bluuebunny/crossref_metadata_2025 huggingface dataset:
MEF2C Silencing Attenuates Load‐Induced Hypertrophy and Energetic Metabolism in Mice Left Ventricle
Authors: Pereira Ana Helena M , Clemente Carolina F.M.Z. , Theizen Thais H , Rocco Silvana A , Souza Jose R.M. , Pascoal Vinicius D.B. , Lopes‐Cendes Iscia , Franchini Kleber G
Published: June 2021

Aim: Myocyte enhancer factor 2C (MEF2C) transcription factor has been suspected to play a critical role in coordinating the proper response of cardiac myocytes to hypertrophic stimuli. However, a better understanding of the role of MEF2C in cardiac hypertrophy has been hampered by the fact the loss‐of‐function models of MEF2C in the heart do not survive to adult life. In the present study, we used an interference RNA strategy to investigate the role of MEF2C in the hypertrophic growth induced by pressure overload in mice left ventricle (LV).

Methods: siRNA targeted to MEF2C was delivery (30 μg) through the jugular vein of mice in the day before the surgery to establish transverse aortic constriction (TAC).

Results: siRNA administration induced prolonged MEF2C silencing (70 to 50% up to 15 days) in both control and TAC mice. MEF2C silencing was also confirmed in myocytes harvested from LV. Myocardial MEF2C silencing was accompanied by a marked attenuation of left ventricular hypertrophy and in the increases of the hypertrophic marker β‐myosin heavy chain but it did not affect the typical myocardial fibrosis of TAC mice. Furthermore, we observed no significant change in LV diameter or fractional shortening in TAC mice treated with siRNA targeted to MEF2C, despite the lack of increase in LV wall thickness in response to pressure overload. MEF2C silencing was also demonstrated to be accompanied by an increase in the AMP:ATP ratio in overloaded LV possibly reflecting the lack of proper mitochondrial biogenesis.

Conclusion: These findings suggest that MEF2C modulates multiple aspects of cardiac hypertrophy induced by mechanical stress, including the expression of sarcomeric genes and the changes in the energetic metabolism.
Genetic deletion of fatty acid amide hydrolase is associated with anti‐inflammatory cardiovascular phenotype in mice
Authors: Batkai Sandor , Mukhopadhyay Partha , Rajesh Mohanraj , Csiszar Anna , Cravatt Benjamin F , Hasko George , Liaudet Lucas , Ungvari Zoltan , Pacher Pal
Published: June 2021

Recent studies have uncovered important cross talk between inflammation, generation of reactive oxygen and nitrogen species, and lipid metabolism in the pathogenesis of cardiovascular aging. Inhibition of the endocannabinoid anandamide metabolizing enzyme, the fatty acid amide hydrolase (FAAH), is emerging as a promising approach for the treatment of various inflammatory disorders. Here, we have investigated the age‐associated decline of cardiac function and changes in inflammatory gene expression, nitrative stress, and apoptosis in FAAH knockout (−/−) mice and their wild‐type (+/+) littermates. There was no difference in the cardiac function between the young (8 weeks old) FAAH(−/−) and wild type mice. In contrast, the decline in cardiac function, increased myocardial gene expression of TNF‐alpha, gp91phox, MMP‐2, MMP‐9, caspase‐3/9; the myocardial iNOS expression, nitrotyrosine formation, PARP cleavage; and the caspase‐3/9 activity observed in the aging (<28 mo‐old) FAAH(+/+) mice, were largely attenuated in the knockouts. There was no difference in the myocardial CB1 and CB2 receptor expression between young and aging FAAH(−/−) and FAAH(+/+) mice. These findings suggest that pharmacological inhibition of FAAH may represent a novel protective strategy against chronic inflammation, oxidative/nitrative stress, and apoptosis associated with cardiovascular aging and atherosclerosis.
Over‐expression of HIV Tat Results in both Systolic and Diastolic Dysfunction in Awake Mice
Authors: Kan Hong , Lewis William , Fang Qiujuan , Xie Sherry , Beto Robert J , Warden Bradford E , Jain Abnash C , Finkel Mitchell S
Published: June 2021

Mechanisms responsible for HIV cardiomyopathy are unknown, but may include direct effects of HIV proteins on the heart. Transgenic mice overexpressing HIV Tat protein targeted to the myocardium, +/−Tat high transgenic mice (TG), have revealed anatomical and biochemical defects in the heart. The present studies were conducted to clarify the effect of Tat on cardiac function. In vivo hemodynamics were measured in awake mice after inserting a catheter tip in the left ventricle under general anesthesia. Under the age of three month, the heart rate (HR) was significantly lower in TG (591¡À47 vs 716¡À45 bpm, TG vs Control, respectively (p<0.05;n=8–12). Other hemodynamic indexes, including left ventricular systolic pressure (LVSP), positive and negative dp/dt (mmhg/s), and left ventricular end diastolic pressure (LVEDP) remained indistinguishable from Control. At 6 mos, however, ventricular dysfunction was evidenced in TG (HR = 580¡À47 vs 673¡À25 bpm, TG vs Control, p<0.05); LVSP (132¡À6 mmhg vs 147¡À6 mmhg, TG vs Control; p<0.05); LVEDP (15¡À4 mmhg vs 8¡À6 mmhg, TG vs Control, p<0.05); +dp/dt= 8872¡À331 mmhg/s vs 10026¡À796 mmhg/s TG vs Control, p<0.01) and .Cdp/dt (7403¡À432 mmhg/s vs 8835¡À368 mmhg/s, TG vs Control, p<0.01; n=8–12 in each group). Thus, targeted myocardial transgenic expression of HIV Tat results in depression in both systolic and diastolic function in mice.
Extensive Downregulation of Matrix Scaffolding Genes and TGF‐beta in Isolated Mitral Regurgitation in the Dog
Authors: Zheng Junying , Chen Yuanwen , Pat Betty , Dell'Italia Louis A , Tillson Michael , Dillon A Ray , Powell Pamela C , Shi Ke , Shah Neil , Denney Thomas , Husain Ahsan , Dell'Italia Louis J
Published: June 2021

