bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of the high‐conductance Ca2+‐activated K+ (BK) channel in pulmonary arterial smooth muscle cells
Authors: Detweiler Neil , Song Li , McClenehan Samantha , Kharade Sujay , Kurten Richard , Rhee Sung , Rusch Nancy
Published: June 2021

Pulmonary hypertension (PH) is associated with a loss of vasodilator influences in the pulmonary circulation, which results in pathogenic vasoconstriction. However, we observed an over‐expression of high‐conductance, Ca2+‐activated K+ (BK) channels in pulmonary arteries (PA) during chronic hypoxia (CH)‐induced PH, providing a potential vasodilator target. Here, we report that BK channels of PA smooth muscle cells (PASMCs) from CH rats retain high Ca2+‐sensitivity, α‐β1 subunit coupling that confers sensitivity to BK channel openers, and hypoxia‐insensitivity as compared to normal (N) rats (n=4‐11). Importantly, these properties may permit pharmacological activation of the BK channel even in a hypoxic environment. In inside‐out membrane patches used to record unitary BK currents in PASMCs from CH rats (n= 4‐7), the BK channel log(Ca2+ EC50(mol/L)) was ‐5.9 in both N and CH rats. Lithocholate (LCA), a BKβ1 subunit‐dependent opener, caused a 26‐fold and 19‐fold increase in BK channel open‐state probability in PASMC from CH and N rats, respectively. Using lung samples from organ donors without diagnosed PH, we also defined BK channel properties in human PASMCs. Similar to rat, the hBK channel exhibited high Ca2+‐sensitivity (log(Ca2+ EC50(mol/L)) = ‐6.1), was activated by LCA, and was not inhibited by acute hypoxia (n=4‐7). Thus, PASMCs from N and CH rats and humans without diagnosed lung disease express a β1 subunit‐coupled, hypoxia‐insensitive BK channel, which is responsive to BK channel openers.

Supported by AHA 13PRE17240055 (NDD), NIH UL1RR029884 (NDD), and the UAMS College of Medicine Dean's Society.
FenfuroTM Attenuates Diet‐Induced Insulin Resistance and Hepatic Fat Accumulation
Authors: Nair Sreejayan , Hua Yinan , Bagchi Debasis , Swaroop Anand
Published: June 2021

Insulin resistance often precedes the onset of type 2 diabetes and offers a potential avenue for early intervention. The objective of this study was to evaluate the effect of furostanolic saponin (FenfuroTM, isolated form the spice fenugreek) either alone, or in combination with Green Coffee Bean Extract (GCB‐70TM, 70% chlorogenic acid) on high‐fat diet (HFD)‐induced insulin resistance in mice. Male C57BL/6J mice (6‐weeks old) were subjected to a normal diet (ND) or HFD and were randomly assigned to receive FenfuroTM, GCB‐70TM or combination for 10 weeks. Metabolic parameters, glucose tolerance, hepatic triglycerides (TG) and cardiac function were evaluated using indirect open‐circuit calorimetry, infrared measurements, intraperitoneal glucose tolerance test (IPGTT), oil‐red O‐staining and echocardiography respectively. Mice that received HFD showed lower oxygen consumption compared to those that received a ND, which was unchanged by the test agents. IPGTT revealed significant glucose intolerance in the mice receiving HFD, which was attenuated by FenfuroTM. Serum and hepatic TG which were elevated following a HFD was reconciled by both FenfuroTM and the combination. High‐fat feeding compromised myocardial contractile function (fractional shortening, peak shortening, maximal velocity of shortening/relengthening) which were unaffected by any of the treatment. Collectively, our data indicate that FenfuroTM counters obesity‐associated whole‐body glucose intolerance and hepatic fat accumulation.
Effect of aqueous stem extract of Momordica charantia on proglucagon and insulin expression in STZ induced diabetic rats
Authors: Kilari Eswar , Kotaiah Prased , Alluri Ramesh
Published: June 2021

To evaluate the In vitro and In vivo antioxidant potential of aqueous stem extract of Momordica charantia (AQMC) and to evaluate the effect of AQMC on proglucagon release and Insulin secretion in STZ induced diabetic rats. The in vitro antioxidant activity of AQMC was estimated by hydroxyl, hydrogen peroxide, DPPH, Nitric oxide radical activity and for determination of reducing power of Fe+3 and inhibition of MO (VI) formation in phosphomolybdinum assay. The in vivo antioxidant activity of AQMC was determined by estimation of protein, catalase, SOD and MDA levels in STZ induced diabetic rats after 28 days. The blood glucose levels and HbA1c levels were also determined. At the end of the study period the gene expression of proglucagon in ileum and Insulin in pancreas were studied. The AQMC showed potential antioxidant activity by In vitro and In vivo studies. The blood glucose levels and HbA1c levels were found to be elevated in STZ induced diabetic rats, however AQMC treatment reduced the elevated blood glucose and HbA1c levels in a dose dependent manner. The STZ induced diabetic rats did not show any gene expression for proglucagon (PLG) in ileum and reduced levels of insulin expression in pancreas. Whereas the treatment with Sitagliptin (55mg/kg) and AQMC (400mg/kg) for 28 days study period showed greater fold change of PLG gene expression in ileum and 3 & 2.2 fold changes of insulin gene expression in pancreas when compared with diabetic control. AQMC shows potent antioxidant, antidiabetic activities and expression of proglucagon in ileum and insulin levels in pancreas. The study reveals that the antidiabetic acivity of the AQMC might be through by influencing the incretin hormones particularly the release of GLP‐I.
Guttiferone K Exhibits Selective Cancer Cytotoxicity through ER Stress Aggravation
Authors: Miu Kai Kei , To Kenneth Kin‐Wah , Xu Hongxi , Lin Ge
Published: June 2021

Guttiferone K (GutK) is a novel phytochemical that was previously reported to show selective cytotoxicity in cancer cells. This study aims to employ different human colorectal cancer (CRC) cell lines as models to confirm endoplasmic reticulum (ER) stress aggravation as the mechanism of GutK to cause apoptosis. Furthermore, normal colorectal epithelial cell line is adopted as a reference to understand how anti‐cancer selectivity occurs.

