bluuebunny/crossref_metadata_2025 huggingface dataset:
Dematin and its interaction with platelet cytoskeleton (833.8)
Authors: Azim Anser , Murungi Terry , Giragandi Abdelrahim
Published: June 2021

Background: Human platelets have spectrin based cytoskeleton that supports the plasma membrane of platelets and plays an important role in its activated and resting stages. The platelet cytoskeleton is also composed of peripheral microtubules. Human platelets have many actin binding proteins like spectrin, α□actinin, vinculin, talin, trpomyosin, adducin, dematin, myosin etc. In platelets monomeric actin is rapidly polymerized into F‐actin upon stimulation with the formation of surface filopodia and lamellipodia and acquires dendritic shape. Myosin forms stress like fibers with actin molecules. Dematin plays a role in activated platelets in the regulation of cytoskeleton. In this study we hypothesize the role of dematin, an ATP binding protein, in the regulation of cytoskeleton and its interaction with actin binding proteins.

Method: We isolated human platelets from anti‐coagulated blood as described earlier (Azim AC et al Blood. 2000, 95(3):959‐64.). Platelets were used after incubating them at 37 0C for half an hour before the experiment.

Results: The present study serially analyzed dematin’s coimmunoprecipitation with platelet myosin. And we believe that this interaction serves as a possible mechanism of ATP loading on the myosin molecule that subsequently plays a role in spreading and shape change.

Discussion: In an attempt to understand the physiological role of dematin in hemostasis its worth knowing the role of this actin bundling protein, dematin, in human platelets. We show for the first time that platelet myosin interacts with dematin. Further studies are needed to know the precise role of this interaction in regulating actin cytoskeleton, shape change and platelet expansion.
NSAIDs inhibit Kv1.3 expression in IEC‐6 cells and rat duodenum (840.8)
Authors: Silver Kristopher , Marsh Elizabeth , Thomas Laurel , Littlejohn Alaina , Lillich James
Published: June 2021

Clinical use of non‐steroidal anti‐inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug‐affected signaling pathways that contribute to the toxic side‐effects of NSAIDs are poorly understood. Previous studies have shown that NSAIDs inhibit cell migration in vitro by depolarizing membrane potential and suppressing expression of voltage‐gated potassium channel subunits. Kv1.3, in particular, is known to play an important role in cell migration, and results from a microarray study suggested that its expression is affected by NSAID treatment. Accordingly, we investigated the effects of NSAIDs on Kv1.3 mRNA and protein expression in a rat duodenal cell line (IEC‐6) or the duodenum of whole rats treated with indomethacin or NS‐398 for 72 h. Treatment of IEC‐6 cells with indomethacin or NS‐398 significantly reduced total and plasma membrane protein expression of Kv1.3. In rat duodenum, our results showed that expression of Kv1.3 mRNA was significantly decreased by NS‐398, but not indomethacin, and that Kv1.3 protein expression was significantly decreased following treatment with either NSAID. These results suggest that inhibition of epithelial cell migration by NSAIDs is associated with a decreased expression of Kv1.3 channels.

Grant Funding Source: Supported by National Institutes of Diabetes and Digestive and Kidney Diseases (1R15DK091791)
Targeted osmotic lysis of metastatic carcinomas by simultaneous blockade of sodium pumps and stimulation of voltage‐gated sodium channels (842.1)
Authors: Paul Dennis , Gould Harry
Published: June 2021

Metastatic carcinomas (MCs) overexpress voltage‐gated sodium channels (VGSCs) by up to 1500‐fold. We reasoned that electrical stimulation of VGSCs with concomitant blockade of Na+, K+‐ATPase (sodium pumps) would produce a rapid increase in intracellular sodium, causing an influx of water. This would cause the MC cells to swell and burst. To test this hypothesis in vitro, we cultured MDA‐MB‐231 cells, which overexpress NaV1.5 VGSCs by 1400‐fold. Treatment with the sodium pump blocker ouabain then electrical stimulation of the cells produced lysis in as little as 16 sec. Ouabain or electric current alone did not lyse the cells. Treatment did not lyse MCF‐10a (normal breast) cells. To test the hypothesis in vivo, we established MDA‐MB‐231 allographs in nude mice. When tumor diameter reached approximately 1 cm, we administered 10 mg/kg ouabain s.c. or saline. 30 and 60 min later, tumors received 60 sec of 10V DC delivered through direct contact stimulation. This single‐treatment produced 70% cell lysis in tumors treated with both ouabain and stimulation. Tumors treated with saline alone and saline plus stimulation averaged 10% lysis. Ouabain alone produced 15% cell death. Treatment did not affect noncancerous tissue. In survival studies, single day treatment attenuated tumor growth by 45%. 3‐Day treatment attenuated growth by 60%. Thus, targeted osmotic lysis can be an effective treatment of MCs that overexpress VGSCs.
Effects of the Gβ5‐RGS7 complex on muscarinic M3 receptor‐induced Ca2+ signaling and insulin secretion (843.12)
Authors: Slepak Vladlen , Karpinsky‐Semper Darla , Wang Qiang
Published: June 2021

The G protein subunit Gβ5 forms obligatory heterodimers with regulators of G protein signaling (RGS) proteins of R7 family (RGS6, 7, 9 and 11). These complexes are expressed in neurons and glands. Previously, we showed that Gβ5‐RGS7 attenuated signaling downstream of muscarinic M3 receptor, M3R. A predicted consequence of Gβ5 ablation on pancreatic beta cells would be an increase in insulin secretion. However, we found that Gβ5‐R7 knockout strongly attenuated, while its overexperession in Min6 insulinoma cells enhanced M3R‐induced insulin secretion. We studied isolated mouse islets, transfected Min6 and CHO‐K1 cells, measuring insulin secretion and changes in intracellular Ca2+ in the presence of various pharmacological agents, muscarinic agonists and functional mutants of Gβ5‐RGS7 and M3R. The results showed that Gβ5‐RGS7 has a dual effect on M3R‐mediated Ca2+ transients. While it attenuated Ca2+ release from internal stores, it stimulated Ca2+ influx across the plasma membranes. This pathway involves Gq, PLC, TRP and L‐type like Ca2+ channels, and may be particularly important in regulating secretory activity in nerve terminals and glandular cells. We also found that distinct muscarinic agonists can not only bias M3R signaling to specific pathways, but also confer differential sensitivity to the Gβ5‐RGS7 complex.