The volume overload of isolated mitral regurgitation (MR) in the dog is characterized by LV dilatation and loss of extracellular matrix. We hypothesize that the MR heart is not only marked by increased matrix metalloproteinase (MMP) expression but also by downregulation of collagen synthesis. Gene arrays were compared from biopsy specimens obtained in 4 dogs prior to MR and 4 months after MR induction. There was a 50% increase in LV end‐diastolic volume by magnetic resonance imaging. MMP‐1 and ‐9 increased 5‐ and 10‐fold, in the face of no change in collagen I, a 1.5‐fold increase in collagen III, but a 35% decrease in LV hydroxyproline levels. Genes associated with cell‐matrix scaffolding including decorin, fibulin, fibrillin, were significantly downregulated. Genes regulating collagen synthesis including thrombospondin, TGF‐beta binding protein, TGF‐beta receptor, connective tissue growth factor, and plasminogen activator inhibitor were also significantly downregulated. LV TGF‐beta activity decreased 30% in MR compared to normal LV. These results indicate that LV collagen loss in MR is chiefly due to post‐translational processing and degradation of collagen I and III. In addition, there is an extensive downregulation of cell‐matrix scaffolding genes controlled by TGF‐beta pathway which is also downregulated. Taken together, this could explain why RAS blockade does not improve LV remodeling in isolated MR.
NOS dependent gene regulation in cardiac tissues of conscious type I diabetic dogs
Authors: Ojaimi Caroline , Kinugawa Shintaro , Hintze Thomas H.
Published: June 2021

We have shown that in conscious dogs with type I diabetes (DM) that baseline CBF was significantlydecreased, endothelium‐dependent (ACh) coronary vasodilation were impaired, and reflex cholinergic NO‐dependent coronary vasodilation was selectivelydepressed. The likely mechanism responsible for the depressedreflex cholinergic NO‐dependent coronary vasodilation was thedecreased bioactivity of NO from the vascular endothelium. Bradykinin reduced oxygen consumption in normal dog heart in vitro (38 ± 1.3%) and this was reduced in type I DM (18 ± 1.6%). The effect of bradykinin was restored by apocynin (30 ± 0.9%), tempol (30 ± 1.1%) or vit C (29 ± 0.7%) indicating a role for NADPH oxidase derived superoxide. Affymetrix Canine Arrays were used to study gene expression in dogs with alloxan‐induced diabetes. Cardiac RNA was extracted from the control and DM (n=4). The data revealed that 797 genes were differentially expressed (P<0.01; fold change of at least ±2). There was no change in eNOS mRNA. There was up regulation of some components of the NADPH oxidase subunits (gp91 (2.2 fold), P<0.03), glutathione peroxidase and down‐regulation of SOD1 (3 fold, P<0.001) and decrease (4–40 fold) in a number of genes encoding mitochondrial enzymes such as cytochrome c oxidase, NADH‐ubiquinone oxidoreductase, ubiquinol‐cytochrome c reductase, ATP synthase, isocitrate dehydrogenase 3, succinate dehydrogenase and aconitase 2. There was down‐regulation of Ca2+ cycling genes (ryanodine receptor; SERCA2 Calcium ATPase), and structural proteins (actin alpha). In conclusion, these data show a direct effect of diabetes on the heart by impairing NO bioavailability through oxidative stress. Supported by HL‐PO1‐43023.
Caloxin 1c2: Experimental Evidence That It Binds Plasma Membrane Calcium Pump
Authors: Pande Jyoti , Szewczyk Magdalena M , Kuner Rajneet , Ghosh Paromita , Grover Ashok K
Published: June 2021

We engineered caloxin 1c2, a novel high affinity peptide (TAWSEVLDLLRRGGGSK‐amide) inhibitor of the Plasma Membrane Ca2+ pump (PMCA). To show that caloxin 1c2 acts by binding to the PMCA, we synthesized a photoreactive derivative 3Bpa‐caloxin 1c2 (TA(Bpa)SEVLDLLRRGGGSK‐biotin), containing the photoreactive residue Bpa (benzoylphenylalanine) and biotin. 3Bpa‐caloxin 1c2 inhibited the Ca2+‐Mg2+‐ATPase activity of PMCA. The human erythrocyte membranes were photolabeled with 3Bpa‐caloxin 1c2 and the proteins were detected in the Western blots using HRP‐streptavidin to identify biotin or PMCA antibodies to detect PMCA. The photolabeled erythrocyte membranes showed a 250–270 kDa doublet with HRP‐streptavidin. The degree of biotinylation of the erythrocyte membranes depended on the length of the cross‐linking time, and concentrations of 3Bpa‐caloxin 1c2 and the membrane protein. Immunoprecipitates from the photolabeled erythrocyte membranes using a PMCA antibody showed a 250–270 kDa doublet with HRP‐streptavidin. Biotinylated proteins isolated from the photolabeled erythrocyte membranes using captavidin also showed a 250–270 kDa doublet with HRP‐streptavidin and PMCA antibodies. Caloxin 1c2 had been selected for binding to the extracellular domain 1 (amino acids 115–131) of PMCA isoform 4. Caloxin 1c2 and the extracellular domain 1 peptide of PMCA 4 competed with 3Bpa‐caloxin 1c2 for photolabeling the erythrocyte membranes. Pieces of de‐endothelialized pig coronary artery cross‐linked to 3Bpa‐caloxin 1c2 and treated with Alexa‐streptavidin were examined by confocal microscopy, using the Na+‐pump inhibitor Bodipy‐ouabain as a positive control. Both the probes bound to the cell surface. Thus, the photoreactive derivative of caloxin 1c2 binds to PMCA at the cell surface.