ER stress markers were probed through immunoblotting and RT‐PCR in CRC cell line HCT116 and HT29 after treatment with GutK. Furthermore, prolonged treatment resulted in induction of pro‐apoptotic proteins, CHOP and DR5 expression. These protein were tested whether they could activate apoptosis by monitoring PARP cleavage after siRNA knockdown. Subsequently, realizing ER stress was aggravated by prolonged treatment with low dose of GutK in cancer cells, the same treatment was adopted on normal colon epithelial cell line CCD‐841 CoN to compare cytotoxicity via SrB assay, to check the ER stress state and to confirm selective toxicity through a normal & GFP‐labelled cancer cells co‐culture model by flow cytometry.

ER stress activation was confirmed as a result of activation of GRP78 & GRP94, eIF2α and XBP‐1 mRNA splicing after GutK treatment. CHOP and DR5 were induced after prolonged treatment yet effects were abrogated after transfected with siRNA. Normal cells were spared after the same treatment, resulting in only a mild ER stress condition which was insufficient to initiate apoptosis.

GutK aggravated ER stress in colorectal cancer cells but not in normal cells to elicit selective cancer cytotoxicity.
Polycystin 2 expression alters calcium‐contraction coupling
Authors: Kuo Ivana , Kwaczala Andrea , Nguyen Lily , Campbell Stuart , Ehrlich Barbara
Published: June 2021

Cardiac disorders are the main cause of mortality in autosomal dominant polycystic kidney disease (ADPKD). However, how the mutations in the polycystins predispose ADPKD patients to cardiac pathologies before development of renal dysfunction is unknown. We investigate the effect of decreased polycystin 2 (PC2) levels, a calcium channel that interacts with the ryanodine receptor, on myocardial function. We hypothesize that heterozygous PC2 mice (Pkd2+/‐) undergo cardiac remodeling due to changes in calcium handling, separate to renal complications. Pkd2+/‐ cardiomyocytes have altered calcium handling, independent of desensitized calcium‐contraction coupling. Paradoxically, in Pkd2+/‐ mice, PKA phosphorylation of phospholamban was decreased, whereas PKA phosphorylation of troponin I was increased, explaining the decoupling between calcium signaling and contractility. In silico modeling supported this relationship. Echocardiography measurements showed that Pkd2+/‐ mice have increased left ventricular ejection fraction after stimulation with isoproterenol (ISO), a ß‐adrenergic receptor (ßAR) agonist. Importantly, the Pkd2+/‐ mice were normotensive and had no evidence of renal cysts. In contrast to the Pkd2+/‐ mice, Pkd2 overexpression mice had decreased ejection fraction in response to isoproternol. Our results showed that PC2 levels change expression of calcium handling proteins, which resulted in altered cardiac contractility. Moreover, we observed alterations in the ß‐adrenergic receptor signaling pathway. We propose that PC2 levels in the heart directly contribute to cardiac remodeling in ADPKD patients in the absence of renal dysfunction.
The adverse effects of Bisphenol A on cardiac mechanical function
Authors: Chandra Akhil , Brooks Daina , Idrees Rabia , Jaimes Rafael , Kay Matthew , Posnack Nikki
Published: June 2021

Biomonitoring studies indicate that 90% of the population is routinely exposed to Bisphenol‐A (BPA), a compound commonly found in household plastics. Environmental exposure varies between 1– 100 nM, while clinical and industrial exposure can reach 10 uM. Epidemiological studies have shown an association between increased BPA exposure and cardiovascular diseases. We aimed to test the direct effects of BPA on cardiac function using a Langendorff‐perfusion model. Excised female rat hearts were treated with 1 nM‐10 uM BPA and the resulting effects on cardiac mechanical function and calcium handling were monitored. For calcium imaging, excised hearts were treated with Blebbistatin to arrest mechanical function, and then stained with Rhod‐2, a calcium indicator dye. Epicardial calcium transients were recorded using an Andor CCD camera equipped with wavelength specific filters (570+/‐ 30 nm), and an LED spotlight (535 nm) was used for dye excitation. Calcium transients were initiated at various pacing frequencies (5Hz, 6.6Hz, and 9Hz). To assess the effect of BPA on the mechanical function of the heart, a latex balloon was inserted into the left‐ventricle to quantitate left‐ventricular developed pressure (LVDP) and maximum contractility. At high pacing frequencies, BPA decreased LVDP by 16% at 1 nM, 24% at 100nM and 36% at 10 uM, while the maximum contractility decreased by 23% at 100 nM and 33% at 10 uM. We conclude that alterations to mechanical function and calcium handling are a sensitive parameter for assessing BPA cardiac toxicity.
CRTC2 Contributes to PGC‐1α Expression in Skeletal Muscle Cells
Authors: Rahnert J , Zheng B , Hudson M , Woodworth‐Hobbs M , Price S
Published: June 2021

Muscle wasting associated with chronic diseases has been linked to decreased expression of the transcriptional coactivator PGC‐1α. Overexpression of PGC‐1α can reduce FoxO‐mediated atrogene expression and counter muscle loss. Previous reports indicate that PGC‐1α transcription is regulated by CREB in a process that requires CREB‐regulated transcription coactivator (CRTC2). We recently found that CRTC2 protein is reduced in skeletal muscle of diabetic rats whereas CREB is highly phosphorylated (i.e., activated). Nuclear translocation of CRTC2 is promoted by dephosphorylation via calcineurin (CnA) while nuclear export occurs by phosphorylation via the salt‐inducible kinases (SIK). Our experiments tested the hypothesis that glucocorticoids decrease PGC‐1α expression, in part, by reducing CRTC2 levels in the nucleus. L6 myotubes were treated ± dexamethasone (Dex, 100nM) for 48hrs. Dex decreased PGC‐1α protein and nuclear level of CRTC2. It also decreased nuclear CnA and increased nuclear SIK, supporting the observed reduction in nuclear CRTC2 levels. Over‐expression of CRTC2 increased PGC‐1α protein and mRNA levels. To assess the role of CRTC2 on PGC‐1α transcription, muscle cells were transfected with a PGC‐1α luciferase reporter plasmid (PGC‐1α‐Luc). Over‐expression of CRTC2 increased PGC‐1α‐Luc activity. Dex decreased PGC‐1α‐Luc activity and deletion of the CRE binding site from PGC‐1α‐Luc (PGC‐1α‐DCRE‐Luc) attenuated the response. These data support the interaction of CRTC2 with phospho‐CREB as a mechanism for increasing PGC‐1α expression. Reduced nuclear CRTC2 may contribute to the loss of muscle mass arising during chronic diseases associated with elevated glucocorticoids.

