds and comparing body mass to healthy animals. Using western blots, levels of HSPs were examined in erythrocytes and plasma homogenates from doves. The results did not support the hypothesis and indicated that differences in expression of HSP 60 and 70 proteins were insignificant (HSP60 p=0.328, HSP70 p=0.783). There were also no significant effects on body mass (p=0.072). Preliminary examination of blood smears created from these animals show increased leukocytes demonstrative of heightened immune system activity. Ongoing analyses of liver, spleen and intestinal segments along with blood samples will further characterize immune responses to the infection.
Electroacupuncture Attenuated Muscle Atrophy Induced by Hindlimb Suspension in Rats
Authors: Lee Sukho , Hong Junyoung , Kim Kijeong , Ni Qingwen
Published: June 2021

Preventing skeletal muscle atrophy is critical for maintaining quality of life and has been a major concern for those who experienced microgravity, aging process, neuromuscular disease, and various injuries.

PURPOSE

To investigate whether acupuncture and/or herb medicine can attenuate muscle atrophy induced by hindlimb suspension.

METHODS

Thirty five female SD rats were randomly divided into 5 groups: 1) Control (CON), 2) Hindlimb Suspension (HS), 3) HS + Herb (HSH), 4) HS + Acupuncture (HSA), and 5) HS + Combination of two treatments (HSHA) (n=7 each). Huang Qi (once a day) and Electro‐Acupuncture (15 mins, 3/wks) were applied for 21 days. The data were analyzed using one‐way ANOVA with Least Significant Difference post hoc test.

RESULTS

Hindlimb suspension induced atrophy of 36.8% in soleus muscle compared to control group.

Group
CON
HS
HSH
HSA
HSHA

Soleus
106±13.6
67±8.4*
68±8.8*
85±8.6*#^
91±14.7*#^

Data (mg): Mean±SD,

p<0.05 from CON.

p<0.05 from HS.

p<0.05 from HSH

Herb itself was not able to attenuate atrophy. However, muscle in HSA and HSHA were significantly higher than HS indicating that acupuncture ameliorated muscle atrophy. Also low‐field nuclear magnetic resonance measurement showed that spin‐spin T2 relaxation times were shorter after HS, then after HSA and HSHA treatments, the relaxation times were longer (recover) than HS, and similar to control group.

CONCLUSION

Acupuncture could be used to prevent (or attenuate) atrophy of skeletal muscle caused by various reasons including aging, space travel, injury and neuromuscular disease.
Preventive effects of premodulated interferential and pulsed currents on muscle atrophy in deep layer of rat hindlimb muscle
Authors: Tanaka Minoru , Fujita Naoto , Fujino Hidemi
Published: June 2021

Objective

Differential effects of premodulated interferential and pulsed currents on atrophy in deep muscle layer were investigated in this present study.

Methods

The rats were randomly divided into four groups: hindlimb unloading for 2 weeks (HU), hindlimb unloading plus electrical stimulation with pulsed current (ES), hindlimb unloading plus electrical stimulation with premodulated interferential current (P‐ES), and weight‐matched control (Cont) groups. One bout of electrical stimulation delivered 3 s (time on: 1 s maximum tetanic isometric contraction; time off: 2 s) for 1 min, followed by 5 min of rest. Finally, the stimulation was provided for 240s in a day during 2 weeks.

Results

For the soleus muscle located in deep layer of hindlimb, the mean muscle fiber cross sectional area in the HU group was significantly less than that in the Cont group. Although there were no significant differences between the HU and ES groups, the value in the P‐ES group was significantly larger than that in the HU and ES groups. The expression level of ubiquitinated proteins in the P‐ES group was significantly less than that in the HU and ES groups.

Conclusion

For prevention of atrophy in deep muscle layer, electrical stimulation with premodulated interferential current was effective compared with pulsed current. It is thought that this result was due to efficient energy transfer by premodulated interferential current.
Docosahexaenoic acid prevents atrophy‐related signaling in palmitate‐treated myotubes
Authors: Woodworth-Hobbs Myra , Zheng Bin , Price Russ
Published: June 2021

Lipid accumulation promotes insulin resistance and dysfunctional protein metabolism in skeletal muscle, resulting in debilitating muscle atrophy. While the saturated fatty acid palmitate (PA) induces muscle insulin resistance and promotes apoptosis and atrophic signaling pathways, omega‐3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) improve insulin sensitivity and may attenuate activation of pathways that lead to atrophy. Therefore, the purpose of this study was to examine the effects of saturated and omega‐3 fatty acids on atrophy‐related signaling pathways in skeletal muscle cells with the hypothesis that DHA prevents the detrimental effects of PA. C2C12 myotubes were treated with 0.1mM DHA and/or 0.5mM PA for 48 h. Insulin (100nM; 15 min) was added to cells to activate signaling pathways. The expression of proteins involved in atrophic and apoptotic signaling pathways was examined by Western analysis. PA reduced myotube size and number. PA also reduced insulin‐stimulated Akt, MEK1/2, and Foxo3a phosphorylation plus induced caspase‐3 and actin‐fragmentation. All of these responses were blocked by the addition of DHA; DHA alone had minimal effects on these pathways. In conclusion, these data suggest that DHA protects C2C12 myotubes from activation of atrophic signaling pathways by high levels of saturated fatty acids by maintaining insulin survival signaling.