Supported by Heart & Stroke Foundation of Ontario.
Ouabain stimulates both proliferation and apoptosis in renal epithelial cells from patients with autosomal dominant polycystic kidney disease.
Authors: Blanco Gustavo , Nguyen Anh‐Nguyet
Published: June 2021

The hormone ouabain binds to its receptor, the Na,K‐ATPase to regulate cell growth and survival. We have studied the effect and intracellular signaling pathways of ouabain in the proliferation and apoptosis of epithelial cells from kidneys of patients with autosomal dominant polycystic kidney disease (ADPKD cells). Physiological levels of ouabain (3 nM) stimulated growth in ADPKD cells. This required the integrity of cell caveolae, and phosphorylation of caveolin‐1. In addition ouabain effect involved phosphorylation of the kinase Src and the epidermal growth factor receptor (EGFR). Downstream signaling of ouabain included activation of B‐Raf and the kinases, MEK and ERK. ERK phosphorylation resulted in translocation of the kinase to the cell nucleus and down‐regulation of expression of the cyclin‐dependent kinase inhibitors, p21 and p27. On the other hand, ouabain also enhanced apoptosis of ADPKD cells through activation of caspase 3. Altogether, these results suggest that ouabain activates both proliferation and programmed cell death, affecting the balance between these processes to favor ADPKD cell growth. Uncontrolled cell growth and abnormal rate of apoptosis play an essential role in kidney cyst formation and development in ADPKD. Therefore, ouabain is an important factor with the potential to enhance progression of the disease. [Supported by NIH grant HD043044 and a grant from the PKD Foundation].
Altered Regulation of Ion Channel Activity by EGF in Polycystic Kidney Disease
Authors: Zhang Zhi‐Ren , Chu Wen‐Feng , Komlosi Peter , Haycraft Courtney J. , Bell Phillip Darwin
Published: June 2021

Cilia are responsible for fluid flow‐induced increases in [Ca2+]i and that this increase in [Ca2+]i may be mediated by member(s) of the transient receptor potential channel family. To elucidate whether there are different cellular ion channel activities in orpk cilia (−) and orpk cilia (+) collecting duct cell lines, derived from the Tg737orpk mouse, we performed single‐channel patch‐clamp recordings in these cells. We observed channels in both cell types having a conductance of ~23 pS but with a different rate of occurrence; channels were observed in 64% of patches from cilia (−) cells vs. 35% of patches from cilia (+) cells. Results of ion selectivity experiments suggest that a non‐selective cation channel mediates these currents. These channels are regulated by epidermal growth factor (EGF). In cilia (−) cells, channel activity was markedly up‐regulated by apical application of EGF. In contrast, channel activity in cilia (+) cells was enhanced by EGF applied only to the basolateral membrane. These results suggest that EGF‐dependent signaling contributes to the regulation of non‐selective cation channels in these cells. Thus, defective cilia structure/function leads to increased apical non‐selective cation channel activity and alterations in membrane coupling to and enhanced sensitivity of this channel to EGF receptor activation.
The role of ubiquitination of the type IIa sodium phosphate cotransporter (Npt2a) in protein trafficking
Authors: Lederer Eleanor DeLand , Ameen Mohammed , Smith Heather , Merchant Michael L , Khundmiri Syed Jalal
Published: June 2021

In the apical membrane of OK cells, Npt2a is associated with the PTH receptor, PKA, AKAP 79/150, NHERF, ezrin, and protein phosphatase 2a. PTH causes phosphorylation of several apical membrane proteins and ubiquitination of Npt2a We hypothesized that PTH‐stimulated phosphorylation of associated proteins was required for dissociation of Npt2a from the protein complex and that ubiquitination was required for Npt2a trafficking to the endosome. To address this hypothesis, we treated OK cells with vehicle or PTH, compared Npt2a‐associated proteins by immunoprecipitation (IP), protein phosphorylation, and Npt2a trafficking. PTH resulted in a rapid (within 15 min) expression of Npt2a in late endosomes (rab7, mannose6phosphate receptor positive), but not early endosomes (rab5 positive). Removal of PTH resulted in insertion of new proteins, not recycled Npt2a, into the apical membrane. Gel filtration of vehicle and PTH‐treated OK cell lysates demonstrated a shift in the fractions expressing NHERF and Npt2a. IP studies showed decreased association of Npt2a with NHERF, ezrin, RII, and the PTH receptor and decreased association of the PTH receptor with NHERF. In contrast, Npt2a continued to co‐associate with AKAP. PTH stimulated ubiquitination of Npt2a and association with VCP, a chaperone for ubiquitinated proteins. Under basal conditions, Npt2a degradation was blocked by inhibitors of either lysosomal or proteasomal degradation, while PTH‐stimulated Npt2a degradation was resistant to proteasomal inhibitors. We conclude that PTH stimulates endocytosis of Npt2a by ubiquitination of Npt2a, resulting in release of Npt2a from its anchoring complex in the apical membrane and targeting to the late endosomal fraction for degradation.
Increased neuronal discharge in the RVLM of rats with chronic heart failure is mediated by AT1R
Authors: GAO LIE , Wang Weizhong , Wang Wei , Zucker Irving H.
Published: June 2021

It is well accepted that sympatho‐excitation is a hallmark of chronic heart failure (CHF). We have recently carried out a series of experiments documenting an important relationship between central AngII and sympatho‐excitation in animals with CHF. However direct neural recordings in the central nervous system of animals with CHF has not been carried out. In the current experiment we determined the spontaneous and AngII induced presympathetic neuronal activity in the RVLM of anesthetized sham and CHF rats. Experiments were performed on 15 sham rats and 18 CHF rats. A total of 39 and 51 neurons were recorded in sham and CHF rats, respectively. We found that (1) baseline neuronal discharge was higher in CHF rats (17.4 ± 1.2 spikes/sec, n = 51) than that in sham rats (9.6 ± 1.1 spikes/sec, n = 39; P < 0.05 vs CHF). (2) picoinjection of AngII into the vicinity of neurons significantly increased the firing rate in both sham (147 ± 11.4%, n = 37; P < 0.05 vs baseline) and CHF (189 ± 12.5%, n = 46; P < 0.05 vs baseline) rats. (3) Losartan significantly decreased the basal neuronal discharge in both sham (63 ± 12.6%, n = 37; P < 0.05 vs baseline) and CHF (32 ± 9.3%, n = 45; P < 0.05 vs baseline) rats. Losartan also completely abolished the AngII induced increased neuronal discharge. These results demonstrate the important role for AngII in the activity of presympathetic neurons in the RVLM of CHF rats. Together with our previous demonstration of an up regulation in AT1 receptors they suggest a pivotal role for central Ang II in the sympatho‐excitation of CHF. Supported by a SDG from AHA: 0635007N, NIH grants #PO‐1‐HL‐62222 and RO‐1‐HL‐38690.
Sympathoexcitation due to angiotensin II in the subfornical organ is increased by pregnancy and requires ionotropic glutamate receptors in the rostral ventrolateral medulla
Authors: Kvochina Lyudmyla I. , Hasser Eileen M. , Heesch Cheryl M.
Published: June 2021

