bluuebunny/crossref_metadata_2025 huggingface dataset:
Individual vasodilatory response heterogeneity during progressive forearm exercise: evidence for vasodilator phenotypes
Authors: Bentley Robert F , Kellawan J Mikhail , Moynes Jackie S , Poitras Veronica J , Walsh Jeremy J , Tschakovsky Michael E
Published: June 2021

PURPOSE

To determine whether vasodilator and/or pressor response phenotypes are present during a perfusion pressure induced perturbation to exercising muscle oxygen delivery (O2D).

METHODS

10 healthy male subjects (19.5±0.4 yrs) completed two trials of progressive handgrip exercise to exhaustion (2.5kg increments every 3.5 mins) in each forearm above and below heart level (forearm arterial perfusion pressure (FAPP) Δ 29.5±0.97mmHg). Forearm blood flow ((FBF (ml/min); brachial artery Doppler and echo ultrasound), mean arterial blood pressure (MAP (mmHg); finger photoplethysmography) and O2D (ml/O2/min; venous effluents) were measured at the end of each work rate (WR).

RESULTS

Group level, Δ FBF was compromised beyond the 5kg WR in above vs. below. There was no vasodilatory (P=0.21) or exercise pressor (P=0.63) response, and submax O2D, submax and peak VO2 and peak WR were compromised by reduced FAPP (all P<0.05). In contrast, individual responses revealed compensatory vasodilators (n=6) and those who did not (n=4). Vasodilators blunted the FAPP‐evoked reduction in submax O2D and VO2 compared to non‐vasodilators (P<0.05), and experienced less of a compromise to peak WR (P<0.05).

CONCLUSIONS

In the current model, vasodilatory response phenotypes exist, which determine hypoperfusion susceptibility and the degree to which aerobic metabolism and exercise performance are compromised. NSERC.
Interaction of mitofilin with respiratory complexes in mitochondrial subpopulations
Authors: Thapa Dharendra , Jagannathan Rajaganapathi , Croston Tara L , Baseler Walter A , Nichols Cody E , Shepherd Danielle L , Lewis Sara E , Hollander John M
Published: June 2021

Mitofilin, a mitochondrial inner membrane structural protein is central to mitochondrial inner membrane organizing system (MINOS) and mitochondrial organizing structure (MitOS). Interactions of mitofilin with outer and inner membrane proteins have been reported to be crucial for mitochondrial membrane organization and morphology. Moreover, MitOS has been shown to function in concert with ATP synthase dimers. However, interaction of mitofilin with the respiratory complex is not known. The goal of this study was to investigate potential interactions of mitofilin with the respiratory complex in cardiac mitochondrial subpopulations. Cardiac mitochondria are comprised of two spatially located mitochondria, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). Using a gel based technique, mitochondrial proteins immunoprecipitated with mitofilin were subjected to LC‐ESI‐MS analysis. Thirty‐six respiratory chain proteins from complexes I, II, III, IV, and V were identified in an immunoprecipitated complex. Furthermore, interaction of mitofilin with F0‐ATP synthase subunits b and d in the immunoprecipitated complex was observed to a greater extent in the IFM as compared to the SSM. Together these results suggest that interaction of mitofilin with the respiratory chain may differ in mitochondrial subpopulations. (Support: NIH DP2DK083095, NIH T32HL090610, AHA 10PRE3420006)
The Increase of Mitochondrial DNA Copy Number Attenuates Eccentric Cardiac Remodeling In Volume Overload Model
Authors: Ikeda Masataka , Ide Tomomi , Fujino Takeo , Hata Yuko , Takehara Takako , Onitsuka Ken , Tobushi Tomoyuki , Sakamoto Kazuo , Saku Keita , Kakino Takamori , Tyynismaa Henna , Suomalainen Anu , Sunagawa Kenji
Published: June 2021

Background

Mitochondrial transcription factor A (TFAM) is essential for the maintenance of mitochondrial DNA (mtDNA). TFAM overexpression increases mtDNA copy number, prevents cardiac remodeling and improves survival after myocardial infarction. However, the role of the increased mtDNA copy number in volume overload (VO) remains unknown.

Methods and Results

We used TFAM and Twinkle (TW) transgenic (TG) mice, both of which nearly doubled the mtDNA copy number in heart. One day after creating the aortocaval shunt to induce VO, left ventricular (LV) end diastolic pressures equally increased in all groups, and TFAM and TW significantly suppressed increased mRNAs of matrix metalloprotease (MMP) ‐9 and connective tissue growth factor. In 8 weeks after operation, both TFAM and TW prevented LV dysfunction (LV ejection fraction: TFAM 53.6±2.5, TW 54.0±1.0, WT 37.1±1.9, 39.1±3.0, p<0.01), dilatation (LV diastolic dimension: TFAM 4.6±0.1, TW 4.3±0.1, WT 5.0±0.1, 4.9±0.1, p<0.01) and perivascular fibrosis. They also suppressed superoxide (TFAM −21%, TW −31% vs. each wild type VO group, p<0.05) and MMP activity (TFAM −31%, TW −23% vs. each wild type VO group, p<0.05).

Conclusion

Overexpressed TFAM or TW equally ameliorated VO‐induced eccentric cardiac remodeling, which suggests that increased mtDNA copy number plays a key role in anti‐remodeling effects of TFAM and TW via suppression of MMP activity and fibrosis.
Selective up‐regulation of genes for MMP3 and MT‐1 MMP by substance P in cardiac fibroblasts
Authors: Dehlin Heather Marie , Monroe Andrew , Reimer Michael , Manteufel Edward , Levick Scott
Published: June 2021

Cardiac scarring or fibrosis results from excessive generation of extra cellular matrix (ECM) by cardiac fibroblasts. Fibrosis increases the stiffness of the heart leading to impaired cellular signaling, cardiac function, and ultimately heart failure. Of particular interest is how the neuropeptide, substance P, contributes to the development of cardiac fibrosis. Substance P is a key neuropeptide in sensory nerves and is localized near the coronary vasculature and in a select population of coronary endothelial cells. This is an ideal location for substance P to be secreted in response to changes in coronary pressure or flow as in a hypertensive condition. Using the spontaneously hypertensive rat model of hypertension, we observed a continual increase in substance P gene expression, Tac1, in the left ventricle corresponding with increasing blood pressure. Substance P up‐regulates the ECM genes, MMP3 and membrane type 1 MMP (MT‐1 MMP) in a concentration dependent manner. Although cardiac fibroblasts express NFKB, this pathway does not appear to be involved. Substance P induces expression of specific, yet necessary, ECMrelated genes in cardiac fibroblasts providing causal evidence that substance P regulates factors relating to the development of fibrosis.