Support: NIH DK007734
The generation of reactive oxygen and nitrogen species (RONS) is not different during injurious lengthening contractions and non‐damaging isometric contractions
Authors: Sloboda Darcee , Brooks Susan V.
Published: June 2021

Neither isometric contractions (IC) nor stretches without activation (passive stretches, PS) cause injury to skeletal muscle. In contrast, when muscles are activated and stretched (lengthening contractions, LC), structural damage occurs that leads to the degeneration of damaged muscle fibers by unknown mechanisms. RONS may contribute to fiber degeneration by damaging proteins and lipids or by initiating signaling pathways, but RONS generation during LC has not been examined. We hypothesized that RONS generation would be greater during LC than IC. Whole lumbrical muscles dissected from mice were subjected to a series of IC or PS that did not injure the muscle or damaging LC that caused a 28 ± 9% deficit in isometric force. RONS were monitored during the protocols with CM‐H2DCF‐DA. CM‐DCF fluorescence increased 6‐fold during IC. CM‐DCF fluorescence also increased during LC, but the increase was not different from that during IC, despite greater force generation during LC (29 ± 2 mN vs 19 ± 2 mN, P < 0.001) and the resultant force deficit. CM‐DCF fluorescence did not increase with PS. We conclude that RONS generation by muscle is associated primarily with activation rather than force and does not change as muscle is damaged by LC. Since RONS generation was not different during contractions whether or not the muscle was damaged, RONS may not be required for the initiation of injury following LC. This work is supported by AG020591.
Muscle RING finger‐1 (MuRF1) inhibits IGF1‐dependent Akt activation and exercise‐induced cardiac hypertrophy
Authors: Wadosky Kristine M. , Rodriguez Jessica E , Willis Monte S.
Published: June 2021

We previously identified that MuRF1 inhibits cardiomyocyte growth in pressure overload‐induced cardiac hypertrophy. To determine if MuRF1 more broadly regulates physiologic cardiac hypertrophy, we tested the hypothesis that MuRF1 regulates insulin‐like growth factor‐1 (IGF‐1)‐dependent cardiomyocyte growth that occurs in response to exercise. Transient MuRF1 knockdown in HL‐1 cardiomyocytes treated with IGF‐1 resulted in a 53.3% increase in cell size, compared to a 30.0% increase in cell size in scrambled siRNA control treated cells. This exaggerated IGF‐1‐dependent cell growth was abolished in cells with increased MuRF1 expression. Since IGF‐1signaling pathway converges on the serine/threonine kinase Akt‐1, we next determined MuRF1's role in IGF‐1 mediated Akt‐1 activation. Decreasing MuRF1 resulted in an increased IGF‐1‐dependent activation of Akt‐1. Since IGF‐1 signaling regulates physiologic cardiac growth, we next subjected MuRF1 −/− mice to voluntary wheel running. MuRF1−/− mice run significantly faster (4.47±0.28 vs. 3.31±0.14 mph) and for longer distances (17.2±1.5 vs. 12.1±0.7 km) compared to sibling‐matched wildtype mice, resulting in an increased left ventricular (LV) mass compared to sham (~40% greater vs. ~30% in MuRF1 +/+ mice). These studies suggest a role of MuRF1 in regulating exercise‐induced cardiac hypertrophy in vivo by its regulation of the IGF‐1 signaling pathway.
Inhibition of Glycolysis and mTORC1 activation in Human Skeletal Muscle with Blood Flow Restriction Exercise
Authors: Gundermann David M , Dickinson Jared M , Fry Christopher S , Walker Dillon K , Volpi Elena , Rasmussen Blake B
Published: June 2021

Low‐intensity resistance exercise (RE) coupled with blood flow restriction (BFR) stimulates an increase in muscle size and strength, however the mechanisms contributing to the anabolic effects of BFR exercise are not well understood. One potential mechanism includes the acidosis‐induced inhibition of glycolysis leading to an accumulation of glycolytic intermediates known to stimulate mTORC1 signaling. The aim of this preliminary study was to compare glycolytic flux from BFR exercise and traditional RE. Young men were divided into two groups. One group performed leg extensions at 70% 1RM (traditional RE). A second group performed leg extensions at 20% 1RM coupled with BFR. The number of repetitions was similar between groups. Phosphorylation of mTOR and muscle protein synthesis in the vastus lateralis was increased to a similar extent in both groups following exercise. Muscle glycogen decreased 37% after traditional RE, whereas it was unchanged following BFR exercise. Lactate production increased in both groups but did not disperse during BFR until after the release of the cuff. The maintenance of muscle glycogen levels in concert with increased lactate production following BFR exercise is in agreement with a proposed mechanism in which accumulated glycolytic intermediates may contribute to increased mTORC1 signaling following BFR exercise.