Baroreflex sympathoexcitation is blunted, but baseline sympathetic tone is elevated in term pregnancy. Circulating angiotensin II (A II), which is increased in pregnancy, activates AT1 receptors in the subfornical organ (SFO). A projection from SFO to paraventricular nucleus (PVN) then to rostral ventrolateral medulla (RVLM) mediates sympathoexcitation. A role for A II at all sites has been proposed, but a recent study found mainly glutamatergic PVN to RVLM projections. We tested the role of RVLM inotropic glutamate (Glu) receptors to responses to microinjection of A II into the SFO of 4 nonpregnant (NP) and 3 term pregnant (P) sinoaortic denervated inactin anesthetized rats. Baseline mean arterial pressure (MAP) was lower (P= 71 ±5; NP= 103 ±1 mmHg) and heart rate higher (P = 312 ±10; NP = 278 ±8 bpm) in P rats. The increase in MAP and lumbar sympathetic nerve activity (LSNA) in response to A II (20 μM, 50 nl) in the SFO was greater in P rats (δMAP: P = +23 ±1; NP = +17 ±3 mmHg. δLSNA: P = +27 ±6; NP = +14 ±1%). Bilateral microinjection of the inotropic Glu antagonist, kynurenate (KYN, 40 mM, 100 nl) into the RVLM had no effect alone, but eliminated responses to SFO A II in both groups (δMAP: P = +2 ±2; NP = + 5 ±1 mmHg. LSNA: P = +2 ±1; NP = +3 ±1 %). Responses to A II in the SFO recovered 45 min later. Thus, RVLM inotropic Glu receptors are required for sympathoexcitation mediated by SFO A II. (NIH HL 36245; MU #URC‐06‐049).
Imaging the mechanics of signal transduction in cell membranes
Authors: Groves Jay T.
Published: June 2021

Signal transduction in living cells is carried out through cascades of chemical reactions, which generally begin on the cell membrane surface. In recent years, there has been growing realization that the large‐scale spatial arrangement of cell surface receptors can regulate the outcome of ensuing signal transduction process. Signaling through the T cell receptor (TCR) in the context of the immunological synapse provides a case in point. Spatial reorganization of TCRs occurs on multiple length‐scales, and apparently with multiple purposes, during antigen recognition by T cells. The cell membrane and cytoskelton, working as an inseparable unit in this case, create the mechanical framework within which TCR signaling processes occur. To better study these phenomena, a new experimental strategy, in which the spatial positions of cell membrane receptors are directly manipulated through mechanical means, has emerged. By physically inducing a ‘spatial mutation’ of the signaling apparatus, the role of spatial organization in signal transduction as well as the mechanisms by which it arises can be illuminated. Specific applications of this strategy to TCR signaling will be discussed.
Latent Fluorophores for Biomolecular Imaging
Authors: Raines Ronald T.
Published: June 2021

Ribonucleases can be cytotoxic because cleaving RNA renders indecipherable its encoded information. We have shown that, with the subtlest of manipulation, an otherwise innocuous ribonuclease can be transformed into a useful cytotoxin. Bovine pancreatic ribonuclease (RNase A) is the most often studied enzyme. We reasoned that this nontoxic enzyme could become cytotoxic if it were able to evade the endogenous ribonuclease inhibitor protein that protects cellular RNA against invading ribonucleases. By changing a single amino acid residue, we made RNase A toxic to human cells, thereby verifying our hypothesis. We have extended this approach to RNase 1, which is the human homologue of RNase A. Most importantly, we have shown that the cytotoxicity of these enzymes is manifested preferentially against cancer cells, both in vitro and in vivo. A variant of RNase 1 is scheduled to enter a Phase I clinical trial as a cancer chemotherapeutic in early 2008. We are now using a variety of tools, especially new small‐molecule probes known as “latent fluorophores” to delineate the mechanism by which our cytotoxic ribonucleases kill human cancer cells.

This work is supported by Grant CA73808 (NIH).
Target specified preautonomic neurones in the PVN receive a GABAergic input
Authors: Pyner Susan , Watkins Nicholas David
Published: June 2021

The paraventricular nucleus of the hypothalamus (PVN) maintains cardiovascular homeostasis. Evidence suggests that within the PVN, GABA and nitric oxide (NO) determine the level of excitability of presympathetic neurones and that an alteration of their action may lead to the sympatho‐excitation evident in heart failure. Anatomically, there is sparse evidence indicating how GABAergic and NO producing neurones might regulate presympathetic neurones. Therefore, the purpose of this study was to identify the location of GABAergic neurones in the PVN in relation two groups of target specified presympathetic neurones that play a role in cardiovascular homeostasis. Male Wistar rats had Fluorogold (FG) injected into the T2 region of the spinal cord. In a further group (male Sprague Dawley) herpes simplex virus expressing green fluorescent protein (HSV‐1‐GFP) was injected into the adrenal medulla (AM) or superior cervical ganglion (SCG). After a suitable survival period, brain tissue was processed to localise FG, HSV‐1‐GFP and GABA. AM and SCG presympathetic neurones were present in the parvocellular nuclei of the PVN. GABAeregic neurones surrounded the lateral boundaries of the dorsal cap. A minority of GABAergic neurones contained HSV‐1‐GFP while no FG neurone was found to be GABAergic. We suggest target specified presympathetic neurones of the PVN receive a GABAergic input.