Grant Funding Source: Supported by National Institutes of Health (NIH) grants RO1EY018666 and RO1GM060019
Influence of M1 muscarinic acetylcholine receptor activation on arousal and cognitive performance using electroencephalography and novel touchscreen cognition assessment (845.5)
Authors: Gould Robert , Nedelcovych Michael , Dencker Ditte , Melancon Bruce , Stauffer Shaun , Wood Michael , Wess Jurgen , Xiang Zixiu , Lindsley Craig , Conn P , Jones Carrie
Published: June 2021

Cognitive impairments associated with neuropsychiatric disorders such as Alzheimer’s Disease and schizophrenia remain largely untreated. Cholinergic hypofunction has been hypothesized to contribute to cognitive deficits yet the role of subtype‐selective muscarinic acetylcholine receptors (mAChR) remains unclear. Electroencephalography (EEG) studies demonstrated that the M1 mAChR partial agonist VU0357017 and positive allosteric modulator (PAM) BQCA induced wake‐promoting effects and a shift from low to high frequency power during wake suggesting enhanced arousal and a pro‐cognitive signature. Cognitive performance of M1 mAChR knockout (M1 KO) mice was examined across a series of computer touchscreen‐based pairwise discrimination tasks assessing susceptibility to interference from previously learned competing sets of information, a cognitive deficit present in neuropsychiatric disorders. M1 KO and wildtype mice acquired the initial discrimination at similar rates yet M1 KO mice showed impaired acquisition of subsequent discriminations. The current data demonstrate M1 mAChR mediation of susceptibility to interference following repeated learning, supporting development of M1 mAChR subtype‐selective compounds as pharmacotherapies for cognitive impairments associated with neuropsychiatric disorders.

Grant Funding Source: Supported By MH086601, MH087965, MH093366, NS065867
Deletion of transient receptor potential canonical channel 1 prevents pulmonary vascular hyper‐permeability and edema formation secondary to cAMP activation of SPHK1 (847.4)
Authors: Tauseef Mohammad , Sukriti Sukriti , Ramaswamy Suresh , Dietrich Alexander , Birnbaumer Lutz , Malik Asrar , Mehta Dolly
Published: June 2021

Transient receptor potential channel (TRPC1) mediates store operated calcium (Ca2+) (SOC) entry in response to endoplasmic reticulum (ER) store depletion by IP3, and thereby signals the loss of endothelial barrier function. We show that pulmonary endothelial cells isolated from TRPC1 null mice failed to increase cytosolic Ca2+ entry, endothelial gap formation and permeability in response to thrombin and thereby rendered the mice resistant to thrombin and thapsigargin‐induced lung vascular barrier dysfunction. cAMP as well as the lipid mediator sphingosine‐1‐phosphate (S1P) stabilize adherens junctions (AJs) and prevent lung vascular permeability and inflammation. Thus, we tested the hypothesis that TRPC1 deletion enhances endothelial barrier function through induction of cAMP and S1P signaling. Intriguingly, loss of TRPC1 in lung endothelial cells augmented sphingosine kinase1 (SPHK1) activity which induces the phosphorylation of sphingosine into S1P preventing AJs disruption. We show that EPAC but not PKA induced the activation of SPHK1 in human lung endothelial cells. EPAC activation enhanced endothelial barrier function in control cells but failed to strengthen barrier function in SPHK1 knockdown cells. Our results suggest that loss of TRPC1 deletion prevents increase in endothelial permeability through EPAC up regulation of SPHK1 activity. Thus, specific inhibitors design to target TRPC1 channel by increasing cAMP and S1P signaling may provide resistance to inflammatory pulmonary diseases such as acute lung injury and acute respiratory distress syndrome.

Grant Funding Source: NIH
Implication of CD38/NAADP‐regulated autophagic flux in the phenotypic plasticity of mouse coronary arterial myocytes (849.3)
Authors: Wang Mi , Xu Ming , Chen Yang , Xia Min , Zhang Yang , Li Pin‐lan
Published: June 2021

We recently reported that CD38 gene deletion inhibits lysosomes trafficking and fusion with autophagosomes (APs) leading to impaired autophagic flux in mouse coronary arterial myocytes (CAMs). However, the pathogenic role of this deranged autophagic flux due to CD38 deficiency remains unknown. Here, we showed that coronary arteries from CD38‐/‐ mice even on the normal diet exhibited marked profibrotic feature with increased collagen deposition compared to CD38+/+ mice. This collagen deposition in coronary arteries was significantly enhanced when CD38‐/‐ mice were fed a Western diet. In primary cultures of CAMs from CD38‐/‐ mice, a marked increase in the level of collagen I protein, but not mRNA was found in comparison with those cells from CD38+/+ mice either under control condition or upon TGF‐β stimulation. By treatment of CAMs from CD38+/+ mice with the inhibitor of CD38 enzyme, nicotinamide or the antagonist of CD38 product‐NAADP, NED‐19, the similar phenotypic changes in CAMs were observed to those cells from CD38‐/‐ mice. This action of CD38 inhibition and gene deletion was also similar to that observed when CAMs were treated by the inhibitor of the AP formation, 3‐methyladenine or the lysosome function disruptor, bafilomycin A1. However, inhibition of proteasome function by MG132 had no effects on increased collagen I levels in CAMs. Further confocal microscopy showed that enhanced collagen I deposition was mainly within lysosomes in CD38+/+ CAMs, which was blocked by CD38 gene deletion or NAADP antagonist, NED‐19. Collectively, we can draw a conclusion from these data that collagen I metabolism is disturbed when CAMs lack CD38 gene or if CD38‐NAADP‐mediated signaling pathway is dysfunctional, which may be associated with impaired autophagic degradation of collagen I. This CD38 regulation of autophagic flux may represent a novel mechanism regulating the phenotypic plasticity of CAMs upon atherogenic stimulation.