Funded by: NIAMS R01AR049877, NIA P30AG024832, NIH T32HD07539
Impact of chronic voluntary resistance training during recovery following hindlimb unloading on rat hindlimb muscles
Authors: Shimkus Kevin L , Shirazi-Fard Yasaman , Hogan Harry A , Fluckey James D
Published: June 2021

Little is known regarding the effects of multiple bouts of microgravity and 1g reloading on skeletal muscle, nor how the application of chronic resistance training during an initial recovery may affect muscle, either immediately or throughout a subsequent unloading. Male Sprague‐Dawley (6mo) were assigned to the following non‐exercise groups: 28d hindlimb unloading (HU), 28d HU session followed by a 56d recovery bout of normal cage ambulation at 1g (1HU+REC), 2 cycles of 28d HU with a 56d recovery period between unloadings (2HU), 2HU followed by an additional 56d recovery at 1g (2HU+REC), and an age‐ and housing‐matched control group (CON). Additionally, two groups of animals following the initial 28d HU session were given 7d recovery at 1g followed by a 7wk (3 sessions/wk) moderate‐intensity, moderate‐volume voluntary resistance exercise program, in which the animals were trained to perform a controlled squat‐like motion with full extension of the lower limb while under loads that gradually increased to additional resistances of ~65% bodyweight. Absolute muscle mass of the hindlimbs of the 1HU+REC/EX and 2HU+EX exercise groups did not differ significantly from their time‐ and mission‐matched counterparts (1HU+REC and 2HU, respectively), but relative mass does indicate a training effect in augmenting muscle recovery as well as a protective effect during an additional unloading exposure.
Altered levels of mitochondrial morphology proteins in skeletal muscle of mitochondrial DNA mutator mice
Authors: Joseph Anna-Maria , Wohlgemuth Stephanie E. , Picca Anna , Wawrzyniak Nick , Kujoth Gregory C. , Adhihetty Peter J. , Prolla Tomas A. , Leeuwenburgh Christiaan
Published: June 2021

The mitochondrial theory of aging proposes that mitochondrial DNA mutations (mtDNA) accumulate with age and this leads to mitochondrial dysfunction and/or damage that contribute to the aging process. mtDNA mutator mice (referred to as PolG) contain a deficient version of the proofreading mtDNA polymerase gamma and consequently incur high mutation rates to mtDNA that lead to mitochondrial dysfunction. As a result of these spontaneous mutations, PolG mice have an accelerated aging phenotype, allowing their use as a model of aging. The present study investigated the effect of increased mtDNA mutations on regulators of skeletal muscle mitochondrial biogenesis and morphology in young (3–6 mo) and old (8–15 mo) PolG and wild‐type (WT) mice. In old animals, mtDNA content was significantly reduced in the PolG compared to WT. Moreover, there was an age‐dependent decrease in PGC‐1α levels exhibited in both PolG and WT animals. However, young mice with the PolG mutation contained a greater level of PGC‐1α when compared to young WT mice. Age‐dependent changes were also observed in morphology proteins, including higher levels of the fusion protein Mfn2 in both WT and PolG mice, and reduced levels of the fission protein Fis1, only in WT and not in PolG mice. These data suggest that alterations in the ratio of fusion to fission proteins may contribute to the aging phenotype that is exhibited in mtDNA mutator mice.
Lifelong wheel running with mild caloric restriction protects against the age‐related disruption of the dystrophin‐glycoprotein complex (DGC) in skeletal muscle
Authors: Hord Jeffrey Mack , Lee Yang , Leeuwenburgh Christiaan L , Lawler John M
Published: June 2021