Supported by British Heart Foundation.
Evaluation of the heart rate changes evoked by air jet stress after inhibition of the right or left sides of dorsomedial hypothalamus
Authors: Peliky Fontes Marco Antônio , Campagnole‐Santos Maria J , Xavier Custódio Carlos Henrique
Published: June 2021

Neurons in the dorsomedial hypothalamus (DMH) play a key role in mediating tachycardia resulting from stressful stimuli. We investigated a possible hemispheric difference in DMH neurons on tachycardia evoked by experimental stress. Changes in heart rate (HR) produced by air jet stress after right (R) or left (L) inhibition of the DMH were evaluated in two separated groups (G1, G2). Under tribromoethanol anesthesia (250 mg/Kg) rats received guide cannulas into DMH and a femoral artery catheter. Air jet stress (1.5 litres/min in a 10 min period) was repeated in 2 consecutive days in conscious rats. G1 (n=5) received injections of GABAA agonist, muscimol, (1nmol / 100nL) into LDMH at the 1st day and into the RDMH at the second day. In the G2 (n=5) the sequence of muscimol injections into the DMH was inverse, i.e. R before L. Strikingly, in both groups the tachycardia evoked by air stress was greatly reduced after RDMH inhibition (G1: L=143±17 bpm, R=58±20 bpm; G2: R=56±13 bpm, L=124±12 bpm P<0.05). In additional control experiments we found that after vehicle (NaCl 0.9%) injections into DMH, the tachycardia evoked by air stress averaged 119±17 bpm (n=10). We conclude that activation of the right DMH is critical to the full cardiac response observed during stress. Support: CNPq, FAPEMIG, CAPES.
Neurokinin‐1 receptor expressing neurons in the nucleus of the solitary tract are required for full expression of post‐exercise hypotension in spontaneously hypertensive rats
Authors: Bechtold Andrea G. , Tabor Jocelyn , Bonham Ann C.
Published: June 2021

A single bout of exercise decreases blood pressure (post‐exercise hypotension; PEH) in both hypertensive humans and spontaneously hypertensive rats (SHR). Our recent studies suggest that in SHR, PEH may be dependent in part on activation of neurokinin‐1 receptors (NK1‐R) in the nucleus of the solitary tract (NTS). The current experiments were performed to test the hypothesis that selective elimination of NK1‐R expressing neurons in the NTS would attenuate PEH in SHR. The NK1‐R targeted neurotoxin, stable substance‐P saporin (SSP‐SAP) was microinjected into the NTS to ablate NK1‐R neurons in adult male SHR implanted with telemetry transmitters to measure blood pressure (BP) and heart rate. SSP‐SAP induced a transient (5 day) increase in BP after ablation relative to control (blank saporin; p<0.01) rats. Blood pressure variability, assessed as the standard deviation of daily systolic BP, increased by the second week after NK1‐R neuron ablation (p<0.05). Two weeks after NK1‐R neuron ablation, rats were subjected to a single bout of dynamic exercise or sham exercise. SSP‐SAP significantly blunted PEH (p<0.005, 2‐way RM ANOVA). For example, by 120 min post‐exercise blood pressure was decreased by −15.2 ± 2 mmHg in control (n=4) versus 0.5 ± 2.7 mmHg in SSP‐SAP (n=6) treated rats. We conclude that ablation of NTS NK1‐R expressing neurons increases blood pressure variability and attenuates PEH in SHR. (Support: HL‐67183)
Serotonin (5‐HT) facilitates ventilation via distinct 5HT2 and 5HT4 receptor‐mediated mechanisms in situ, in the arterially perfused rat brainstem preparation
Authors: Corcoran Andrea E , Richerson George B , Harris Michael B
Published: June 2021

Studies in vitro suggest that serotonin (5HT) neurons may be important for CO2 chemosensitivity. Their roles in modulating breathing in vivo, however, are unclear. We use a perfused in situ rat brainstem preparation that exhibits patterns of phrenic nerve discharge akin to breathing. Neuroventilation (NV) in this preparation is sensitive to the levels of CO2 equilibrating perfusion solutions (ranging between 3.5 and 9% CO2). We have shown that changes in NV associated with mild hypercapnia are abolished by inhibiting 5‐HT neurons. The present study was designed to identify post‐synaptic receptor subtypes involved in the control of breathing, and test the hypothesis that specific serotonergic mechanisms are critical for normal ventilatory control. Treatment with a 5HT2 antagonist (ketanserin; 5 and 10 uM) depresses NV under low CO2 conditions, indicating a 5HT2 mediated facilitation of NV under hypocapnea. Elevating CO2 (3.5 to 7%) reverses ketanserin‐induced NV depression, indicating CO2‐sensitive NV excitation, mediated through a mechanism other than 5HT2 receptor activation. Preliminary results suggest the balance may be due to 5HT4 activation in response to CO2, supporting a role for diverse serotonergic mechanisms in CO2 chemosensitivity in this preparation.

Research supported by the NIH, NIMH and NINDS, Office of Minority Health Research SNRP program; grant number 5 U54 NS41069‐06A1
NITRIC OXIDE (NO)‐INDUCED RELAXATION IN PULMONARY VESSELS OF EWES EXPOSED TO CHRONIC HIGH ALTITUDE HYPOXIA IS THROUGH A cGMP‐INDEPENDENT, Na+‐K+‐ATPase‐MEDIATED MECHANISM
Authors: Chai Edward Ekmaha , Negash Sewite , Gao Yuasheng , Longo Lawrence , Raj Usha
Published: June 2021

Purpose. To determine alterations in NO mediated relaxation in pulmonary vessels of pregnant ewes exposed to chronic high altitude hypoxia (HAH). Methods. Isolated intrapulmonary vessels from pregnant ewes exposed to HAH or from matched sea‐level controls were preconstricted with endothelin‐1 and relaxed to DETA‐NONOate, a nitric oxide donor. Roles of guanylate cyclase; cGMP‐dependent protein kinase, PKG; Na+‐K+‐ATPase; and sarcoplasmic reticulum Ca2+‐ATPase, SERCA; were assessed by determining DETA‐NONOate‐mediated relaxation in the presence of the various inhibitors. Results. We found that relaxation to DETA‐NONATE in HAH vessels was decreased compared to controls. In the presence of ODQ, relaxation of HAH vessels to the NO donor was decreased in arteries but unchanged in veins, indicating that in the HAH veins, NO was mediating relaxation by a cGMP‐independent mechanism. The cGMP‐independent relaxation in veins was abolished with the addition of ouabain but not by thapsigargin, indicating that NO was mediating relaxation by activating Na+‐K+‐ATPase. Western blot analysis showed increased protein expression of Na+‐K+‐ATPase. Conclusion. Following exposure to chronic high altitude hypoxia, the relaxation responses to NO in intrapulmonary vessels of pregnant ewes are differentially altered. In veins, NO induced relaxation is decreased and is no longer mediated by activation of guanylate cyclase but occurs through activation of Na+‐K+‐ATPase. In arteries, NO‐mediated relaxation is also decreased but is still mediated via cGMP.
Beneficial effects of sildenafil to alleviate pulmonary hypertension after 2 and 4‐week chronic hypoxia
Authors: Milano Giuseppina , Joncic Ana , Morel Sandrine , Samaja Michele , Vassalli Giuseppe , von Segesser Ludwig Karl
Published: June 2021