Supported by NIH RO1 DK95610, NIH T32 DK007656 and VA Merit X01BX001456
The Force Dependency of Potentiation in Mouse Skeletal Muscle With and Without Myosin Light Chain Kinase (skMLCK)
Authors: Gittings William , Aggarwal Harish , Stull James , Vandenboom Rene
Published: June 2021

The isometric potentiation associated with myosin phosphorylation is force dependent. The purpose of this study was to assess the influence of a pre‐existing period of isometric force on the concentric force potentiation displayed by mouse muscles with and without the ability to phosphorylate myosin. We tested isometric (ISO) and concentric (CON) potentiation, as well as concentric potentiation after isometric force (ISO‐CON), in muscles from wildtype (WT) and skeletal myosin light chain kinase deficient (skMLCK‐/‐) mice. A conditioning stimulus increased (i.e. potentiated) mean concentric force in the ISO‐CON and CON conditions to 1.31±0.02 and 1.35±0.02 (WT) and to 1.19±0.02 and 1.21±0.01 (skMLCK‐/‐) of pre‐stimulus levels, respectively (data n = 6‐8, p < 0.05). No potentiation of mean isometric force was observed in either genotype. The potentiation of mean concentric force was inversely related to relative tetanic force level (P/Po) in both genotypes. Moreover, concentric potentiation varied greatly within each contraction type and was negatively correlated with unpotentiated force in both genotypes. Thus, although no effect of pre‐existing force was observed, strong and inverse relationships between concentric force potentiation and unpotentiated concentric force may suggest an influence of attached and force generating crossbridges on potentiation magnitude in both WT and skMLCK‐/‐muscles.
Interferon‐γ is a Potent Activator of Caspase 12 in C2C12 cells HL112085, HL113494
Authors: Callahan Leigh Ann , Wang Lin , Song Xiao‐Hong , Supinski Gerald
Published: June 2021

We recently found that pharmacologic activation of endoplasmic reticulum (ER) stress in vivo induces skeletal muscle contractile dysfunction. However, it is not known if cytokines are capable of inducing ER stress in muscle. The purpose of the present study was to determine if interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), or interleukin1 beta (IL1β) activates ER stress and the unfolded protein response (UPR) in skeletal muscle. C2C12 myoblasts were grown to 80% confluence, differentiated for 5 days, and mature myotubes exposed to either PBS+0.1% BSA (control), IFNγ (25ng/ml),TNFα (20ng/ml), or IL1β (10ng/ml). Cells were harvested at 30 minutes, 1, 2, 4, 8, and 24 hours post‐exposure (n=5/condition/time point). Using Western blots, we assessed cleavage of caspase 12, an early marker of ER stress; we also measured BiP, Grp94, calreticulin, PDI and CHOP, proteins which are late markers of ER stress and the UPR. At 24 hours after exposure, caspase 12 cleavage robustly increased in C2C12 cells exposed to IFNγ, while TNFα and IL1β had little effect to activate caspase 12 (e.g. the 38 kDa caspase 12 degradation product increased 7‐fold with IFNγ, when compared to control, TNFα , or IL1β, p<0.001). We did not detect significant changes in late markers of ER stress at 24 hours. Our findings indicate that IFNγ is a potent activator of ER stress in skeletal muscle. Future studies are needed to determine the role of IFNγ –mediated ER stress activation in clinical conditions which produce skeletal muscle dysfunction (e.g. sepsis, cancer, and aging).
Proteome Dynamics Rapidly Detect Protein Synthetic and Fibrogenic Changes Following Eccentric Contraction‐Induced Muscle Damage in mdx Mice
Authors: Stimpson Stephen , Kramer Fritz , Shearer Todd , Billin Andrew , Russell Alan , Hellerstein Marc , Turner Scott , Evans William , Shankaran Mahalakshmi
Published: June 2021

Turnover rates of the skeletal muscle proteome (proteome dynamics) may reveal pathophysiologic mechanisms and identify markers that change rapidly, for clinical monitoring in disease. Here, we used high resolution LC/MS/MS of tryptic peptides to compare the fractional synthetic rates (FSR) of hundreds of proteins in gastrocnemius (gastroc) muscles of 9‐11 month‐old wild type (wt) and mdx mice. One day after damage‐inducing hind‐limb eccentric muscle contraction that resulted in 40‐60% reduced contractile force, mice were labeled with heavy water (D2O) for 12 days. We also measured the FSR and content of hydroxyproline (OHP), representing total collagen, in the gastroc and diaphragm. Basal (non‐injured) mdx gastroc and diaphragm muscles had significantly higher FSRs for most proteins compared to wt mice. Basal OHP content and collagen abundance were also significantly increased from mdx vs wt. During the repair phase in response to injury, wt and mdx gastrocs had distinct dynamic proteomic signatures across several myofibril, extracellular matrix, cytosolic and mitochondrial protein ontologies. OHP and collagen FSR were also significantly increased in the damaged gastroc compared to contralateral limb. Thus, proteome dynamic measurements reveal increases in fibrosis and generalized skeletal muscle protein FSR in mdx vs wt mice, and repair following eccentric muscle damage is associated with unique dynamic proteomic signatures that include increased fibrogenesis. These findings hold promise for monitoring the response of muscle damage to therapy in muscle diseases, including muscular dystrophies.
Preventive effects of middle frequency electrical stimulation on cachexia‐induced muscle atrophy
Authors: Tanaka Minoru , Tanaka Kohei , Takegaki Junya , Fujino Hidemi
Published: June 2021