Grant Funding Source: HL057244, HL091464 and HL075316
ADAM17 cleaves sphingosine 1 phosphate receptor 1 and regulates downstream signaling in endothelial cells (851.1)
Authors: Sukriti Sukriti , Desai Bhushan , Malik Asrar , Mehta Dolly
Published: June 2021

Sphingosine‐1‐ phosphate receptor 1 (S1PR1) is a high affinity seven‐membrane G protein coupled receptor for bioactive lipid sphingosine‐1‐ phosphate (S1P). S1PR1 plays a critical role in regulating several endothelial cell functions such as proliferation, migration and importantly enhances endothelial barrier function. However, mechanism regulating S1PR1 cell surface retention remains unclear. Members of the ADAM (a disntintegrin and metalloproteases) family of proteases are known for cleaving the ectodomain of a variety of cell surface receptors thereby regulating their activities. In our present study, we addressed the possibility that ADAM family of proteases target S1PR1 and thereby downregulate it’s signaling in endothelial cells. We found that treatment of human pulmonary arterial endothelial (HPAE) cells with phorbol‐12‐myristate 13‐acetate (PMA), an activator of ADAM proteases, induces cleavage of S1PR1 within 2 hrs, while treatment with a broad range MMP inhibitor (TAPI‐0), stabilized S1PR1 expression. Additionally, PMA‐cleaved S1PR1 failed to localize on the cell surface rendering it refractive to exogenous S1P ligation and in enhancing the endothelial barrier. Also, Transendothelial resistance after ADAM17 deletion clearly shows enhanced barrier function. Over expression or depletion of ADAM17, but not ADAM10, in HPAE cells altered S1PR1 cell surface expression and endothelial barrier function by S1P identifying a novel role of ADAM17 as the protease responsible for targeting S1PR1.
cAMP‐signaling regulates the expression of atheroprotective genes in vascular endothelial cells exposed to differential fluid shear stresses (851.14)
Authors: Rampersad Sarah , Hubert Fabien , Brzezinska Paulina , Freitag Silja , Umana M , Wudwud Alie , Maurice Donald
Published: June 2021

Fluid‐mediated shear stress is an important, but often overlooked, mediator of vascular endothelial cell (VEC) health. VECs in the linear portions of macrovascular structures experience high laminar shear stress and exhibit an anti‐inflammatory and anti‐thrombotic phenotype. However, at vascular bifurcations and large curvatures fluid shear stress dramatically declines. Exposure to this low shear stress reduces VEC expression of vasculoprotective genes, making these areas preferential targets of atherosclerosis development. cAMP‐signaling is known to regulate a diverse array of VEC functions. Using various methodologies, we investigated the role of cAMP, and its downstream effectors, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC)‐1, in regulating VEC health under differential fluid shear stresses. We report a novel role for cAMP, where modulation of this signaling pathway alters the expression of both atheroprotective and inflammatory genes in high and low shear stress conditions. This work has very important therapeutic implications for diseases characterized by endothelial cell dysfunction and atherosclerosis.

Grant Funding Source: Canadian Institutes of Health Research
Label‐free proteomic analysis uncovers early molecular link to abnormal cardiac neurotransmission in pro‐hypertension rats (856.2)
Authors: Burton Rebecca‐Ann , Schmidt Carla , Lu Chieh‐Ju , Larsen Hege , Gallone Giuseppe , Li Dan , Hao Guoliang , Nikiforova Natalia , Bub Gil , Kramer Holger , Robinson Carol , Paterson David
Published: June 2021

Sympathetic hyper‐responsiveness is an early hallmark of hypertension and has been linked to early metabolic and oxidative stress. We used label‐free quantitative proteomics to compare cardiac sympathetic neurons from the neonatal pro‐hypertensive rat (SHR) with neurons from an age matched normotensive control (WKY). Canonical protein interaction pathways and networks were generated using Ingenuity Pathway Analysis (IPA) to study protein regulation. We also performed functional association network analysis using STRING and the Girvan‐Newman fast greedy algorithm to produce protein clusters. Marked differences in proteins coupled to calcium signalling, metabolic processes, tissue architecture and cell migration were seen in the diseased neurons. In particular, we found abnormally high levels of non‐muscle myosin 9 in SHR neurons. This protein has been implicated in cytokinesis, vesicular trafficking, neurotransmitter release and familial human cardiovascular and neurological disease. Our findings indicate that direct modulation of myosin 9 could present a novel therapeutic target to manage abnormal sympathetic activation.