Aim. We investigated whether sildenafil alleviates the cardiovascular and pulmonary alterations induced by 2‐ or 4‐week chronic hypoxia (CH).

Methods. Rats were exposed to room air (21% O2, N), CH (10% O2) for 2 and 4 weeks, with and without treatment with Sildenafil (1.4 mg/Kg/day, ip) (n=7/group). At the end of the exposure, we measured right ventricular hypertrophy (right to left plus septum weight, RV/LV+S), pulmonary wall thickness index (WTI), right and left systolic ventricular pressures (RVSP and LVSP, respectively) and endothelial nitric oxide synthase (eNOS) proteins.

Results. CH increased RV/LV+S (p<0.05) and WTI (p<0.01) after 2 and 4 weeks. Treatment with sildenafil attenuated both parameters after 2 weeks (p<0.05) and after 4 weeks hypoxia (p=0.01). RVSP was higher in CH than N rats (p=0.01). Treatment with sildenafil attenuated this parameter irrespectively of the duration of hypoxia. By contrast, CH did not change LVSP after either 2 or 4 weeks. The phosphorylated form of eNOS decreased in CH hearts (p<0.05). Treatment with Sildenafil increased the phosphorylated form of eNOS to normoxic values irrespectively of duration of hypoxia.

Conclusion. Treatment with sildenafil provides an efficient strategy for the management of cardiac and pulmonary hypertrophy during CH, irrespectively of the duration until 4 weeks, presumably via increased phosphorylation of eNOS.
Effect of Adenosine Receptor Antagonists on Augmented Vasodilation During Hypoxic Exercise
Authors: Wilkins Brad W. , Casey Darren P. , Pike Tasha L. , Eisenach John H. , Dietz Niki M. , Joyner Michael J.
Published: June 2021

The augmented skeletal muscle vasodilation during hypoxic exercise is due, in part, to an intensity dependent contribution of beta‐receptor activation. We tested the hypothesis that an adenosine mediated vasodilation increases with hypoxic exercise intensity. Subjects (N=10) breathed hypoxic gas to titrate arterial O2 saturation to 80% while remaining normocapnic (re‐breath system). Consecutive bouts of forearm exercise (10% and 20% of maximum) were performed under normoxia and hypoxia during phentolamine (alpha‐receptor antagonist) alone and the combination of phentolamine / aminophyline (adenosine receptor antagonist). Exogenous adenosine administered before and after aminophyline established effective receptor blockade. Forearm vascular conductance (FVC; ml/100mmHg/min) was calculated from blood flow (ml/min) and blood pressure (mmHg). During hypoxic exercise, the rise in FVC (Δ from baseline) during phentolamine was 314±30 and 470±37 (10% and 20% respectively). ΔFVC was similar at both 10% (349±45; P=0.3) and 20% (536±47; P=0.2) exercise during hypoxia with combined phentolamine / aminophyline. This occurred despite significant reduction in ΔFVC to exogenous adenosine following phentolamine / aminophyline (P<0.03). Our data suggests that in the absence of overlying vasoconstriction, adenosine does not contribute to the augmented hypoxic exercise hyperemia in humans.
Superoxide dismutase mimetic tempol inhibits hypoxic pulmonary vasoconstriction in rats independently on either nitric oxide production or basal tone of pulmonary vessels
Authors: Hodyc Daniel , Snorek Michal , Brtnicky Tomas , Herget Jan
Published: June 2021

Hypoxic pulmonary vasoconstriction (HPV) is regulated by changes in the production of and interactions among reactive oxygen species (ROS). There is a controversy as to whether HPV is mediated by an increase or a decrease in ROS production. Also, the role of nitric oxide (NO) in HPV remains unclear. We investigated whether the inhibition of HPV by the antioxidant tempol was dependent on the concentration of NO, and how its effect was influenced by increased basal pulmonary vascular tone.

In isolated rat lungs we measured vasoconstrictor responses to acute hypoxia before and after administration of tempol during perfusion with or without L‐NAME and after increase of basal vascular tone in the perfused lungs by increasing the potassium ion concentration in the perfusate by 15 mmol/l.

Administration of tempol significantly decreased vasoconstriction response to acute hypoxic challenge both in controls (89% less) and in L‐NAME group (100% less), the vasoconstrictor response did not differ significantly between these groups. The administration of tempol significantly decreased HPV both in K+ treated (96% less) and in K+ non‐treated group (68% less), the vasoconstrictor response did not differ significantly between these groups.

Inhibition of HPV by the SOD mimetic tempol does not depend on either NO production or basal tone of the pulmonary vessels.