The purpose of the present study is to evaluate the effect of electrical stimulation using middle frequency (ES) on muscle atrophy induced by cachexia. Mice were randomly divided into the control, lipopolysaccharide (LPS) for 4 days, LPS plus ES (LPS+ES) groups. The animals in the LPS groups were induced sepsis‐derived cachexia by LPS‐injected during the intervention period (10 µg/g body weight/day, i.p.). The animals in the ES group were stimulated electrically using middle frequency current twice a day during the intervention period. The LPS‐injected animals for 4 days exhibited the significant loss of muscle mass, decreased cross‐sectional area of muscle fibers, and overexpression of ubiquitinated protein in the tibialis anterior muscle compared with the control. ES attenuated the cachexia‐induced loss of muscle mass, decreased cross‐sectional area of muscle fibers in the tibialis anterior muscle. In addition, the LPS+ES attenuated the overexpression of ubiquitinated protein in the tibialis anterior muscle. These results suggest that the electrical stimulation might contribute to prevent cachexia‐induced muscle atrophy.
Apoptosis Signal‐regulating Kinase 1 and c‐Jun N‐terminal Kinase Affect Tumor Necrosis Factor Alpha Signaling by Independent Mechanisms in Vascular Smooth Muscle Cells
Authors: Choi Hyehun , Dikalova Anna , Lamb Fred
Published: June 2021

Tumor necrosis factor‐α (TNFα) is linked to cardiovascular risk, including inflammatory vascular lesions. TNFα signaling occurs at the plasma membrane and in endosomes, a site of reactive oxygen species (ROS) production. Apoptosis signal‐regulating kinase 1 (ASK1) is an MAPKKK, it activates MAPKKs and in turn c‐Jun N‐terminal kinase (JNK) and p38. We hypothesized that mitogen‐activated protein kinases (MAPK) site‐specifically modulate TNFα‐induced superoxide (O2‐.) production and nuclear factor‐kappa B (NF‐κB) activation in vascular smooth muscle cells (VSMC). We measured TNFα‐induced endosomal O2‐. (OxyBurst green), total O2‐. (TMH spin probe), and NF‐κB activation (luciferase reporter). TNFα‐induced endosomal O2‐. production was Nox1‐dependent (absent in Nox1 null). siRNA targeting ASK1 decreased endosomal O2‐. by 80 ± 0.1% and NF‐κB activation by 66±0.7%. siRNA targeting JNK1 enhanced endosomal O2‐. production by 199±1.5%, total superoxide production by 143±1.6% and NF‐kB activation by 154±1%. Dominant‐negative dynamin (Ad‐DynK44A) inhibits endocytosis. It impaired TNFα‐induced ASK1 phosphorylation (T845), but augmented JNK phosphorylation. These data demonstrate that ASK1 and JNK modulate Nox1‐dependent O2‐. production and NF‐κB activation through independent mechanisms that are both modified by TNFα receptor endocytosis. Thus, ASK1 enhances, but JNK down regulates the inflammatory response to TNFα by reducing endosomal ROS production. These kinases may represent novel targets for pharmacologic modulation of TNFα signaling.
Chronic Hypoxia Attenuates cGMP‐dependent protein kinase (PKG)‐Mediated Vasorelaxation by Depressing BK Potassium Channel Activity in Cerebral Arteries
Authors: Thorpe Richard , Pearce William
Published: June 2021

OBJECTIVE

Chronic hypoxia attenuates the ability of PKG to induce vasorelaxation in serotonin‐contracted ovine carotids, and more so in fetal than adult arteries. The present investigation examines the hypothesis that alterations in the BK channel and other PKG kinase substrates contribute to hypoxic attenuation of PKG‐dependent vasorelaxation in term fetal and adult ovine cerebral arteries.

METHODS

Adult and fetal sheep were maintained under normoxic (140±2 days at sea level) or hypoxic (140±2 days at 3820 meters elevation) conditions. PKG‐induced vasorelaxation was quantified by measuring changes in contractile force of middle cerebral artery segments using graded doses of serotonin in the presence or absence of the BK channel inhibitor iberiotoxin (100 nM) and/or the PKG activator 8‐pCPT‐cGMP (30 μM).

RESULTS

Total PKG‐induced vasorelaxation was significantly less in hypoxic (8.9% fetal, 8.1% adult) than in normoxic (33.3% fetal, 26.3% adult) cerebral arteries. Corresponding values for BK‐independent PKG‐induced vasorelaxation were also less in hypoxic (6.3% fetal, 10.0% adult) than normoxic (28.7% fetal, 18.2% adult) cerebral arteries. For BK‐dependent PKG‐induced vasorelaxation, these values again were less in hypoxic (2.2% fetal, 1.8% adult) than normoxic (4.7% fetal, 8.1% adult) arteries.

CONCLUSION

Hypoxic attenuation of PKG‐induced vasorelaxation in ovine cerebral arteries was attributable to a reduced influence of PKG on the BK channel. Non‐BK channel mediators of vasorelaxation exhibited similar attenuation but of lesser magnitude. Fetal cerebral arteries were most affected by hypoxic stress.

This work was supported by NIH Grants HL‐54120, HD‐31266, HL‐64867 and NS‐076945.
Effect of Desferroxamine Infusion on Cerebral Autoregulation in Elderly Individuals: A Randomized Placebo Controlled Trial
Authors: Purkayastha Sushmita , Sorond Farzaneh , Tan Can Ozan
Published: June 2021

Objective

Cerebrovascular disease results from chronic hypoxia induced by reduced perfusion. Animal studies have shown iron chelators such as desferroxamine (DFO) reduce brain damage and promote functional recovery after transient focal ischemia. The present study was designed to investigate the effects of DFO infusion on cerebral autoregulation (CA) in elderly individuals.

Methods

Nineteen volunteers (64±7 yrs) were enrolled in a randomized double‐blind placebo‐controlled crossover study. Subjects were randomized to either DFO or placebo (PLB) infusion for 9 hours. Arterial blood pressure and middle cerebral artery blood velocity were obtained using finger photoplethysmography and transcranial Doppler ultrasound for 5 min while subjects were seated upright at 0, 6 & 9 hours infusion. A non‐linear projection pursuit regression was used to examine the effectiveness of dynamic CA with DFO and PLB infusion.