This work is supported by project number T32 HL007792.
Doxycycline Attenuates Cardiac Injury and Improves Cardiac Function with Inhibition of Myocardial Matrix Metalloproteinase (MMP)‐2 in a Swine Model of Hypoxia‐ Reoxygenation (H‐R)
Authors: LaBossiere Joseph R. , Pelletier Jean‐Sebastien , Ali Mohammad , Bigam David , Schulz Richard , Cheung Po‐Yin
Published: June 2021

Background

Cardiac dysfunction is common in asphyxiated neonates. Although small animal studies support attenuation of cardiac injury with the known MMP inhibitor doxycycline in H‐R, this effect has not been tested in a clinically relevant large animal model of H‐R. We hypothesized that doxycycline would improve the recovery of cardiac function in newborn piglets with H‐R.

Methods

Piglets were instrumented for hemodynamic monitoring and subjected to 2 hr of hypoxia followed by 4 hr of normoxic reoxygenation. Piglets were blindly randomized to receive i.v. saline or doxycycline (3, 10, or 30 mg/kg) 5 min into reoxygenation (n=7/group). Sham piglets (n=5) received no H‐R. Markers of myocardial injury (serum and myocardial tissue troponin; myocardial lactate) and oxidative stress (lipid hydroperoxides) were measured by ELISA and Western blot. Myocardial MMP‐2 activity was quantified by gelatin zymography.

Results

Doxycycline dose‐dependently improved cardiac (CI) and stroke volume (SVI) index (30mg/kg: 84±3 (SEM)% and 78±5% of baseline vs. 65±3% and 50±5% in controls [p<0.05]). Markers of myocardial injury, oxidative stress and MMP‐2 activity were improved in doxycycline groups vs. control [all p<0.05]. Significant correlations were found between markers of myocardial injury and CI and SVI recovery (r =−0.5, p<0.01), with a negative correlation also observed between myocardial tissue and serum troponins (r =−0.4, p=0.02).

Conclusions

Doxycycline attenuates cardiac injury and improves functional recovery in newborn pigs with H‐R.

Funded by: Canadian Institutes of Health Research and the Women and Children's Health Research Institute
Effect of aerobic interval training on erythrocyte rheological and hemodynamic functions in heart failure patients with anemia
Authors: Wang Jong‐Shyan
Published: June 2021

This study investigated whether aerobic interval training (AIT) influenced aerobic capacity by modulating hemorheological/hemodynamic functions in heart failure (HF) patients with/without anemia. Sixty HF patients were divided into non‐anemic (HF‐NA, n=30) and anemic (HF‐A, n=30) groups, and 30 normal counterparts were enrolled as a control group. These HF patients performed AIT (3‐minute intervals at 40% and 80%VO2peak) for 30 minutes/day, 3 days/week for 12 weeks. In both HF‐NA and HF‐A groups, the AIT regimen 1) reduced blood senescent/spherical erythrocyte counts, 2) diminished the values of critical shear stresses for disaggregation and half‐maximal deformation of erythrocytes, 3) enhanced cardiac output during exercise, and 4) decreased plasma myeloperoxidase and interleukin‐6 levels. However, AIT increased the amounts of blood distributed to frontal cerebral lobe and vastus lateralis muscle during exercise in HF‐NA group but not in HF‐A group. Additionally, HF‐A group exhibited fewer the enhancements of VO2peak by AIT than HF‐NA group did. Hence, we conclude that AIT improves aerobic capacity by depressing aggregability and enhancing deformability of erythrocytes in patients with HF. However, anemic comorbidity attenuates the adaptations of cerebral/muscular hemodynamic responses to exercise following this regimen.
Aerobic interval training improves mobilization and migratory capacity of endothelial progenitor cells in sedentary men
Authors: Tsai Hsing‐Hua , Wang Jong‐Shyan
Published: June 2021

Endothelial progenitor cells (EPCs) enhance vascular endothelial repair and neovasculogenesis. This study investigated the effects of aerobic interval (AIT) and moderate continuous training (MCT) on mobilization and migratory capacity of endothelial progenitor cells in sedentary men. Forty sedentary males were randomized to perform either aerobic interval training (AIT; 3‐minute intervals at 40% and 80%VO2peak) or moderate continuous training (MCT; sustained 60% VO2peak) for 30 minutes/day, 5 days/week for 5 weeks. Circulating levels of various EPC subsets were detected by three‐color flow cytometry. The experimental results showed that both AIT and MCT increased circulating level of CD34+/KDR+ cell, whereas only AIT elevated concentrations of CD34+/KDR+/CD133+ and CD34+/KDR+/CD31+ cells in blood. Moreover, AIT, but not MCT, significantly enhanced in vitro migratory capacity of CD34+/KDR+ cells. Hence we conclude that AIT effectively enhances mobilization and migratory capacity of EPCs in sedentary men.
Low‐volume high intensity interval training induces a modest inflammatory response in young active males
Authors: John Casey Scott , Lawrence Marcus M , Shanely R. Andrew , Battista Rebecca A , Zwetsloot Kevin A
Published: June 2021