GACR 305/05/0672, GAUK 7415/2007
Does Dietary Cholesterol Increase Cardiovascular Risk in Exercising People? A Randomized Placebo‐Controlled Trial
Authors: Lee Chang Woock , Gasier Heath G , Chikani Gentle , Riechman Steven E
Published: June 2021

Background: There is no clear evidence that dietary cholesterol increases cardiovascular risk (CVR), likely due to the lack of control for physical activity. We hypothesized in the presence of a standardized exercise program that varying dietary cholesterol would not change CVR. Methods: 3 groups of 50–69 year old subjects (N=30) underwent 12 weeks of resistance exercise training (RET, 3×/week, 2–3 sets, 8–12 reps, 70% of max strength). Each group consumed 3.5 mg/kg lean/day (<200 mg/day, LC), 7.0 mg/kg lean/day (∼400 mg/day, MC), or 14.0 mg/kg lean/day (∼800 mg/day, HC) of cholesterol. Results: No significant differences were found among 3 groups in regard to changes of blood cholesterol (mg/dl, LC: −1.9±15, MC: 4.7±38, HC: 5.9±12, P=.784), HDL (LC: 0.8±2, MC: −0.2±7, HC: 2.1±6, P=.684), LDL (LC: −0.4±13, MC: 12.9±23, HC: 7.5±11, P=.247), triglycerides (LC:−11.8±16, MC:7.6±41, HC:−10±47, P=.479), cholesterol/HDL ratio (LC: −0.08±0.3, MC: 0.08±0.9, HC: −0.02±0.4, P=.846), and systolic/diastolic blood pressures (mmHg, LC: 1.1±5, MC: −2.1±6, HC: −2.1±6, P=.340 / LC: −0.8±4, MC: 0.03±4, HC: 0.1±5, P=.859). Conclusion: The results suggest that variability of dietary cholesterol may not increase CVR in the context of RET which is supported by the weak link of dietary cholesterol to CVR in studies that did not control physical activity.

This study was supported by US Poultry and Egg Association
Does mitochondrial oxidative stress limit skeletal muscle function?
Authors: Lustgarten Michael S. , Remmen Holly Van
Published: June 2021

To determine whether skeletal muscle‐specific oxidative stress is sufficient to limit muscle performance. Skeletal muscle‐specific oxidative stress was induced via a fast twitch skeletal muscle‐specific Cre Recombinase‐Lox P knockout of MnSOD (TnICreSod2Lox/Lox). Superoxide content (measured via EPR) was higher in mitochondria from the KO (11.6 ± 1.02 vs. 5.8 ± 1.14 pmol O2–), using Complex I linked substrate (Glutamate + Malate). Aconitase activity was reduced by 63% (indicative of increased intramitochondrial superoxide) in the KO (67.7 ± 8.7 vs. 181.2 ± 12.0 U/min/ug protein). The rate of mitochondrial ATP production was unchanged using Complex I linked substrate; however, using Complex II linked substrate (Succinate + Rotenone), ATP production was 40% higher in wild type when compared to TnICreSod2Lox/Lox mice (61.2 ± 4.5 vs.37.6 ± 6.6 nmol/min/mg mito protein). Plasma glucose levels in the KO were significantly lower starting at 20 minutes of running (96.4 ± 9.9 vs. 171.1 ± 8.5 mg/dL), and continued to decline at a greater rate in the KO, when compared to Wt, until exhaustion. Plasma lactate levels were higher in the KO starting at 10 minutes of running (7.5 ± 0.8 vs. 5.6 ± 0.5 mMol), and, continued to increase at a greater rate in the KO such that at 40 minutes, lactate levels were 2‐fold elevated. When the KO animals are run on a treadmill to exhaustion, TnICreSod2Lox/Loxran 47% less distance compared to wild type mice (287 ± 31 vs. 608 ± 127 meters) mice. We conclude that a skeletal muscle‐specific reduction in mitochondrial matrix antioxidant defense is sufficient to impair mitochondrial function and hence, muscle performance, and, deficits in Complex II activity may play a causative role in this process.
The role of human milk oligosaccharides (HMOs) in the establishment and proliferation of Bifidobacterium infantis in the infant gut
Authors: LoCascio Riccardo G , Paulino Gabriel , Freeman Samara L , Lebrilla Carlito B , German J. Bruce , Raybould Helen E , Mills David A
Published: June 2021

The molecular basis by which human breast milk supports the development of a protective intestinal microbiome in infants is unknown. Human milk oligosaccharides (HMOs) are quantitatively the third largest and most diverse component of breast milk. HMOs are believed to provide protection against pathogens and prebiotic enrichment of beneficial commensals such as bifidobacteria. Bifidobacterium infantis ATCC 15697, an isolate from the infant gut, preferentially consumes four milk oligosaccharides representing nearly 70% of all HMOs present in milk. The purpose of this study was to understand the role of HMOs in establishing and promoting a Bifidobacteria‐rich gut microflora in infants. When B. infantis is pre‐grown on HMOs versus lactose, there is a two‐fold increase in its binding ability to Caco2 cells. To further understand this interaction we have measured the global changes in gene expression of Caco2 cells in the presence of HMOs and B. infantis. This approach will serve as model for understanding how nutrients in a diet (HMOs) influence the interactions between gut microbiota and epithelial cells to effect human health. Research support provided by the California Dairy Research Foundation and UC Discovery Grants.
Genetically diverse invasive E. coli from IBD patients induce high concentrations of TNF‐α
Authors: Klapproth Jan‐Michael Axel , Ribot Efrain , Gerner‐Smidt Peter , Garrett Nancy , Sasaki Maiko
Published: June 2021

Increased number of invasive E. coli has been identified in patients with Crohn's disease (CD). Our goal was to characterize genotype and phenotype of invasive E. coli in American population of patients with CD, ulcerative colitis (UC) and normal controls. Significant genetic similarities, as determined by pulse field gel electrophoresis and cluster analysis, were identified for 114 E. coli strains from the same patient, regardless of whether they were isolated from macroscopically inflamed or normal appearing tissues (cluster II 95.5%, cluster III 81.3%). Invasive isolates from different patients were not genetically related but all IBD E. coli strains induced high mean concentrations of TNF‐α (CD 2,607.6 pg ±450/1 X 105 cells, UC 2,374.7 pg ±54/1 X 105 cells) and IL‐8 (CD 850.1 pg ±107/1 X 105 cells, UC 1,089 pg ±54/1 X 105 cells) in macrophage and epithelial cell cultures respectively. In comparison to isolates from normal tissues, invasive E. coli isolated from inflamed CD samples induced more TNF‐α (inflamed 3,081.4 ±235/1 X 105 cells, normal CD 2,476.5 pg ±448/1 X 105 cells), but significantly less IL‐8 (inflamed 672.2 ±59/1 X 105 cells, normal 992.2 pg ±94/1 X 105 cells) in cell cultures. TNF‐á expression was similar in TLR‐4 deficient and wild type macrophage cultures. Even though genetically diverse, phenotypically, invasive E. coli from patients with CD induce high concentrations of TNF‐α.
Bicarbonate Enhance Secretagogue induced Mucus release in Mouse Small Intestine
Authors: Yang Ning , Quinton Paul M.
Published: June 2021