Grant Funding Source: British Heart Foundation
Mitochondrial ROS generation and function in skeletal muscle from older subjects (863.5)
Authors: Sakellariou Giorgos , Lightfoot Adam , Pearson Timothy , Wells Nicola , Gilmore Alastair , Griffiths Richard , McArdle Anne , Jackson Malcolm
Published: June 2021

Oxidative damage is reported to be involved in loss of tissue function with aging and the potential role of mitochondria as a source of increased ROS generation has been attributed to the impaired mitochondrial function and oxidative damage to mitochondrial components that occurs with advancing age. Data have predominantly been obtained from rodent studies and there is limited information on the role of mitochondrial ROS generation in muscle from older humans. To determine the role of mitochondrial ROS in the muscle decline that occurs with aging, muscle biopsies from young (20‐30 years) middle‐aged (45‐55 years) and older(>60 years) healthy volunteers were taken from the vastus lateralis muscle. Analyses were performed in small bundles of permeabilized fibers including mitochondrial ROS changes in the presence of various ETC substrates and inhibitors, changes in mitochondrial respiration, membrane potential and changes in maximum calcium‐activated tetanic force. No differences in fiber cross‐sectional area or force generation in response to calcium stimulation were observed in fibers between age groups. Respiratory function, mitochondrial membrane potential and ROS generation, assessed via changes in superoxide and hydrogen peroxide were not significantly different between age groups. Our data challenge the concept that mitochondrial ROS generation plays a key role in human skeletal muscle aging.

Grant Funding Source: Funded by the Medical Research Council, UK (Grant No: G1002120)
Regulation of ion channels in cystic fibrosis (869.8)
Authors: Henry Katherine , Schiffhauer Eric , Lee Seakwoo , Zeitlin Pamela
Published: June 2021

Cystic Fibrosis (CF) is an autosomal recessive disease, characterized by mutations in the CFTR gene, incorrect protein folding, and impaired chloride secretion. In addition to the maintenance of chloride secretion, CFTR is also suggested to be an important regulator of epithelial sodium channel (ENaC) activity. We previously identified the chloride channel ClCN2 as coexpressed with the mutant CFTR and hypothesized that in addition to restoring chloride secretion, ClCN2 can act as a negative regulator of ENaC. Coimmunoprecipitation analysis shows a novel physical interaction between ClCN2 and ENaC in pulmonary epithelium, which is accompanied by changes in ClCN2 and ENaC protein localization, as well as transepithelial resistance. Furthermore, ClCN2 has been shown to play an important role in the regulation of epithelial cell junctions and to this end, we show that changes in ClCN2 localization and activity may also affect cell junctions in CF cells. In conclusion, our data is the first to identify a novel interaction between ClCN2 and ENaC subunits. We also show potential changes in junction formation which may be regulated by changes in ClCN2 activity and localization. Therefore, when CFTR is nonfunctional, as in Cystic Fibrosis, ClCN2 may regulate ENaC activity and tight junction formation, which suggests that modulation of ClCN2 activity might be an advantageous target for treatment of the disease.

Grant Funding Source: Supported by 5T32HL72748‐11: Ruth L Kirschstein National Research Service Award
Rostral ventrolateral medullary catecholaminergic neurons contribute to the hypoxic ventilatory reflex in unanesthetized rats (872.5)
Authors: Barna Bárbara , Damasceno Rosélia , Takakura Ana , Moreira Thiago
Published: June 2021

The catecholaminergic (TH) neurons that reside in the rostral half of the ventrolateral medulla (VLM), also called C1 neurons, regulate blood pressure, the CRF/ACTH/corticosterone cascade and autonomic glucoprivic responses. The stimuli that most commonly activates the C1 neurons (nociception, hypotension, and hypoxia) also increases breathing. Although pharmacological evidence suggests that catecholaminergic neurons regulate breathing, a specific contribution of the C1 neurons to respiratory control has not been demonstrated, particularly in vivo. Here, we evaluate the role of the C1 cells in the control of breathing under hypoxia (8% O2) or hypercapnia (7% CO2) condition in unanesthetized rats. Bilateral injection of the immunotoxin anti‐dopamine β‐hydroxylase‐saporin (anti‐DβH SAP) was done in adult unanesthetized male Wistar rats (270‐300g, N = 5/group). Bilateral injections of anti‐DβH SAP (1.2 ng/100 nl) into VLM destroyed TH neurons. Selective inhibition of the C1 cells with bilateral injection of anti‐DβH‐SAP into the VLM reduced the increase in respiratory frequency (fR) (120 ± 8, vs. saline 136 ± 5 bpm), tidal volume (VT) (6.5 ± 0.4, vs. saline: 10.2 ± 2.7 ml/kg), and minute ventilation (MV) (818 ± 82, vs. saline: 1387 ± 325 ml/kg/min) elicited by hypoxia (8% O2). Hypercapnia challenges (7% CO2) caused graded increases in fR, VT, and MV, however, the changes in breathing elicited by hypercapnia was not altered after selective destruction of C1 cells in the VLM. The present study indicates that C1 neurons could be involved in the regulation of breathing and provide evidence that they may contribute to the hypoxic ventilatory reflex.

Financial support: FAPESP, CNPq, CAPES/PROEX.

Grant Funding Source: Supported by FAPESP, CNPq, CAPES/PROEX.
Contribution of the carotid body chemoreceptors to the modulation of heart rate variability (873.6)
Authors: Johnson Maja , Johnson Blair , Schlader Zachary , Crandall Craig , Joyner Michael
Published: June 2021