Results

Decrease in gain (P=0.0341) within CA range was observed following 9 hours DFO compared to PLB suggesting effective CA although DFO infusion reduced the blood pressure range within which CA was effective compared to the PLB (P=0.004). The latter effect could be due to vasodilatation‐induced reduction in mean arterial pressure with DFO.

Conclusion

DFO infusion resulted in improvement in CA in elderly individuals and may be a potential therapeutic target for improving cerebral blood flow regulation.

Research supported by K23AG030967 R01NS085002 (FAS)
Neurovascular Coupling Across the Menstrual Cycle
Authors: Davenport Margie , MacKay Christina , Skow Rachel , Steinback Craig
Published: June 2021

Vascular function is modulated in response to the changing hormonal patterns of the menstrual cycle. Sex hormones may also act on non‐vascular cells regulating blood flow, including astrocytes and neurons, which may alter cerebral blood flow responses. However, the impact of menstrual phase on cerebral neurovascular coupling has not been investigated. We hypothesized that the visually evoked cerebral blood flow response would be higher in the mid‐luteal phase of the menstrual cycle, corresponding to elevated estrogen levels.

Nine normally menstruating women (Age = 28 ± 6 yrs, BMI = 25 ± 4kg/m2) underwent a visual stimulation test in the early follicular (EF) and mid‐luteal (ML) phase of the menstrual cycle. Beat‐by‐beat mean arterial pressure (MAP) was derived using photoplethysmography (Finometer) and posterior cerebral artery blood flow velocity (PCAVP) was measured using transcranial Doppler ultrasound (Multigon).

At rest, MAP (EF: 86.8 ± 7.4mmHg, ML: 87.7 ± 6.2mmHg, p=0.78) and cerebral blood flow (EF: 36.5 ± 5.5cm/s; ML: 37.6 ±11.3cm/s, p=0.80) were similar between phases. Further, the magnitude (EF: 25.0 ± 6.0%, ML: 26.5 ±8.5%, p=0.38) and the timing (EF: 12.0 ± 1.4s, ML: 11.8 ± 1.9s, p=0.78) of the cerebral blood flow response to visual stimulation was similar between phases.

These data suggest that contrary to our hypothesis, neurovascular coupling assessed by visual stimulation was not different across the menstrual cycle. Supported by the University of Alberta ‐ Human Performance Scholarship Fund.
Structural Basis for Ion Conductance and Selectivity in Sodium and Calcium Channels
Authors: Catterall William
Published: June 2021

Voltage‐gated sodium and calcium channels generate rapid electrical signals and local calcium transients that initiate key physiological events in many cell types. High selectivity for sodium and calcium is essential to this signaling process. The high‐resolution structure of a bacterial ancestor of voltage‐gated sodium and calcium channels (NavAb) at 2.7 Å resolution reveals the structural basis for ion conductance and selectivity. The NavAb selectivity filter is short, ~4.6 Å wide, and water‐filled, with four glutamate side‐chains that form a high field‐strength site surrounding the entry to the ion‐conduction pathway. NavAb is highly selective for sodium over calcium, with P(Na):P(Ca)=30. Molecular dynamics studies show that sodium is conducted as a hydrated cation and interacts sequentially with three sites in the ion selectivity filter during conduction. Addition of two more negative charges on the extracellular side of the ion selectivity filter increases P(Ca):P(Na)=400, a 12,000‐fold change. Addition of a single negative charge on the extracellular side of the high field‐strength site accounts for a 1000‐fold increase in calcium selectivity by itself. Structural analysis reveals bound calcium ions in the extracellular vestibule and at three sites in the ion selectivity filter. These results define the mechanism of ion conduction and selectivity for calcium through interactions of the hydrated cation at 3 sequential ion coordination sites within the pore. Binding sites for calcium and the pore‐blocking ions cadmium and manganese are illuminated at atomic resolution by x‐ray crystallography. Overall, these results reveal the molecular basis for electrical signaling and give a high‐resolution view of the ion conductance and selectivity of sodium and calcium channels.
Cellular and Respiratory Responses to Inhalation of Traffic Derived Particles in a Rat Model of High Fructose Diet‐Induced Cardio‐Metabolic Syndrome
Authors: DIAZ EDGAR , Vasileios Papapostolou , Coull Brent , Harkema Jack , Godleksi John
Published: June 2021

METHODS

Male Sprague Dawley rats 250‐350g (Taconic Farms ‐ Rensselaer, NY), were fed for 8‐weeks prior to their first day of exposure with a high‐fructose diet (HFrD; 60% fructose by mass; TD.89247; Harlan Laboratories) to induce the metabolic syndrome phenotype, and continued on the diet until the end of the experiment. Animals were exposed to Aged Primary plus Secondary Organic Aerosol (P+SOA) tunnel emissions (Air Qual Atmos & Health 2013;6(2):419‐429). In all studies, control (Sham) exposures were conducted simultaneously.

Animals were implanted with C50‐PXT blood pressure, temperature and ECG telemeters, Continuous respiratory data were collected at the time of exposure. Effects of each exposure were assessed using linear mixed models containing random animal effects and within exposure serial correlation in the form of a first‐order autoregressive process. In addition, we controlled for each day, so comparisons are between exposed and unexposed on a given day, with this difference related to the exposure level.

RESULTS

Average exposure concentration was 20.41±6.18υg/m3.