Prolonged, high intensity aerobic exercise induces a substantial systemic inflammatory response. High intensity interval training (HIIT) is a time‐effective exercise strategy for improving physiological systems; however, the inflammatory response to short‐term HIIT is not well known. Recreationally active, males (18–25 yrs) underwent 2 weeks of low‐volume HIIT (6 sessions at 8–12 intervals; 60 sec intervals) at a power output equivalent to 100% of their pre‐determined VO2max. Serum samples were analyzed for inflammatory cytokines at rest, immediately after, and 15, 30, and 45 min after completion of an acute HIIT session before and after 2 weeks of HIIT, using a bead‐based multiplex cytokine assay. HIIT induced 2–3‐fold increases in IL‐6 (main effect of time; p<0.001), a 2‐fold increase in IL‐8 (main effect of time; p=0.038), a 2‐fold increase in IL‐10 (main effect of time; p=0.02), a 55% increase in TNFα (main effect of time; p=0.028), and a 30% increase in MCP‐1 (main effect of time; p=0.018), compared to rest. The concentrations of IFNγ, GM‐CSF, and IL‐1β were unaltered with HIIT. These results suggest that young healthy subjects undergoing an acute session of low‐volume HIIT induces a modest, yet significant inflammatory response; however, 2 weeks of HIIT does not appear to alter this response.
Endothelium‐dependent cutaneous vasodilation in young smokers: role of endothelial‐derived hyperpolarizing factors
Authors: Fujii Naoto , Brunt Vienna E , Francisco Michael A , Minson Christopher T
Published: June 2021

In young humans, endothelium‐dependent cutaneous vasodilation (EDCV) relates to both nitric oxide (NO) and cyclooxygenase (COX) pathways. We have recently shown that endothelial‐derived hyperpolarizing factors (EDHFs) are also involved, and that young smokers have impaired EDCV due to diminished NO‐ and COX‐dependent vasodilation. In this study, we tested the hypothesis that EDHFs greatly contribute to EDCV in young smokers to compensate for the lack of NO‐ and COX‐dependent vasodilation. We evaluated the increase in cutaneous vascular conductance (CVC) from baseline to peak value (CVCΔpeak) during acetylcholine (ACh) infusions of 1, 10, and 100mM at four intradermal microdialysis sites: 1) Ringer (control), 2) tetraethylammonium (TEA, a calcium‐sensitive potassium channel blocker which prevents EDHF‐induced vasodilation), 3) Nω‐Nitro‐L‐arginine (L‐NNA, NO synthase inhibitor) + Ketorolac (Keto, COX inhibitor), and 4) combination of TEA, L‐NNA, and Keto. At 100mM of ACh, CVCΔpeak at Ringer sites (54.4±14.2%max) was higher than that at the TEA sites (37.8±17.8%max), but was similar to that at the L‐NNA + Keto sites (57.5±1.3%max). Similar results were also obtained with 1 and 10 mM ACh. These data suggest that NO and COX pathways are deficient in EDCV of young smokers, but that a significant EDHF component remains.

Grant: NIH HL081671
Effects of Prior Exercise on Skin Blood Flow and Insulation during Subsequent Moderate Cold Exposure
Authors: Spitz Marissa Gayle , Castellani John W. , Alinovi Martha J , DeGroot David W
Published: June 2021

Exercise (EX), prior to cold air exposure (CAE), increases heat flow (HF) and reduces insulation (It) to a greater extent compared to passive warming (HEAT). It is not known if this is due to an attenuated skin blood flow (SkBF) response and/or changes in superficial shell and muscle It. We hypothesized that skin vasoconstriction and insulation would both be reduced during CAE following EX. Nine men sat in 19°C air for 120‐min following either: 1) cycle EX in 37°C water immersion (WI) at 60% VO2peak (rectal temperature (Tre) reached ~38.5°C), and 2) HEAT conducted during a 39°C WI. Cutaneous vascular conductance (CVC; SkBF/mean arterial pressure) and skin/subcutaneous fat/muscle temperatures were measured throughout CAE. The following observations were made during CAE in EX: 1) CVC was not different (p = 0.22) between EX vs. HEAT (12.0 vs. 5.6 % maximum CVC); 2) overall tissue insulation (It) was significantly lower (p < 0.05) in EX vs. HEAT (0.089 vs. 0.103 °C·m2·W−1); 3) superficial shell It was lower (p < 0.05) in EX vs. HEAT (0.008 vs. 0.0129 °C·m2·W−1); and 4) muscle It was similar (P >; 0.05) in the thigh (0.025 vs. 0.029 °C·m2·W−1) and trapezius (0.033 vs. 0.038°C·m2·W−1) for EX and HEAT, respectively. These results demonstrate that following acute EX, CVC is not attenuated during moderate CAE. However, tissue insulation is reduced, primarily through the superficial/subcutaneous fat layer.
The origin of air breathing in vertebrates: turn (on) your head and cough
Authors: Hoffman Megan , Taylor Barbara E. , Harris Michael B.
Published: June 2021

Air breathing in tetrapods involves a centrally generated rhythmic activation of respiratory muscles to ventilate a lung, and modulation of this rhythm by central CO2‐sensitive chemoreceptors that match ventilation to metabolic demands. The tetrapod lung arose in primitive air‐breathing fish. The origins of the central rhythm generator and chemoreceptor(s) are unknown. We propose that these critical elements were present in basal vertebrates and were exapted for air breathing with the evolution of the lung. Lamprey represent the basal vertebrate form and as larvae (ammocoetes) are aquatic burrow dwelling filter feeders. We propose that a behavior characterized as “cough” in lamprey is the basis for subsequent evolution of air breathing, and test the hypothesis that the centrally generated cough is stimulated by CO2. Ammocetes in vivo and isolated brainstems in vitro were exposed to hypercapnia. Ammocetes responded with a dose dependant increase in the frequency of spontaneous cough. Brainstems in vitro also produced the cough motor pattern, illustrating that cough results from a centrally CO2‐sensitive rhythm generator. We conclude that a central CO2‐sensitive chemoreceptor and central rhythm generator were present in basal vertebrates. We propose that, with the evolution of the lung, CO2‐sensitive cough is exapted to form the basis for air breathing common to air‐breathing tetrapods. Supported by NSFIOS‐1022442
Changes in expression and activity of MnSOD and CuZnSOD in carotid body and adrenal medullary cells of rats exposed to chronic intermittent hypoxia.
Authors: Moya Esteban , Varela Carlos R , Arias Paulina , Iturriaga Rodrigo
Published: June 2021