We tested the hypothesis that administering low‐dose dopamine (2 mcg/kg/min) to desensitize the carotid body chemoreceptors would alter metrics of heart rate variability (HRV). Electrocardiogram (ECG) recordings were obtained from 18 healthy adults (age = 31 ± 2 years; BMI = 24.6 ± 0.6 kg/m2) during two randomized study days where they received an IV infusion of saline (S) or dopamine (DA). HRV was analyzed during five minute segments at baseline and during infusions of S and DA. Time‐domain analysis of the root mean square of the successive differences (RMSSD) along with spectral domain analysis of low (LF) and high frequency (HF) were obtained. There were no differences (P > 0.05) in respiration rate or blood pressure at any time point. Heart rate increased from baseline during DA (60 ± 2 vs. 63 ± 2 bpm; P < 0.01) and during infusion it was greater in DA vs. S (63 ± 2 vs. 58 ± 2 bpm; P < 0.01). RMSSD decreased from baseline to infusion during DA (68.2 ± 7.6 vs. 51.6 ± 6.0 ms; P < 0.01) and during infusion it was lower in DA vs. S (78.3 ± 10.3 vs. 51.6 ± 6.0 ms; P < 0.01). HF was reduced during DA vs. baseline (1733± 301 vs. 870 ± 171 ms2; P < 0.01), and during infusion DA was also lower than S (1936 ± 471 vs. 870 ± 171 ms2; P = 0.02). There were no changes (P > 0.05) in LF, LF/HF or normalized HF and LF values. These data indicate that during resting conditions the carotid body chemoreceptors likely modulate parasympathetic components of HRV in healthy adults.

Grant Funding Source: Supported by NIH R01DK090541 and RO1HL61388
Aspirin augments carotid‐cardiac baroreflex sensitivity during muscle mechanoreflex and metaboreflex activation in older humans (875.8)
Authors: Drew Rachel , Blaha Cheryl , Herr Michael , Stocker Sean , Sinoway Lawrence
Published: June 2021

Low‐dose aspirin augments carotid‐cardiac baroreflex (CBR‐HR) sensitivity during muscle mechanoreflex and metaboreflex activation in young humans. However, whether aspirin has a similar effect in older humans is unknown. Therefore, we examined the effect of inhibiting thromboxane (TX) A2 via low‐dose aspirin on CBR‐HR sensitivity during mechanoreflex and metaboreflex activation in older humans. Twelve older, healthy subjects (62±1y, 6 men) underwent 3min passive calf stretch (mechanoreflex) with limb occlusion after performing 1.5min 70% MVC calf exercise and post‐exercise circulatory occlusion for 3.5 min (metaboreflex). This occurred twice, after 7 days of either low‐dose (81mg) aspirin (Asp) or placebo (Pla). Asp decreased baseline TX B2 by 83±4% (P<0.05) but did not affect 6‐keto prostaglandin F1α. Resting CBR‐HR maximal gain (GMAX) was similar between Asp and Pla (‐0.18±0.05 vs. ‐0.25±0.05 b.min‐1.mmHg‐1 for Asp and Pla, respectively). CBR‐HR GMAX was significantly augmented during stretch and metabolite accumulation with Asp compared to Pla (‐0.22±0.03 vs. ‐0.15±0.02 b.min‐1.mmHg‐1 for Asp and Pla, respectively; P<0.05). These results suggest that low‐dose aspirin augments CBR‐HR sensitivity during muscle mechanoreflex and metaboreflex activation in older humans. This could be due to increased cardiac vagal activity caused by reduced TX sensitization of muscle mechanoreceptors.

Grant Funding Source: Supported by NIH P01 HL096570 (LIS) and UL1 RR033184 (LIS).
Platelet levels and activity are decreased in hibernating 13‐lined ground squirrels which may prevent venous thromboembolism (879.17)
Authors: Cooper Scott , Howard David , Koch Tony , Lin Xingxing , Johnston Nate , Dobbs Kathryn , Zuberbuehler Matt , Cooper Amy , Lamont Christine , Mortimer Sarah , Hying Skyler , Bernhardt Kaley , Sell Shawn , Nelson Luke , Hawes Jennifer , Willems Heather , Morgan Leah , Macapugay Gizelle , Krus Catherine , Bowman Melissa , Lloyd Sarah
Published: June 2021

The objective of this study is to determine how a hibernating mammal avoids the formation of blood clots under periods of low blood flow. Hibernating ground squirrels have dramatically decreased heart rates (3‐5 beats per minute) and blood flow, which should put them at risk of forming deep vein thrombi. In response, they have several adaptations during hibernation that prevent blood clotting, including 3‐fold decreases in Factors VIII and IX, and 10‐fold decreases in platelets. During hibernation, platelets are maintained at temperatures of 4‐8°C for up to six months. Within two hours of arousal, platelet levels return to normal, are not rapidly cleared from circulation, and are still functional. We have shown that in addition to decreased clotting factors, ground squirrels have a 10‐fold decrease in von Willebrand factor (vWF) protein and a 3‐fold decrease in vWF mRNA during hibernation. Platelets adhere to exposed collagen in a wound through binding to vWF, and unlike other mammalian platelets, cold storage of ground squirrel platelets does not increase binding of vWF. Ground squirrel platelets are also atypical in that their microtubules are cold‐resistant and change in shape from a circumferential ring to an equatorial rod once they reach 20ºC. This shape change was originally thought to trap the platelets in the spleen, but splenectomized and sham operated ground squirrels had the same platelet levels during torpor and interbout arousal, suggesting storage in an extrasplenic location. Finally, no evidence of irreversible cardiac ischemic damage was observed during hibernation through either histological staining or blood markers including troponin T, lactate dehydrogenase and creatine kinase. Taken together, these results suggest that ground squirrels avoid deep vein thrombosis by reversibly decreasing platelet levels and activity during hibernation.