Sarcopenia is the progressive loss of skeletal muscle mass and strength, associated with aging. Compromised integrity of sarcolemmal proteins including the dystrophin‐glycoprotein complex (DGC) are linked to dystrophic myopathies, and recent data suggest a role in sarcopenia. A critical key to compromised “mechanotransduction” of the DGC appears to be loss of neuronal nitric oxide synthase (nNOS) from the DGC and sarcolemma. Previously, we reported that life‐long wheel running (WR) and mild‐caloric restriction (CR) attenuate sarcopenia. Therefore, we tested the hypothesis that lifelong WR and CR would attenuate age‐related disruption in sarcolemmal localization of key DGC proteins in the plantaris muscle. Fischer 344 rats were assigned to four groups: young ad libitum (YAL), old ad libitum (OAL), old CR (OCR), and life‐long WR plus CR (OWRCR). OAL displayed dislocation of nNOS, α‐syntrophin, and dystrophin from the sarcolemma. nNOS and DGC disruption was attenuated in OWRCR, with lesser effect in OCR. Matrix metalloproteinase‐9 (MMP‐9), which can cleave DGC subunits, was elevated in the extramyocyte space in OAL compared with YAL, an effect diminished in OCR and OWRCR. Results are consistent with the hypothesis that lifelong exercise and mild caloric restriction may be effective countermeasures against age‐related disruption of the DGC. Supported by NIH (AR054084).
Skeletal muscle fibroblast collagen expression is negatively regulated by satellite cells
Authors: Fry Christopher Schaffer , Jackson Janna R , McCarthy John J , Peterson Charlotte A
Published: June 2021

Interaction between satellite cells and components of the extracellular matrix (ECM) of skeletal muscle has been demonstrated, and recent evidence suggests that satellite cells may regulate ECM remodeling following overload in muscle. We utilized mouse primary myoblasts and fibroblasts in co‐culture in addition to a genetically modified mouse model (Pax7‐DTA) to investigate the role of satellite cells in regulation of skeletal muscle ECM. The Pax7‐DTA mouse allows for the conditional ablation of >90% of satellite cells in adult mice following the administration of tamoxifen. Control/tamoxifen treated animals were randomized to sham or synergist ablation surgery, leading to overload of the plantaris muscle for 2 weeks. Microarray analysis was performed to determine if the loss of satellite cells altered plantaris gene expression. The loss of satellite cells under rest and overload conditions was associated with increased expression of a number of different collagens, as well as ECM structural and remodeling genes. Co‐culturing mouse myoblasts with fibroblasts led to a 40% decrease in the mRNA expression of Col VIa2 and Fibronectin compared to fibroblasts co‐cultured with fibroblasts. These results suggest that satellite cells are involved in the regulation and maintenance of the ECM both under rest and during muscle hypertrophy.

Funding: NIAMS R01AR060701 and the Jeane B. Kempner Postdoctoral Fellowship.
Alterations in the myogenic capacity of satellite cells in a mouse model of ALS
Authors: English Samuel Andrew , Gupta Sumeet , Clermont Daphney , Chen Dapeng , Mazala Davi , Chin Eva R
Published: June 2021

Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig's disease, is an invariably fatal progressive neurodegenerative disorder that results in pervasive muscle wasting. The purpose of this study was to characterize a possible defect in the myogenic capacity of the skeletal muscle resident satellite cell population in the SOD1G93A ALS mouse. Satellite cells were obtained from the flexor digitorum brevis (FDB) muscle of wild‐type control (CON) and SOD1G93A (ALS) mice and allowed to differentiate into cultured myotubes. In 90d old pre‐symptomatic mice, we observed significant decreases in the number of nuclei per myotube (4.97 ± 1.7 vs. 2.48 ± 1.1, p=0.051; CON vs. ALS), number of myotubes (5.58 ± 0.37 vs. 3.39 ± 1.7, p=0.049) and fusion index (% of total nuclei fused into myotubes; 32.1 ± 3.6 vs. 17.4 ± 5.4, p<0.01) in cultures from wild‐type CON vs. SOD1G93A ALS. These data suggest that the myogenic capacity of the satellite cell population in a mutant mouse model of ALS is impaired in a pre‐symptomatic stage of the disease, and thus may contribute to the ongoing atrophy process.
Pyrene exposure induces activation and differentiation of cultured satellite cells in Northern Leopard frogs
Authors: Goertzen Ned J. , Stabenau Erich K.
Published: June 2021

It has been demonstrated that pyrene exposure negatively affects muscle contractility and exercise performance in Northern Leopard frogs. In light of the fact that depuration can reverse the detrimental effects of pyrene exposure, it is possible that pyrene leads to proliferation and differentiation of satellite cells, a conserved niche of stem cells able to form myotubes. In the present study, satellite cells were isolated from control and pyrene‐exposed frogs. A significant cell activation and differentiation was observed in cells cultured from muscles of pyrene‐exposed frogs. Comparable results were seen following an in vitro exposure of cultured cells to pyrene. Moreover, western blot analyses from isolated muscle tissue revealed that the concentrations of PAX3/PAX7 and Myogenin were significantly altered following pyrene exposure. These data suggests that pyrene, and other polycyclic aromatic hydrocarbons, are myotoxins that may lead to activation of muscle repair mechanisms via proliferation and differentiation of satellite cells. Funding was provided by Bjorklund Research Funds, and the Bradley University Biology Department and LAS Dean's Office.