Nitro‐oxidative stress is associated with hypertension and enhanced carotid body (CB) chemosensory responses induced by chronic intermittent hypoxia (CIH). Since, ROS levels are regulated by cytoplasmic (CuZnSOD) and mitochondrial (MnSOD) superoxide dismutases; it is plausible that changes in their expression or enzymatic activity due to tyrosine nitration may contribute to the chemosensory potentiation. We studied effects of CIH on 3‐nitrotyrosine (3‐NT), SODs levels and activities, and MnSOD nitration in CB and adrenal medullary cells from Sprague‐Dawley male rats exposed to CIH (5% O2, 12 times/h for 8 h/day) for 7 days. To determine the contribution of mitochondrial ROS to the chemosensory potentiation, we studied the effects of the mitochondrial targeted antioxidant MitoTEMPO on the CIH‐induced CB chemosensory potentiation. CIH increased the 3‐NT levels in the CB and adrenal cells, the MnSOD level and activity in the CB, but not in adrenal medullary cells. CuZnSOD levels did not increase in any tissue. MitoTEMPO treatment prevented the CIH‐induced CB chemosensory potentiation. These results indicate that mithochondrial ROS are crucial for the chemosensory potentiation, while the increased CB MnSOD level and activity may be the result of a compensatory mechanism to the increased oxidative stress.

Supported by FONDECYT 1100405 & AT 24110172
Adaptive Immune Cells Contribute to Chronic Hypoxia‐Induced Pulmonary Hypertension
Authors: Maston Levi David , Resta Thomas C , Cannon Judy L , Giermakowska Wieslawa , Bosc Laura V González
Published: June 2021

Inflammation is a prominent pathologic feature in pulmonary arterial hypertension (PAH) as demonstrated by infiltration of inflammatory cells, including T and B lymphocytes, in the pulmonary vasculature of both patients and animal models. However, the contribution of the adaptive immune system is not well characterized in pulmonary hypertension caused by chronic hypoxia (CH) exposure. Therefore, we hypothesized that a lack of inflammatory cells, specifically T and B cells, protects mice from CH‐induced pulmonary hypertension. To test this hypothesis, we assessed indices of pulmonary hypertension in normoxic and CH (PB=380 mmHg, 3 wk) C57BL/6 wild‐type (WT) and RAG1 knockout (KO) mice, which lack mature T and B cells. Consistent with our hypothesis, CH RAG1 KO had a significantly lower peak right ventricular systolic pressure (RVSP) compared to CH WT mice (27.2 vs 37.7 mmHg, n=5 p<0.05). In contrast, RVSP was not different between normoxic RAG1 KO and WT animals (20.95 vs 21.8 mmHg). Additionally, CH RAG1KO mice displayed significantly reduced right ventricular hypertrophy (right ventricle/left ventricle+septum weight of 32.7 vs 40.15 % for CH WT, p<0.05) and pulmonary arterial remodeling (arterial wall thickness of 22.4 vs 28.1 % for CH WT, p<0.05) compared to CH WT mice. Our data suggest that adaptive immune cells contribute to the development of CH‐induced pulmonary hypertension.
TRPC1 and TRPC6 Regulate Chronic Hypoxia‐induced Vascular Tone, Vasoreactivity and Pulmonary Hypertension
Authors: Xia Yang , Yang Xiao‐Ru , Paudel Omkar , Fu Zhenzhen , Birnbaumer Lutz , Sham James S.K.
Published: June 2021

Pulmonary hypertension (PH) due to chronic hypoxia (CH) exposure is associated with increased vascular tone, altered reactivity to agonists and profound vascular remodeling, which are associated with alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells. We have previously demonstrated that transient receptor potential channel 1 and 6 (TRPC1 and TRPC6) channels were upregulated in CH rats. However, whether TRPC1 and TRPC6 genes contribute to elevated pulmonary pressure and how these cation channels affect vascular tone, reactivity and remodeling remain unclear. Here we found that CH caused a significant increase in the vascular tone of intrapulmonary arteries (IPAs) of wild‐type (WT) mice, but the enhancement was attenuated in TRPC1 null (trpc1−/−) mice and TRPC6 null (trpc6−/−) mice. Moreover, the enhanced vascular reactivity to serotonin observed in endothelium denuded IPAs of hypoxic mice was obliterated in trpc6−/− mice. Morphological analysis indicated that the muscularization in small PAs (<100 μm) of CH mice was significantly less in trpc1−/− and trpc6−/− mice compared to WT. In extension of these results, the elevated pulmonary arterial pressure and right heart hypertrophy were significantly suppressed in trpc1−/− mice. Our results hence provide strong evidence supporting the pathophysiological roles of TRPC1 and TRPC6 gene in the development of PH. (Supported by NIH)
Quantification of reduced blood flow through intrapulmonary arteriovenous anastomoses in healthy humans during exercise breathing 100% O2
Authors: Laurie Steven , Elliott Jonathan E. , Duke Joseph W , Goodman Randall D , Gladstone Igor M , Fish Mathews B , Lovering Andrew T
Published: June 2021

Saline contrast echocardiography (SCE) data suggest that breathing 100% O2 during exercise reduces blood flow through large diameter intrapulmonary arteriovenous anastomoses (QIPAVA), however this has not been quantified. We hypothesized that during exercise when breathing 100% O2, QIPAVA would be reduced compared to exercise breathing 21% O2 as quantified using Technetium‐99m macroaggregated albumin (99mTc‐MAA). We filtered MAA through a 20μm filter prior to labeling with 99mTc; quality control determined a binding efficiency >;99.8% prior to injection. Six subjects underwent whole body Gamma camera imaging post injection of filtered 99mTc‐MAA on 3 separate visits: A) rest breathing 21% O2; B) 85% max exercise breathing 21% O2; and C) 85% max exercise breathing 100% O2. Shunt fractions were quantified as the ratio of counts outside the lung divided by total counts from the whole body, less the fraction due to freely circulating 99mTc determined from a venous blood sample. During exercise breathing 21% O2 the shunt fraction increased from 0% at rest to 1.1% during exercise, but was reduced during exercise breathing 100% O2 to 0.4%. These data obtained using solid MAA suggest that breathing 100% O2 during exercise reduces QIPAVA, supporting data obtained using SCE. Funding: Eugene & Clarissa Evonuk Memorial Graduate Fellowship in Environmental, Exercise, or Stress Physiology.
Proteomic analysis of the lung proteome after chronic ethanol exposure
Authors: Alli Abdel A. , Montgomery Darrice S. , Ghant Marcus S. , Eaton Douglas C. , Helms My N.
Published: June 2021