Grant Funding Source: Supported by NIH grant 2R15HL093680‐02
Integrating physiology into a scheme‐based curriculum (88.2)
Authors: Janssen Herbert , Osborne David
Published: June 2021

The scheme‐based curriculum model used at the Paul L Foster School of Medicine delivers the same basic science topics covered in other curriculum models; however, these topics are related primarily to a patient’s presenting condition and secondarily to an organ system. This model is designed to provide students the opportunity to examine basic science content as it relates to each condition with the belief that this will encourage critical thinking, content integration, and ultimately improved diagnostic skills. The model, originally developed at the University of Calgary has been modified to take advantage of the strengths at our University. Each scheme provides a framework that could be followed when evaluating data used in the diagnosis and treatment of a patient. Anatomical consideration are often considered first, followed by physiology, pathology, pharmacology with other disciplines providing the underpinnings of the diagnostic process. Close collaboration among clinician and basic science educators is essential. The order of basic science topic presentation is scheme dependent; thus, teaching materials available in traditional textbooks often becomes cumbersome to follow. Students graduating from this new school have performed above the national average on standardized exams.
Integrating pathology education: current practice and coming changes at Harvard Medical School (88.3)
Authors: Lichtman Andrew
Published: June 2021

Pathology education in the current HMS pre‐clerkship M.D. curriculum starts with general pathology in an integrated 11 week year 1 course, which also includes immunology and microbiology. Anatomic pathology is also sporadically included in other year 1 courses. Year 2 pathology is taught in lectures and small group exercises within several of the organ system pathophysiology blocks. Nonetheless, there is little pathology coordination between the year 2 blocks, and variable integration with other content within each block. A new HMS curriculum is now being developed, which will limit pre‐clerkship course work to about 1 year. General pathology will be covered in an intro foundations block, and pathology will also be highly integrated in the organ system courses that combined physiology and pathophysiology. Importantly, the new curriculum will include a post core‐clerkship year 3 with specialized courses in combined basic and medical science. These courses will be designed to take advantage of the clinical experience and more developed interests of the students. Pedagogic methods that deemphasize content delivery in the lecture hall, and encourage active learning will be applied throughout. How pathology learning will take place in this new environment is an important focus of the reform task force, and several exciting opportunities exist for pathology education in the context of the new year 3 courses.
Voluntary exercise during 6 weeks induces pathological cardiovascular responses in MAS‐deficient mice (881.4)
Authors: Santos Daisy , Santos Robson , Oliveira Marilene , Mosienko Valentina , Plehm Ralph , Todiras Mihail , Qadri Fatimunnisa , Poglitsch Marko , Alenina Natasha , Bader Michael
Published: June 2021

Physical training induces numerous physiological adaptations in the cardiovascular and skeletal muscle system related to changes in gene expression including modulation of the renin angiotensin system (RAS) components. Thus, the aim of this study was to evaluate the effect of running wheel training in knockout mice for the angiotensin‐(1‐7) receptor (MAS‐KO). For this we used C57Bl6 male mice (8‐12 weeks old) WT and MAS‐ KO divided into voluntary exercise group or sedentary control (N = 4‐8 per group). The aerobic training lasted 6 weeks and the amount of exercise was measured by the number of rotations per day. The gene expression responses of factors related to physiological and pathological adaptations and RAS components in the left ventricle, skeletal muscle and adipose tissue were evaluated by real‐time PCR (qPCR). Blood pressure (BP) was measured before and during the period of physical training using radio‐telemetry. The plasma levels of angiotensin peptides were determined by mass spectrometry. The results showed that in WT and MAS‐KO mice aerobic training promotes cardiac hypertrophy but in MAS‐KO it induced the expression of genes related to pathological conditions (ANP and BNP) and high plasmatic AngII / AngI ratio (0.204 vs. 0,126 pg/ml). The chronic aerobic exercise promoted an increase in skeletal muscle expression of FNDC5, irisin precursor, only in WT. In contrast, angiotensin‐(1‐7) receptor deficiency mice showed reduced FNDC5 expression in skeletal muscle (gastrocnemius and soleus) and no differences in inguinal adipose tissue UCP1 expression, opposite responses to that observed in WT mice. The MAS‐KO group presented slightly elevated baseline mean BP (105.5 ± 6.55 vs. 98.48 ± 7.65 mmHg for MAS‐KO and WT). A paradoxal increase in BP after 4 weeks of voluntary running was observed im Mas‐deficient group. It was concluded that voluntary exercise during 6 weeks induces pathological cardiovascular responses in MAS‐KO mice.

Grant Funding Source: CAPES/PROBRAL
Oxygen cost of exercise hyperpnea is greater in women compared to men (882.3)
Authors: Dominelli Paolo , Render Jacqueline , Molgat‐Seon Yannick , Foster Glen , Romer Lee , Sheel A
Published: June 2021

We tested the hypothesis that the O2 cost of exercise hyperpnea is greater in women compared to men. Healthy subjects (8M, 7F) first performed a maximal step cycle exercise test with esophageal and gastric balloon instrumentation followed by a hyperpnea familiarization trial on a separate day. Cardiorespiratory parameters were measured and flow‐ and pressure‐volume loops were constructed from the same breaths (~n=10) for each exercise stage. Days 3 and 4 involved exercise hyperpnea mimicking over 5 ventilatory levels while in the cycling position. Mimicking trials were repeated (n=4) for each ventilatory level. End‐tidal CO2 was controlled to a level corresponding to each exercise stage. Respiratory muscle O2 uptake (VO2rm) was linearly related to work of breathing (WOB) in men (r=0.94, P<0.001) and women (r=0.88, P<0.001). The VO2rm to VE relationship was assessed by fitting a regression accounting for viscous and resistive work. There was no difference in viscous work (P=0.45); however, the resistive component was ~13x greater in women (P<0.05). At maximal ventilation the absolute VO2rm was similar between sexes (0.39 vs. 0.42 l min‐1, P=0.52) despite men having a greater VE (108 vs. 169 l min‐1); but the VO2rm represented a greater fraction of whole‐body VO2 in women (14.6 vs. 9.8%, P<0.05). In conclusion, the O2 cost of exercise hyperpnea is greater in women owing to a greater resistive WOB.