Parameter
Δ Exp/Sham ± SE
p‐value

Systolic BP
6.40±3.31
0.055

Diastolic BP
5.01±2.28
0.030

Heart Rate
4.86±3.30
0.143

Frequency
7.30±1.27
<0.001

Tidal Volume
5.34±2.0
0.001

Inspiratory Time
‐9.56±5.33
0.078

Expiratory Time
‐35.73±9.89
<0.001

Peak Inspiratory Flow
1.57±0.36
<0.001

Peak Expiratory Flow
1.13±0.25
<0.001

CONCLUSIONS

The diet‐induced metabolic syndrome model was successfully produced with the high fructose diet. Our toxicological results for blood pressure outcomes behaved similar to our prior exposure with this aerosol in normal animals. Respiratory airflows differed suggesting parasympathetic influences, which had been observed with much higher exposure concentrations.
When Mbytes are not enough: long term, multiple channel, physiological telemetry implantable datalogger
Authors: Rotar Viorel , Pitsillides Koullis , Axelsson Michael
Published: June 2021

Telemetry monitoring is an established tool that allows measurements from unrestrained animals and most telemetry systems for large animals have a typical range of less than 6‐8m in air. When an experimental design requires that instrumented animals are released into a large outdoor environment or mesocosm settings or when the animals are water living or have an amphibious lifestyle, this precludes the use of an RF link to transfer the implanted data to the computer and requires local storage means such as a datalogger.

We have developed an implantable, low power datalogger that interfaces directly to a bio‐telemetry system (EG1‐V2S3T, Transonic EndoGear Inc). The datalogger has the capacity to store up to 6 channels of data at 100 Hz which results in about 125MB/day of continuous data. The datalogger has a capacity of 8GB and is capable of continuous operation for over 8 weeks. Datalogging is controlled via the same computer interface controlling the implant and can be started either during implantation or at a later stage after post‐surgical recovery period before the animals are released from the lab environment.

These dataloggers were field tested in six crocodiles (apprx. 22‐42kg, 1.4 ‐2.2m long) instrumented with the EG1‐V2S3T implants for blood flow, pressure, ECG and temperature and connected to a datalogger (Model EG1‐DTL). The animals were released in a mesocosm setting at Mongena Game Reserve in South Africa and were allowed to remain undisturbed for 4 weeks. Video cameras were used to correlate physiological data with behavioral data and external time depth recorders were used to monitor diving events.
Does Left Atrial Size Predict Clinical Outcomes in Heart Transplant Recipients?
Authors: Shizukuda Yukitaka , Ahmad Saad , Naz Tehmina , Dunlap Stephanie , Gujja Pradeep
Published: June 2021

We have previously reported that larger post‐heart transplant (HT) left atrial (LA) size is associated with decreased ventilatory efficiency indicating lesser exercise capacity. Therefore, we hypothesized that larger LA size negatively influenced clinical outcomes in heart transplant (HT) recipients by limiting exercise capacity. To test this, clinical outcomes over 5 years after HT were evaluated in 35 HT recipients who had a LA size measurement with echocardiography at 1 year after HT at the University of Cincinnati Medical Center. The LA size was assessed as LA volume derived from a stacked disc method. The average LA volume normalized to BSA was 38.3±9.9 ml/m2 (mean±SD) at 1 year after HT. Two deaths and one drop‐out occurred during 5‐year follow up. A total of 552 cardiac symptom‐related hospitalizations occurred in the recipients. The average time to first hospitalization was 166±279 days and average number of hospitalizations of each recipient was 15±16. The indexed LA volume failed to correlate with the time to first hospitalization and number of hospitalizations of each recipient (Spearman's p‐value; 0.141 and 0.519 respectively). In summary, our results implicate that LA size is not a significant predictor of cardiac symptom‐related hospitalizations in HT recipients. Therefore, despite its association with exercise capacity, LA size may not predict clinical outcomes in this population.
Rho‐kinase Mediates Biventricular Coronary Arterial Remodeling During Pulmonary Arterial Hypertension in Fischer 344 Rats
Authors: Crockett Edward , Stevens Troy , McMurtry Ivan
Published: June 2021

Rationale: Fischer 344 rats exposed to Sugen‐5416 and hypoxia, then normoxia (Su/Hx/Nx) develop severe pulmonary arterial hypertension (PAH). Unique to this strain, PAH is accompanied by significant death within 8 weeks. Coronary remodeling and acute heart failure is suspected. Here, we test the hypothesis that PAH is accompanied by biventricular Rho‐kinase mediated coronary arterial remodeling, ischemia and heart failure.
Methods: Rats were injected with Sugen‐5416 followed by 3 weeks of hypoxia (10% O2), then 6 weeks of normoxia (21% O2). Survival was plotted as a Kaplan‐Meier curve. Histology examined biventricular coronary remodeling and matrix deposition. Hypertensive rats were treated with fasudil, a Rho‐kinase inhibitor, added to the drinking water.
Preliminary Results: 3 out of 4 non‐treated PAH rats died between 5 and 8 weeks of Su/Hx/Nx exposure, while 3 out of 3 fasudil‐treated PAH rats survived to 9 weeks, when sacrificed. Echocardiography at 5 weeks showed right ventricular dilation, septal deviation and decreased cardiac output. RV/(LV+S) confirmed significant hypertrophy of the right ventricular wall regardless of treatment. Histology revealed that Rho‐kinase inhibition normalized biventricular coronary remodeling and perivascular fibrosis (Figure 1).

Conclusion

During PAH in Fischer rats, Rho‐kinase mediates coronary arterial wall thickening in both ventricles, which primes the heart for ischemia and failure.

Funding: NIH‐ HL060024 and HL066299

Figure 1: Coronary arteries during PAH with or without fasudil treatment compared to normotensive. Red arrows‐ smooth muscle medial hypertrophy; yellow star‐ perivascular fibrosis.

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Exercise Training Reverses Age‐induced Coronary Microvascular Dysfunction
Authors: Muller‐Delp Judy , Behnke Bradley , Delp Michael , Chen Bei , Ghosh Payal , Stabley John , Hotta Kazuki , Cowan Morgan , Lucero Tiffani , Morgan Hannah
Published: June 2021