The lungs are among several organs that can be adversely affected by chronic alcohol consumption. We used a proteomic approach to identify changes in lung proteins in female C57Bl/6 mice chronically ingesting 20% EtOH for 6 weeks and in similarly maintained caloric control‐fed animals. Proteins from the lungs of EtOH‐and control‐mice were separated by 2‐D gel electrophoresis. Coomassie stained gels revealed many protein spots that shifted and densitometry showed these proteins either increased or decreased in abundance in response to EtOH exposure. Protein spots were excised from the gels, destained, and digested with trypsin before being identified by LC‐MS/MS. Chronic alcohol consumption affected the protein expression levels of a tight junction protein, kinases, and actin‐binding proteins. The differentially expressed protein spots were analyzed using the PDQuest software which revealed 31 unique protein spots in the control group, 49 unique protein spots in the EtOH group, and 54 unique protein spots that overlapped between the two groups. Western blot analysis was used to confirm proteins up or down regulation by chronic EtOH exposure. Chronic alcohol consumption and alcohol metabolism are linked to tissue damage and various diseases. Herein, we identified specific proteins that may be involved in the etiology of these diseases. Funding from NIH R00HL09226 awarded to MNH and NIH NRSA awarded to AAA.
The Effect of Allopurinol Administration on Xanthine Oxidoreductase and Mitochondrial Respiration in Broiler Chickens.
Authors: Settle Tabatha Lynn , Falkenstein Elizabeth , Blemings Kenneth , Klandorf Hillar
Published: June 2021

As the end‐product of purine degradation, uric acid is generated in the xanthine/hypoxanthine reactions catalyzed by xanthine oxiodreductase (XOR). The purpose of this first study was to determine the effects of allopurinol and inosine on XOR activity in liver.In the first study, Cobb x Cobb (n=15; 5weeks old) were separated into three treatments (n=5); control (CON), INO (inosine at .6M/kg feed), or INOAL (inosine and allopurinol 50mg/kgBW). INOAL birds had a significantly lower (p=0.005) uric acid concentration in the liver. In the second study, Cobb x Cobb broilers (n=12; 4 weeks old) were separated into two treatments (n=6); control (CON) and AL (allopurinol 35mg/kg BW). The purpose of this study was to assess mitochondrial function in broiler chickens in response to potential oxidative stress from administration of allopurinol. Mitochondria were freshly isolated from liver tissue and assessed for State III and State IV respiration using polarography. There was a significant reduction in State III respiration (p=0.01) and State IV respiration (p=0.007) in allopurinol‐treated birds compared to the control. The reduced uric acid concentration in the liver suggests that there is a residual effect of allopurinol, which potentially results in lowered antioxidant activity in this tissue thereby causing an increase in oxidative stress within this tissue and can result in mitochondrial dysfunction.
N‐acetylcysteine administration modulates NADPH oxidase in skeletal muscle of rats with heart failure
Authors: Martinez Paula F , Fernandes D C , Bonomo C , Cezar M D M , Oliveira S A , Zornoff L A M , Okoshi K , Laurindo F R , Okoshi M P
Published: June 2021

This study aimed to evaluate the influence of antioxidant N‐acetylcysteine on NADPH oxidase (NOX) activity and gene expression in soleus muscle of rats with myocardial infarctioninduced heart failure.

Methods

Myocardial infarction (MI) was induced by left coronary occlusion. Four months later, rats were assigned to three groups: Sham (n=8), MI‐C (MI without treatment, n=8), and MI‐NAC (MI treated with N‐acetylcysteine, 120 mg/kg/day, n=8). Rats with small MI were excluded. Six months after surgery, NOX activity was assessed by HPLC analysis of dihydroethidium oxidation fluorescent products in soleus muscle. Gene expression of NOX complex subunits was evaluated by RT‐PCR.

Results

MI size did not differ between MI‐C and MI‐NAC groups (p>;0.05, Student's t test). NADPH oxidase activity was not different between MI‐C and Sham, but it was reduced in MI‐NAC compared to MI‐C group. Table shows the RT‐PCR results (ANOVA and Bonferroni; *p<0.05 vs. Sham, #p<0.05 vs. MI‐C):

Conclusion

N‐acetylcysteine administration reduces NADPH oxidase activity and gene expression in soleus muscle during heart failure. Support: FAPESP and CNPq.
Ketone Supplementation Reduces Superoxide Production in Cultured Primary Human Dermal Fibroblasts: Comparison of Young vs Aged Cells
Authors: Kesl Shannon Lynn , Moor Andrea Nichole , Jung Michelle Yung , Poff Angela M , Gould Lisa , D'Agostino Dominic Paul
Published: June 2021