Grant Funding Source: Natural Sciences and Engineering Research Council of Canada
Development of a rat model of exercise under anesthesia capable of reproducing abnormal respiratory responses in chronic heart failure (882.5)
Authors: Nishizaki Akiko , Miyamoto Tadayoshi , Saku Keita , Ueda Shinya , Oga Yasuhiro , Kuwabara Yukimitsu , Ikeda Masataka , Kakino Takamori , Ide Tomomi , Kishi Takuya , Sunagawa Kenji
Published: June 2021

Background: Abnormal ventilation during exercise is one of the major manifestations of chronic heart failure (CHF). However the lack of good animal models under anesthesia makes it difficult to investigate underlying mechanisms of respiratory abnormality. In this study, we developed a rat model of cardio‐respiratory exercise testing (CPX) under anesthesia.

Methods: We used 16 weeks old rats with/without CHF. CHF was created by myocardial infarction. We induced muscle contraction by stimulating bilateral distal sciatic nerves (SNS) and mimicked exercise under anesthesia. We recorded breath‐by‐breath respiration with gas analysis. We ran a ramp protocol and a step protocol while recording respiratory responses.

Results: In the ramp protocol, SNS increased minute ventilation (VE) more in CHF in normal rats (NRM). The increase in VE with the production of carbon dioxide (VE/VCO2 slope) was 23.0% more in CHF than in NRM (p<0.05).The increase in tidal volume with the respiratory rate was 16.5% less in CHF than in NRM reflecting the rapid and shallow breathing in CHF. In the step protocol, the time constant (TC) of oxygen uptake (VO2) was 78.7% longer in CHF than in NRM. Conclusion: These data indicated the model simulated respiratory abnormalities in CHF reasonably well. This novel CPX model will provide us an opportunity to clarify the mechanisms of exercise‐induced abnormal ventilation in CHF.
The purine nucleotide cycle: a cardioprotective pathway induced by HIF‐1α (887.4)
Authors: Wu Joe , Bond Cherie , Li Ying , Wright Gary
Published: June 2021

Overexpression of hypoxia inducible factor 1α (HIF‐1α) confers robust ischemic cardioprotection. Previously we showed that HIF‐1α induces the ability to use fumarate as a terminal electron acceptor to sustain mitochondrial anaerobic electron transport chain (ETC) activity. The source of fumarate for ETC activity was established to be the purine nucleotide cycle (PNC). Here, we report that mRNA, protein, and activity of the rate‐limiting enzyme in the PNC, AMP deaminase, is induced by HIF‐1α. Thus, in addition to providing fumarate for anaerobic ETC activity, we hypothesized that induction of the PNC might serve as a mechanism that conserves the nucleotide pool during ischemia. The AMP that accumulates during ischemia can be metabolized by AMP deaminase to IMP, a membrane impermeable metabolite. Alternatively, AMP can be degraded to adenosine, which can diffuse into the interstitial space leading to a reduction of the cardiomyocyte's nucleotide pool. Consistent with our hypothesis, we show that HIF‐1α‐expressing hearts accumulate significantly less adenosine than wildtype hearts during ischemia. Collectively, our findings indicate that the PNC is a novel cardioprotective mechanism induced by HIF‐1α that preserves ETC activity while conserving the nucleotide pool.

Grant Funding Source: Supported by NIH grant R01HL084302
Using of oligodendrocyte progenitors from integration‐free human induced pluripotent stem cells in treatment of spinal cord injury (89.4)
Authors: Gharibani Payam , Kerr Candace , Gupta Siddharth , All Angelo
Published: June 2021

Since the first successful derivation of induced pluripotent stem (iPS) cells, the field of regenerative medicine has grown exponentially and the goal of ‘personalized’ therapy using autologous transplants is emerging as a feasible approach. While prior studies demonstrate that human embryonic stem cells can be used to derive oligodendrocyte progenitors, there are no reports describing these cells derived from human iPS cell lines. Here, we demonstrate for the first time that oligodendrocyte progenitors (OPs) can be derived from iPS cells generated using either an episomal, non‐integrating plasmid approach or standard integrating retroviruses. The efficiency of OP differentiation ranged from 40 to 60% of total cells, which is comparable to that of human embryonic stem cells. In addition, the iPS cell lines derived using episomal vectors or retroviruses generated a similar number of early neural progenitors (NPs) and glial progenitors (GPs). We also found that the episomal plasmid‐derived iPS generated OPs expressing late OP markers (O1 and GALC), as compared to early markers (O4 and NG2), indicating that they differentiate and express mature OP markers. Moreover, we discovered that OPs derived from iPS cells engraft for over 60 days following implantation in a rat model of contusive spinal cord injury (SCI). Although further investigation is needed, these results suggest that patient‐specific, iPS‐derived OPs have potential for regenerating damaged tissue following SCI.
Malignant hyperthermia: a runaway heat production futile cycle via the sodium channel (893.2)
Authors: Williams Charles
Published: June 2021

MHis a runaway heat production futile cycle mediated via the sodium channel at the myoneural receptor site. Non depolarizing muscle relaxants do not trigger MH episodes. Depolarizing muscle relaxants, i.e. succinylcholine, are potent triggers of MH. Acetycholine, a natural depolarizer, is capable of triggering MH during transport stress, fighting activity, breeding activity and other stressors. Halothane apparently destabilizes the myoneural sodium channel which results in a rapid induction of MH. The heat released by this activity drives the further release of heat accelerating MH.

The MHS pig has an increased action potential amplitude at the myoneural junction that lasts longer than normal. This is thought to induce hypertrophy of muscle mass, increased metabolic rate, and other effects. The increased metabolic activity in muscle causes the post mortem release of heat in muscle, accumulation of low acidity, which denatures the muscle proteins, resulting in pale, soft, exudative, pork.