The risk for diastolic dysfunction increases with advancing age. We tested the hypotheses that 1) microvascular dysfunction underlies age‐related diastolic dysfunction, and 2) exercise training (ET) reverses age‐related microvascular and diastolic dysfunction. Old and young male Fischer 344 rats underwent 10 weeks of daily ET or remained sedentary cage controls. Cardiac function was assessed before and after ET. At the end of training, coronary blood flow was measured at rest and during treadmill exercise. The reactivity of the coronary resistance vasculature was assessed in isolated coronary arterioles. E/a ratio decreased with age and was partially restored by ET. Isovolumic relaxation time increased with age and this increase was reversed by ET. Resting blood flow to the left ventricular (LV) endocardium and the right ventricular free wall was decreased in old rats; however, ET increased resting blood flow to the septum, papillary muscles, LV endocardium and epicardium. Exercise hyperemia was reduced by age in the septum, and the LV endocardium and epicardium, but these age‐related reductions were reversed by ET. Age‐induced impairments of both contractile and relaxation responses of coronary arterioles were reversed by ET. Thus, microvascular dysfunction contributes to development of diastolic dysfunction with advancing age; however, ET at an advanced age reverses microvascular dysfunction and improves diastolic function.
Both Peroxidase and Phospholipase A2 Activities of peroxiredoxin 6 Protect Against Oxidative Stress by Reduction of Peroxidized Cell Membranes
Authors: Li Haitao , Feinstein Sheldon , Zhou Suiping , Dodia Chandra , Fisher Aron
Published: June 2021

Peroxiredoxin 6 (Prdx6), a protein with both peroxidase and PLA2 activities, plays a critical role in antioxidant defense of lung and other organs. We investigated the relative importance of these 2 activities of Prdx6 in the “repair” of peroxidized cell membranes in pulmonary microvascular endothelial cells (PMVECs) and isolated mouse lungs. Lipid peroxidation was evaluated by measurement of thiobarbituric acid reactive substances (TBARS) or oxidation of diphenyl‐1‐pyrenylphosphine (DPPP). PMVECs were treated with tert‐butyl hydroperoxide (tBOOH, 0.2 mM) or Cu2+ /ascorbate for 4 h and then evaluated at 0,2,4 and 6 h after removal of the oxidant. Values for lipid peroxidation returned to control levels in 2 h with wild type (WT) PMVECs but were unchanged at 6 h after oxidant removal in Pxdx6 null PMVECs. An intermediate degree of repair was observed with lentivirus transfected C47S or D140A mutant Prdx6 PMVECs; the former express only PLA2 activity and the latter only peroxidase activity. Similar results were obtained with the isolated mouse lungs from WT, Prdx6 null and Prdx6‐C47S or ‐D140A “knock‐in” mice that were observed for 3 h following 60 min of perfusion with 15 mM tBOOH. These results indicate that the peroxidase and phospholipase A2 activities of Prdx6 both play a critical role in the repair of peroxidized phospholipids in cell membranes and the recovery of lung cells from peroxidative stress.
Changes in Endothelial Cell Membrane Morphology and Elasticity with Exposure to Polyunsaturated Free‐Fatty Acids
Authors: Mabry Brittany , Lau Elese , Hunt Brian
Published: June 2021

Background

The purpose of this preliminary study is to determine the effect of incubating human endothelial cells in PUFA's on their nanomechanical properties.

Methods

Human aortic endothelial cells (Invitrogen) were cultured in endothelial cell medium containing fetal bovine serum, growth supplement and penicillin/streptomycin (ScienCell Research Laboratories) and incubated in the presence of 5% CO2 at 37°C for 24 hours. Cells were then incubated for another 24 hours in medium containing either 400 mM cholesterol, normal cell medium, or 400 mM docosahexaenoic acid (DHA), or eicosapentanoic acid (EPA). Differences in membrane morphology and elasticity were measured using atomic force microscopy (AFM; Bruker BioScope) in cell medium at room temperature. Data were analyzed using NanoScope.

Results

Cells incubated in in EPA or DHA were flatter (height =2667nm and 2372 nm respectively) compared to control cells (3141nm) and cholesterol cells (height = 5390nm). The elastic modulus of DHA‐incubated cells (3.63 log(Pa)) differed compared to control cells (3.95 log(Pa)) and cholesterol cells (4.14 log(Pa)). Deformation also differed between DHA cells (323 nm) and cholesterol cells (249 nm).

Conclusion

PUFA incubated cells differ in their nanomechanical properties compared to control and cholesterol cells, as measured by AFM. Measures of elastic modulus and deformation suggest a more fluid membrane in PUFA incubated cells, which may enhance glucose uptake in these cells.
Sex differences in the open probability of TRPV4 channels in mouse mesenteric artery endothelium
Authors: Sonkusare Swapnil , Villalba Nuria , Raza Abbas , Bonev Adrian , Freeman Kalev , Wellman George , Nelson Mark
Published: June 2021

Activation of TRPV4 (transient receptor potential vanilloid 4) channels leads to endothelium‐dependent vasodilation of resistance‐sized mesenteric (MAs) and cerebral arteries (CAs). In CAs, lower channel open probability results in reduced vasodilations to the TRPV4 channel agonist GSK1016790A (GSK101) compared to the MAs (Sonkusare et. al., 2014) suggesting that the activity of endothelial TRPV4 channels can be modulated by their environment. Further, TRPV4 channels have been proposed to be male‐specific regulators of physiological functions. We therefore used optical recordings of TRPV4 channel function–TRPV4 Ca2+ sparklets, to evaluate fundamental differences in TRPV4 properties in MAs from male and female mice. Interestingly, there was a remarkable similarity between TRPV4 sparklets from male MAs and female MAs, i.e. a four‐channel metastructure, same single‐channel quantal levels, similar coupling strength of sparklets), indicating that the fundamental channel properties were not different between the sexes. However, the open probability (NPo) of TRPV4 channels in ECs from female MAs was 4‐fold lower than the NPo in male MAs under basal conditions and in the presence of GSK101 (1‐10 nM). Further, GSK101 (1‐3 nM)‐induced dilations were significantly decreased in the MAs from female mice compared to the male mice. These results indicate that an unknown endogenous modulator may differentially regulate TRPV4 open probability in MAs from male and female mice.
Impact of Moderate Zinc Deficiency During Fetal Life and Postnatal Growth on Cardiac Nitric Oxyde System, Inflammatory and Oxidative Markers in Adult Male Rats
Authors: Juriol Lorena , Gobetto Natalia , Mendes Garrido Facundo , Pineda Gonzalo , Dasso Marina , Toblli Jorge , Carranza Andrea , Elesgaray Rosana , Tomat Analía , Arranz Cristina
Published: June 2021