In the United States, it is estimated that there are 1.8 million new cases of chronic wounds annually, with the majority occurring in the elderly. Reactive oxygen species (ROS) damage is augmented in aged patients leading to redox imbalance and is a key characteristic in impaired wound healing. Ketones are an energy substrate that reduce ROS production in the brain and heart, and thus may augment wound healing. We hypothesized that Beta‐Hydroxybutyrate (BHB; 5mM) would reduce cytosolic and mitochondrial ROS production in both young and aged primary human dermal fibroblasts. Primary human dermal fibroblasts (HDFs, passaged 2–8) from young and aged donors (24 to 88 years) were incubated in the absence or presence of BHB for 72 hours prior to stress with 100uM tert‐buytl‐hydrogen peroxide for 3 hours. ROS production was measured fluorescently with dihydroethidium (DHE, cytosolic) and Mitosox Red (mitochondrial) in triplicate, N=6. In the cytosol, aged HDFs produced 28% (p=<0.001)) more ROS basally compared to their young counterparts. When incubated in BHB for 72 hours, ROS production in the young HDFs decreased by 40% (p=<0.0001) and by 41% (p=<0.0001) in the aged. In the mitochondria, aged HDFs produced 27% (p=<0.0001) more ROS at baseline than the young HDFs, consistent with observations in cytosol. When incubated in BHB, ROS production was reduced in the young by 29% (p<0.0001) and in the aged by 51% (p<0.0001); the aged ROS production was no longer significantly different from the young. Ketone supplementation did not significantly change proliferation in the HDFs, but did increase migration in both young and aged HDFs. We conclude that ketone supplementation may protect against redox stress and augment wound healing, shifting redox homeostasis in HDFs, especially in the elderly. Funding: ONR, VA Merit Review.
ERM proteins colocalize with the Na+/H+ exchanger NHE1 in MCF‐7 breast cancer cell invadopodia and affect invadopodia number
Authors: Roest Gemma Anna Brigitta , Pedersen Stine Falsig
Published: June 2021

Invadopodia are actin‐rich membrane protrusions which have been assigned essential roles in cancer cell invasiveness. Here, we investigate the roles of ezrin, radixin and moesin (ERM), three closely related F‐actin‐plasma membrane linker proteins implicated in metastasis, and their binding partner the Na+/H+ exchanger isoform 1 (NHE1), in the formation and function of invadopodial rosettes in MCF‐7 human breast cancer cells. ERM proteins colocalized strongly with NHE1 in invadopodia in MCF‐7 cells +/− constitutively active, N‐terminally truncated ErbB2 (ΔNErbB2). In contrast, the tumor suppressor Merlin which is more distantly related to the ERM proteins, did not. siRNA‐mediated knockdown of NHE1 increased the average number of invadopodia per cell in ΔNErbB2‐expressing cells but not in vector controls. Overexpression of either dominant negative (DN) or constitutively active (CA) ezrin decreased the number of invadopodia per cell, in vector control cells only. Average invadopodial rosette diameter was unaffected by knockdown of ezrin or NHE1, or by expression of DN‐ or CA‐ezrin. Thus, in MCF‐7 cells, NHE1 colocalizes strongly with ERM proteins in invadopodial rosettes and both NHE1 and ezrin appear to affect their formation. Studies are ongoing to determine the generality and roles of these interactions in cancer invasiveness.

Funding: Danish Cancer Society, Danish Research Council
Cortactin and the actin cytoskeleton control the lateral membrane mobility of Kv1.3 channels
Authors: Martin Geoffrey V , Hajdu Peter , Chimote Ameet A , Takimoto Koichi , Szilagyi Orsolya , Conforti Laura
Published: June 2021

The proper Kv1.3 membrane motility is necessary for T cell polarization and activation. The exact mechanisms regulating Kv1.3 membrane movement, however, remain unclear. Increased activity of actin has been found during T cell activation. Additionally, multiple protein complexes including cortactin and PDZ proteins like Dlg1 have been found to bind both Kv1 channels and actin. The purpose of the present investigation was to determine if actin and cortactin/PDZ proteins modulate Kv1.3 membrane motility. HEK293 cells were transfected with EGFP‐tagged Kv1.3 wild‐type or C‐terminal mutants lacking either cortactin or the PDZ binding sites, and their membrane diffusion (D) and mobile fraction (Mf) was determined by FRAP. Wild‐type and mutant channels exhibited similar baseline D and Mf. Upon induction of F‐actin polymerization by jasplakinolide wild‐type and PDZ mutants showed a 40% reduction in Mf and no change in D, while cortactin mutants exhibited no change in Mf and an increase in D. Kv1.3 wild‐type and mutants displayed similar biophysical and electrophysiological properties. Furthermore, the ability of Kv1.3 to bind to cortactin and Dlg1 was confirmed by immunoprecipitation. These data indicate that Kv1.3 lateral membrane motility is regulated by the actin cytoskeleton and cortactin as only the deletion of the cortactin binding site inhibited the actin‐mediated Kv1.3 mobility. (NIH 2R01CA095286)
The role of the Arp2/3 complex in the cytoskeleton organization and actin‐mediated sodium reabsorption in kidney epithelial cells
Authors: Ilatovskaya Daria V. , Chubinskiy‐Nadezhdin Vladislav , Shuyskiy Leonid S. , Pavlov Tengis S. , Palygin Oleg , Negulyaev Yuri A. , Staruschenko Alexander
Published: June 2021

Actin cytoskeleton is an important mediator of a variety of functions in cells, including regulation of the epithelial Na+ channel (ENaC). One of the factors that control nucleation of actin filaments is the actin related protein 2/3 (Arp2/3) complex that potentiates actin polymerization. We have shown that treatment of principal cell line M‐1 with Arp2/3 complex inhibitors CK‐0944666 (CK‐666) and CK‐869 results in reorganization of F‐actin and decreases cell motility and lamellipodia formation. Compound concentrations used in this study neither caused loss of cell viability nor influenced the shape of the cells or the integrity of the monolayers. Furthermore, the action of the described compounds was not due to structural side effects since inactive analog of CK‐0944666, CK‐689, lacked the effect on cell motility. Inhibition of the Arp2/3 complex by CK‐666 precluded the effect of cortactin on ENaC; furthermore, it has been revealed that treatment with CK‐666 does not result in the loss of association between ENaC and cortactin. This finding was proved by patch‐clamp studies that showed that only a cortactin mutant unable to bind Arp2/3 had no influence on ENaC activity. Thus, Arp2/3 complex plays an important role in cell motility and F‐actin organization in the kidney cells. Moreover, Arp2/3 complex mediates renal Na+ transport via cortactin‐mediated regulation of ENaC. Supported by NIH (HL108880) and OPTEC.
Osteoblast Differentiation of Adipose Tissue‐Derived Mesenchymal Stem Cells (MSCs) is Enhanced by Bone Marrow MSC‐Conditioned Medium
Authors: Beloti Marcio Mateus , Abuna Rodrigo Flores , de Oliveira Fabiola Singaretti , Kato Rogerio Bentes , Rosa Adalberto Luiz
Published: June 2021