Inhalation anesthetics trigger MH by degree. Sevoflurane is a very weak trigger of MH and requires several hours of exposure to initiate an episode of MH. The transition from Halothane to Sevo lead to a 11 fold decrease in the incidence of MH in the Greater Kansas City Area from 1:50,000 to 1:550,000.

Organon 9426, is a nondepolarizing muscle relaxant that prevents MH when MHS pigs are challenged with halothane or succinylcholine.
Expression of intersectin 1/2 is repressed by aldosterone through microRNAs in the CCD to alter ENaC‐mediated Na+ transport (893.4)
Authors: Butterworth Michael , Liu Xiaoning , Edinger Robert
Published: June 2021

Aldosterone is a well characterized regulator of the epithelial sodium channel (ENaC), and increases transepithelial Na+ transport in the kidney CCD. Here we report on two related scaffold proteins, intersectin (Itsn) 1 and 2 that are aldosterone repressed via microRNA regulation. Itsns are involved in coordinating vesicle trafficking events and modulating several signaling pathways. In a cultured mCCD‐cl1 cell line, Itsn expression is reduced by aldosterone (50nM, from 6hrs), which in turn increases ENaC activity. Exogenous depletion of Itsn 1&2 by siRNA increases ENaC activity (175±18% over control), while overexpression of Itsns reduces ENaC activity (40±6% of control, n=8). Immunolocalization of Itsn demonstrates a vesicular staining pattern consistent with a role in mediating ENaC endocytosis. To confirm this, we performed transepithelial capacitance recordings to assess changes in membrane surface area and demonstrate an increase in membrane retrieval with Itsn overexpression. Similar to its reported role in ROMK internalization we demonstrate here that Itsn is an aldosterone‐repressed protein (via microRNAs), involved in the altering of ENaC surface expression by regulating channel endocytosis. The molecular mechanism that underlies this function is currently under investigation.

Grant Funding Source: Supported by DK078917
Molecular impact of a human divalent metal‐ion transporter‐1 (DMT1) mutation (G212V) found in two compound heterozygotes with microcytic anemia (893.40)
Authors: Shawki Ali , Illing Anthony , Mackenzie Bryan
Published: June 2021

DMT1 is a H+‐coupled divalent metal‐ion transporter that is critical for intestinal iron (Fe) absorption and erythroid Fe utilization. Beaumont et al (Blood 107, 4168 [2006]) and Bardou‐Jacquet et al (Blood Cells Mol Dis 47, 243 [2011]) have described two patients compound heterozygous for mutations in DMT1 associated with hypochromic–microcytic anemia and hepatic Fe overload. The unrelated probands have in common a G212V substitution; the first proband also possesses a V114 deletion and the second, an N491S substitution. The probands’ siblings and parents each possess one mutated allele but were reportedly asymptomatic. We expressed mutant and wildtype (wt) DMT1 in Xenopus oocytes and used voltage‐clamp and radiotracer assays to examine the molecular impact of these mutations. The V114 deletion and N491S substitution abolished 55Fe2+ transport activity whereas 55Fe2+ transport mediated by G212V‐DMT1 did not differ from wtDMT1. G212V had no effect on the pH dependence of 55Fe2+ transport, presteady‐state kinetics, or apparent affinity for Fe2+ (K0.5 = 1.2 ± [SE] 0.4 µM cf. 1.5 ± 0.2 µM in wtDMT1; P = 0.55, n = 6 oocytes); however, G212V mediated much smaller Fe2+‐evoked currents than did wtDMT1. From simultaneous measurement of currents and 55Fe2+ fluxes in wtDMT1, we determined the H+/Fe2+ coupling ratio to be 17 ± 1 which greatly exceeds that expected for a tightly coupled system, i.e., most of the wtDMT1‐mediated current arises from H+ slippage. In contrast, the H+/Fe2+ coupling ratio for G212V was 1 ± 0.3. Slippage in cotransporters is normally thought of in terms of the energetic penalty; however, our observation that H+ slippage, but not Fe2+ transport, is disrupted in a mutant associated with disease raises the possibility that H+ slippage serve a physiological role.

Grant Funding Source: Supported by PHS Grant DK080047
LAT1 (Slc7a5) null mice are embryonic lethal and exhibit neural tube defects (896.5)
Authors: Poncet Nadege , Shi Yun‐Bo , Taylor Peter , Storey Kate
Published: June 2021

The System L1‐type amino acid transporter (LAT1; catalytic subunit Slc7a5) mediates transport of large neutral amino acids (LNAA) and thyroid hormone (TH). Some LNAA are required for full activation of the mTOR‐S6K pathway promoting protein synthesis and tissue growth. TH signalling is an important regulator of embryonic development.

We generated transgenic mice with functional deletion of Slc7a5. Inter‐crossing Slc7a5+/‐ mice failed to produce Slc7a5‐/‐ offspring, indicating that Slc7a5‐knockout is embryonic lethal. Fifty‐nine embryos from such crosses analysed at E9.5 to 11.5dpc (days post coitus) showed normal Mendelian distribution: 25.4% Slc7a5‐/‐, 54.2% Slc7a5+/‐, 20.4% Slc7a5+/+. At 11.5dpc, Slc7a5‐/‐ embryos were dead, but earlier stages had beating hearts. Slc7a5‐/‐ embryos exhibited defects in neural development at 9.5/10.5dpc: 20% show a “flat‐top” brain phenotype similar to that seen in mTOR deficit embryos, while 80% exhibit a failure of anterior neural tube closure and a cruciform morphology in the caudal neural tube indicative of a neurulation defect.

These preliminary data suggest an essential role of Slc7a5 in embryonic development. Further studies are now required to establish the exact cause of death, the relative importance of LNAA and TH transport by Slc7a5 for embryonic development and the mechanism by which such signalling regulates neurulation.

Grant Funding Source: Supported by the Wellcome Trust