Moderate zinc deficiency during fetal and postnatal growth programs cardiovascular dysfunction and hypertension. Male zinc deficient rats showed reduced left ventricular (LV) wall thickness and ejection fraction. Aim: to evaluate nitric oxide (NO) system, pro‐inflammatory cytokines and oxidative state in LV of adult male rats exposed to this deficiency. Wistar rats received during pregnancy up to weaning low (L:8 ppm) or control (C:30 ppm) zinc diet. After weaning, male offspring fed low (l) or control (c) zinc diet during 60 days (Cc, Ll, Lc). At day 81, we evaluated in LV: Interleukin‐6 and Tumor Necrosis Factor‐α (IL‐6;TNF‐α), basal NO synthase (NOS) and endothelial, neuronal and inducible (eNOS;nNOS;iNOS) isoforms activities; eNOS and Ser1177 eNOS phosphorylation protein expression (eNOS/β‐actin; pSer1177eNOS/eNOS), mRNA expression (eNOS/GAPDH) and lipid peroxidation end products (TBARS). Means±SEM, n=6/group. One way ANOVA, Bonferroni post‐test.

Cc
Ll
Lc

TNF‐α (immunohistochemistry;% of positive staining/area)
1.4±0.4
20.4±2.0*
1.7±0.3

IL‐6 (immunohistochemistry; % of positive staining/area)
1.8±0.3
21.1±1.7*
2.8±0.8

Basal NOS Activity (pmol14C L‐citrulline/g tissue.min)
204±6
164±10*
157±11*

Basal NOS+iNOS inhibitor (Aminoguanidine; pmol14C L‐citrulline/g tissue.min)
216±7
166±8*
163±9*

Basal NOS+nNOS inhibitor (7‐nitroindazole; pmol 14C L‐citrulline/g tissue.min)
209±10
166±8*
164±9*

Basal NOS+Ca2+‐calmodulin inhibitor (Calmidazolium; pmol 14C L‐citrulline/g tissue.min)
47±2#
52±2#
49±2#

pSer1177eNOS/eNOS (Western Blot; optical density relative to Cc)
1.00±0.02
0.74±0.03*
1.02±0.16

TBARS (nmol/mg protein)
0.21±0.02
0.78±0.08*
0.47±0.02*†

No differences were found in eNOS protein and mRNA expression.

*p<0.01vsCc;†p<0.01vsLl;#p<0,01vs basal NOS.

Zinc deficiency during fetal and postnatal life programs a lower production and bioavailability of cardiac NO due to decreased activity and pSer1177 of eNOS and increased TBARS. These alterations, jointly with higher levels of IL‐6 and TNF‐α, could contribute to the cardiac disorders previously observed. Supported by:UBA‐CONICET
Ventricular Repolarization Pattern and Ischemic Arrhythmia Susceptibility at Different Follow‐Ups of Diabetes Mellitus
Authors: Azarov Ian , Ovechkin Alexey , Vaykshnorayte Marina , Sedova Ksenia , Bernikova Olesya , Arteyeva Natalia , Shmakov Dmitry
Published: June 2021

The ventricular distributions of activation‐repolarization intervals (ARIs) were determined in control and diabetic (DM) rabbits (alloxan model, 1 and 2 months). In DM (1 month), the epicardial ARIs were prolonged heterogeneously, being longer on the right than on the left ventricle (LV) and on the LV apex than on the LV base. In DM (2 months), the ARIs lengthened ubiquitously. Coronary occlusion led to the repolarization shortening and repolarization dispersion increase in the control and DM (2 months) groups. However, the repolarization dispersion did not increase in the DM (1 month) rabbits since the ARIs shortened predominantly in the areas where they were prolonged in the preischemic conditions. The ventricular tachyarrhythmias were induced more frequently in the DM (2 months) group as compared to the DM (1 month), whereas the latter did not differ from the controls. The DM‐related precordial T wave flattening was associated with the apicobasal repolarization gradient changes. Thus, the enhanced arrhythmogenicity in DM was associated with the pattern of the overall prolongation of repolarization observed at the 2 months but not 1 month follow‐up, which predisposed to the increase of repolarization dispersion under myocardial ischemia. Grants ‐ Ural Branch of RAS (12‐I‐4‐2059, 13‐4‐032‐KSC) and RFBR (14‐04‐31070 young_a).
TBC1D1, a Rab‐GTPase Activating Protein, is Critical for Maintaining β‐cell Mass and Glucose Homeostasis
Authors: Paglialunga Sabina , Simnett Genevieve , Robson Holly , Houad Jacy , Jain Swati , Herbst Eric , Snook Laelie , Arkell Alicia , Pillai Renjitha , Dyck Davide , Simpson Jeremy , Bonen Arend , Joseph Jamie , Holloway Graham
Published: June 2021

Understanding the mechanisms regulating islet function is crucial for establishing novel therapeutic modalities to combat diabetes. TBC1D1, a Rab‐GTPase activating protein involved in skeletal muscle GLUT4 trafficking events, was recently identified withinβ‐cells and implicated in cell proliferation. However, the in vivo function of TBC1D1 remains to be elucidated. Therefore, we addressed the role of TBC1D1 in the pancreas utilizing a rat knockout (KO) model. Wild‐type and TBC1D1 KO rats were maintained on a chow or high‐fat diet. We demonstrated that TBC1D1 KO rats were glucose intolerant and displayed reduced insulin levels during a glucose challenge, suggesting that their phenotype prominently manifests within the pancreas. Next, we examined islet function and β‐cell mass. While, glucose‐stimulated insulin secretion from harvested islets was increased in TBC1D1 KO rats, we observed an overall decrease in β‐cell mass by ~30%. In addition, consumption of a high‐fat diet did not influence these changes observed in the KO rats. Altogether, our data suggests that in vivo impaired glucose homeostasis observed in TBC1D1 KO rats is a consequence of altered islet mass, thereby establishing a fundamental in vivo role for TBC1D1 in maintaining β‐cell mass. Therefore, pancreatic TBC1D1 may represent an attractive target to improve β‐cell function and stability to treat and prevent diabetes.