Mesenchymal stem cells (MSCs) derived from several adult tissues may be driven to differentiate into distinct cell lineages, making them the focus of a plethora of studies on cell therapy and tissue engineering. Recently, we have noticed that adipose tissue‐derived MSCs exhibit a lower osteogenic potential compared with bone marrow MSCs. As MSCs secrete many growth factors that could affect cells in terms of genotype and phenotype expression, here, we investigated the effect of conditioned medium from bone marrow MSCs on osteoblast differentiation of adipose tissue‐derived MSCs. Rat bone marrow MSCs were cultured in osteogenic medium, which was collected every 48 hours during 14 days and stored at −80ºC. This conditioned medium was used to culture adipose tissue‐derived MSCs for periods of up to 17 days and non‐conditioned osteogenic medium was used as a control. Real‐time RT‐PCR for key bone markers was carried out in quintuplicate (n=5) and data were compared by Mann‐Whitney test (p<0.05). Conditioned medium upregulated gene expression of Runx2 and bone sialoprotein at 4 and 10 days, collagen, type I, alpha 1 at 10 and 17 days, and osteocalcin at 4, 10 and 17 days when compared with non‐conditioned medium. Our results indicate that bone marrow MSCs synthesize paracrine factors that favor osteoblast differentiation of adipose tissue‐derived MSCs. Thus, this approach is feasible for obtaining an osteoblast‐enriched cell population from adipose tissue‐derived MSCs to be employed in cell therapy and bone tissue engineering research.
Activation of electrogenic K secretion requires BK channels in guinea pig distal colon
Authors: Halm Dan R , Zhang Jin , Halm Susan T
Published: June 2021

A variety of hormones and neurotransmitters activate electrogenic K+ secretion in guinea pig distal colon, generally together with Cl− secretion. Blockers of BK channels (KCa1.1, Kcnma1), iberiotoxin (IbTx) and paxilline, inhibited the short‐circuit current (Isc) associated with K+ secretion. Consistent with this K+ secretion occurring via apical membrane BK, mucosal addition of IbTx inhibited epinephrine (epi) activation with an IC50 for Isc (epiIsc) and for transepithelial conductance (epiGt) of ~200 nM. However, maximal inhibition was only ~50%. Mucosally added paxilline [10 μM] also inhibited epiIsc and epiGt by ~50%. IbTx and paxilline each inhibited Isc activated by mucosal ATP, supporting apical BK as an absolute requirement for this K+ secretion. Sensitivity to IbTx and paxilline demonstrated K+ secretion during activation of Cl− secretion by prostaglandin‐E2 and a cholinergic agonist. Distal colonic epithelial cells expressed BKα mRNA with the ZERO splice variant and 3 splice variants for the C‐terminus. These cells also expressed the regulatory β‐subunits BKβ1 and BKβ4. Immuno‐localization demonstrated BKα in apical and basolateral membranes of surface and crypt cells. Together these results support a cellular mechanism for electrogenic K+ secretion involving activation of apical membrane BK, but epi activated K+ secretion also required opening of other K+ channel types. [NIH DK65845]
Deletion of claudin‐7 in renal collecting duct cells impairs paracellular chloride permeability
Authors: Fan Junming , Tatum Rodney , Hoggard John , Jeansonne Beverly G , Chen Yan‐Hua
Published: June 2021

We have reported previously that claudin‐7 knockout mice (Cldn7−/−) display severe salt wasting and chronic dehydration phenotypes. To investigate the role of claudin‐7 in ion permeability in renal epithelial cells, we generated stable collecting duct (CD) cell lines isolated from Cldn7+/+ and Cldn7−/− mouse kidneys. Deletion of claudin‐7 significantly increased the transepithelial electrical resistance (TER) and decreased the absolute permeability for Cl− (PCl) and Na+ (PNa). The ratio of PCl/PNa was <1, suggesting an impairment of Cl− permeability along with a suppression of Na+ permeability in Cldn7−/− CD cells. TER and dilution potential values were unchanged after inhibition of Cl− and Na+ channels and the current‐voltage curves were linear in Cldn7+/+ and Cldn7−/− CD cells, indicating that the resulting ionic flux changes were through the paracellular pathway. The impairment of paracellular Cl− permeability can be rescued by transfecting Cldn7−/− CD cells with claudin‐7 cDNA. In addition, we also found that the expression level of WNK4 was dramatically reduced in Cldn7−/−CD cells, which was consistent with our previous finding that claudin‐7 is a substrate of WNK4 and can be phosphorylated by WNK4. Collectively, our findings indicate that deletion of claudin‐7 in CD cells impairs Cl− permeability, and that claudin‐7 could be one of the key factors in WNK4 mutations‐induced hypertension. This research is supported by NIH grant HL085752.
ENaC gating is coupled to the distance between the α‐and βENaC subunits at the base of the thumb and wrist domains
Authors: Collier Daniel M , Snyder Peter M
Published: June 2021

The extracellular domain of ENaC functions as a sensor that modulates channel activity in response to changing extracellular conditions. However, the mechanism by which the extracellular domain influences channel gating is not known. In previous work, we determined that αK477 plays a role in ENaC pH sensing. Based on the ASIC1a crystal structure, we predict that αK477 forms an interface with βV85 and hypothesize that movement at this interface is coupled to channel gating. In the current work, we investigated potential intersubunit interactions at the single channel level. In Xenopus oocytes expressing αK477CβV85CγENaC, treatment with a long cysteine reactive cross‐linking reagent (MTS‐14‐MTS) resulted in an increase in open probability (Po) from 0.4 (± .06) to 0.6 (± .04, p = .04). Because ENaC Po varies widely, we introduced a mutation in the γENaC subunit that prevents endogenous cleavage and causes the channel to favor the closed state (Po = 0.03 ± .06). MTS‐14‐MTS increased the Po of these channels four fold to 0.12 (± .06, p = .02). To test the effect of a short cross‐linking reagent (MTS‐2‐MTS), we performed single channel studies in low extracellular Li+ conditions to cause the channel to favor the open state (Po = 0.67 ± .13). Under these conditions, treatment with MTS‐2‐MTS decreased ENaC Po to 0.2 (± .08, p = .01). The data suggest that movement at the interface between αK477 and βV85 influences channel gating, providing a potential link between extracellular pH sensing, extracellular domain conformational changes, and channel gating.