The intestinal mucosa is covered by a mucus gel layer that has the important functions of lubrication and protection. Physiologically, HCO3− and mucus appear concurrently to protect the mucosal lining of the gastrointestinal tract as well as in other tissues. The present study investigates the effect of bicarbonate on secretagogue induced mucus release in mouse small intestine. Segments of small intestines of CL57BL/6 wild‐type mice were continously perfused (bath) with phosphate‐containing Ringer's solution (control) or bicarbonate‐containing Ringer's solution (experimental). Serial samples of luminal perfusates were collected and assayed for mucus content using a lectin‐HRP‐DAB reaction (dot Blot). Basal rates of mucus release in the experimental and control groups were not statistically significant. However, upon stimulation with serotonin (10 μM) and PGE2 (1 μM), the average rate of mucus release into the perfusate over 9 mins in the presence of HCO3− was 1.63 and 1.92 time higher than in the absence of HCO3−, respectively. Serotonin induced mucus release even after PGE2 stimulation indicating that different cells and/or signaling pathways for serotonin and PGE2 may be involved. These results suggest that HCO3− may be play an important role in the expansion, solubilization, and transportability of mucins, in general.
Enriched expression of lipid biosynthetic genes in follicle‐depleted mouse ovaries
Authors: Rivera Zelieann , Hoying James B , Hoyer Patricia B
Published: June 2021

An ovary‐intact mouse model for menopause has been developed using the chemical 4‐vinylcyclohexene diepoxide (VCD) which causes gradual ovarian failure (OF) by accelerating follicular atresia. This study was designed to compare gene expression in residual ovarian tissue of VCD‐treated mice to that of cycling controls while taking into consideration potential direct effects of VCD dosing. Mice were dosed daily (20d) with vehicle (sesame oil; n=12) or VCD (i.p; n=30). On d105, VCD‐treated mice were divided into 2 groups (n=10/group): follicle‐depleted (FD) and follicle‐depleted + VCD (VCD) and dosed for 15d. Ovaries were collected 24h after last day of the second VCD dosing and processed for microarray analysis (n=4/group). The abundance of mRNAs encoding fatty acid binding protein 3 (Fabp3), branched chain aminotransferase 2 (Bcat2), panthotenate kinase 4 (Pank4), enoyl CoA hydratase 1 (Echs1) and acetyl‐CoA dehydrogenase (Acadl) was greater (P<0.05) in FD and VCD compared to cycling controls. There were no differences (P>0.05) in expression of all genes between FD and VCD. Enriched expression of genes involved in lipid biosynthesis suggests a role for residual ovarian tissue in the regulation of cholesterol, lipids and fatty acids. Moreover, there was no evidence for direct effects of VCD on residual ovarian tissue, thus furthering the VCD‐treated mouse as a relevant model for menopause (AG021948).
Altered expression of iron‐management proteins in the brain microvasculature of Restless Legs Syndrome
Authors: Ponnuru Padmavathi , Wang Xinsheng , Earley Christopher , Allen Richard P. , Connor James R.
Published: June 2021

Restless Legs Syndrome (RLS) is a common movement related sleep disorder affecting 5–10% of the general population. Although the pathogenesis of RLS is still unclear, MRI, postmortem brain tissue and CSF analyses consistently indicate that brain iron insufficiency is associated with RLS. Given the consistent evidence of insufficient brain iron in RLS, we hypothesized that the profile of iron management proteins in the brain microvasculature would be altered in RLS. To test the hypothesis, microvessels were isolated from the temporal cortex of five RLS and five control brains. The concentrations of H ferritin, L ferritin, mitochondrial ferritin, transferrin receptor (TfR), transferrin, divalent metal transporter 1, ferroportin, and prohepcidin were determined by immunoblot assay. Our results reveal a significant decrease in the expression of HFRT in the microvessels from RLS compared to the controls indicating that there is low iron status in the endothelial cells of the blood brain barrier (BBB). There was also a significant decrease in the expression of transferrin and TfR in the microvasculature from the RLS brains. Normally, when a cell is iron deficient, TfR are elevated. The data are consistent with our position that there is a misregulation of TfR expression in RLS and imply that brain iron deficiency in RLS could begin at the level of transport across BBB.
Segmental segregation of Na/H Exchanger‐3 (NHE3) and Cl/HCO3 exchanger SLC26A3 (DRA) in rodent proximal colon
Authors: Lytle Christian , Talbot Colleen
Published: June 2021

The colon is believed to absorb NaCl through the coupled operation of the apical exchangers NHE3 and DRA. We determined the density of these proteins within the absorptive microdomain (apical margin of surface epithelial cells) by quantitative confocal immunofluorescence microscopy in colon segments from 5 mice and 4 rats fed a normal diet. Axial position was defined as the distance from the cecal/colon junction (0%) to the rectum (100%). In both rodents, NHE3 abundance was highest at 5–60% length, decreased steeply at 60–70% length, and was nearly absent at 70–100% length. In inverse fashion, DRA was absent at 0–20% length but increased steadily to maximal levels at ~60% length. The cecum contained abundant NHE3 and DRA in mouse, but only DRA in rat. The pH and [Na+] in the fluid overlying the colonic mucosa in fed anesthetized rats was measured using esophageal‐type microelectrodes. Surface pH profile correlated closely with positional variations in DRA abundance: pH was 7.6±0.2 in the cecum, decreased sharply in the proximal colon to 6.1±0.2, and returned to 7.5 by 60–100% length. Surface [Na+] was ~60 mM in the cecum, declined steadily to ~20 mM by 20% length, and declined further to ~10 mM over the rest of the colon. Our results indicate that NHE3 and DRA do not spatially co‐exist in the rodent proximal colon. Uncoupled operation of NHE3 without DRA could contribute to the luminal acidification distinctive of this segment.