DMC was supported by NIH 2T32HL007121–36 and PMS by HL072256
Paracellular urate transport in the placenta
Authors: Sakurai Hiroyuki , Kimura Toru , Tsukada Ai , Ohtsuki Sumio , Uehara Ichiro , Tanigaki Shinji , Iwashita Mitsutoshi , Ichida Kimiyoshi
Published: June 2021

Serum urate level is elevated in pregnancy induced hypertension or multiple pregnancies; however, how urate is handled in the placenta is still unknown. Blood samples from pregnant women who underwent caesarian section were examined. There were no significant differences in serum urate levels between maternal and umbilical cord blood, suggesting that urate is freely moved through the placenta. By RT‐PCR, mRNA of possible urate transporters, OAT4, OAT10, URATv1 and ABCG2, was expressed in the placenta and BeWo cells, a cell line derived from choriocarcinoma. BeWo cells did not take up urate despite the presence of urate transporters. Instead, urate moved through BeWo cell monolayers from apical to basal as well as reverse direction when the cells were incubated at 37 or 4 degree, suggesting that urate moves through paracellular route. Several claudins were commonly expressed in BeWo cells and in the placenta by RT‐PCR and LC/MS. It is likely that such claudin(s) are responsible for paracellular transport of urate in the placenta.
Acetylation Modulates ENaC Degradation
Authors: Butler Phillip L , Snyder Peter M
Published: June 2021

The epithelial Na+ channel (ENaC) forms a pathway for Na+ absorption across epithelia in the renal collecting duct. Dysregulation of ENaC results in defective blood pressure regulation (e.g. Liddle's syndrome and pseudohypoaldosteronism type I). ENaC is regulated in part through ubiquitination of lysines by Nedd4–2, which targets ENaC for endocytosis and degradation. Here we found that ENaC lysines are also substrates for acetylation. The histone deacetylase (HDAC) inhibitior trichostatin A (TSA) increased acetylation of lysines in α‐, β‐, and γENaC. This resulted in increased ENaC abundance in the total cell lysate and at the cell surface. Moreover, TSA increased ENaC current in FRT and mpkCCD epithelia. TSA increased ENaC surface expression and current by reducing its degradation. By screening candidates, we identified HDAC7 as a regulator of ENaC. Knockdown of HDAC7 (siRNA) recapitulated the effects of TSA on ENaC abundance and current, whereas overexpression had the opposite effects. We observed a reciprocal relationship between ENaC acetylation and ubiquitination; maneuvers that increased acetylation reduced ubiquitination, whereas reduced acetylation increased ubiquitination. Based on the data, we propose a model in which acetylation promotes epithelial Na+ absorption by protecting the channel from ubiquitination. Supported by NIH HL058812.
Heart rate, blood pressure and baroreflex gain in three species of anurans from different environments
Authors: McNew Kadi A , Hedrick Michael S
Published: June 2021

Anurans from different environments vary with respect to lymph mobilization capacity which is linked to blood pressure/volume status. We hypothesized that anurans from different environments might also vary in their ability to regulate blood pressure and heart rate through the baroreflex. We altered mean arterial blood pressure (MAP) pharmacologically and measured changes in heart rate (HR) to determine baroreflex gain. Experiments were conducted at 20 °C with three species of anurans from terrestrial (cane toad, Rhinella marina), semi‐aquatic (North American bullfrog, Lithobates catesbeianus) and aquatic (African clawed frog, Xenopus laevis) environments. MAP was increased with phenylephrine (Phe; 20–200 μg/kg, i.v.) and decreased with sodium nitroprusside (SNP; 20–200 μg/kg, i.v.). Heart rate ranged from 27 ± 3 to 31 ± 2 beats min−1 (P>;0.05), but MAP was significantly higher in R. marina (5.3 ± 0.2 kPa) compared with X. laevis (3.6 ± 0.2 kPa) or L. catesbeianus (4.2 ± 0.2 kPa). The baroreflex gain (ΔHR/ΔMAP) ranged from was −6.5 ± 0.5 to −7.2 ± 0.8 beats min−1 kPa−1 but was not significantly different. These data indicate that baroreflex gain does not vary in three species representing a wide environmental range, but the more terrestrial species (cane toad) operates at a higher arterial blood pressure suggesting that baroreflex resetting has occurred in this species. Supported by NSF‐IOS 0843082 and HHMI.
Environmentally‐Induced Phenotypic Plasticity in Embryonic Reptiles
Authors: Crossley Dane Alan , Elsey Ruth , Marks Christopher , Eme John
Published: June 2021

In response to chronic developmental stress, embryonic reptiles exhibit phenotypic plasticity resulting in morphological and physiological modifications. Utilizing chronic hypoxia, we investigated plasticity of cardiovascular regulatory maturation in two species, American alligator and common snapping turtle. Both exhibit phenotypic plasticity, resulting in increased relative heart mass and depressed heart rate. However, they differ in their capacity to modify the timing of cardioregulatory ability and the strength of each regulatory mechanism during development. We also determined that the cardiovascular system appeared most susceptible to environmentally‐induced phenotypic change during the last third of embryonic development. Relocation of chronically hypoxic‐incubated (10% O2) American alligator embryos to normoxia (H to N) at 70% of incubation returned heart mass to control values measured at 90% of development; the opposite manipulation (N to H) did not increase heart mass. Physiological phenotype was also altered, resulting in a reduced intrinsic heart rate in the N to H shift group compared to the H to N group. These data indicate that the degree of cardiovascular developmental phenotypic plasticity is species dependent and may require exposure during finite (‘critical’) windows of development to produce a given response. NSF CAREER IBN IOS‐0845741 to DAC