bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of isocapnic hypoxia on reflex cutaneous vasoconstriction
Authors: Simmons Grant H. , Minson Christopher T. , Halliwill John R.
Published: June 2021

We have previously shown that isocapnic hypoxia increases skin blood flow in thermoneutral humans, but the effect of hypoxic vasodilation on reflex cutaneous vasoconstriction is unknown. In this study, we tested the hypothesis that reflex cutaneous vasoconstriction is blunted during isocapnic hypoxia. Ten healthy volunteers (7 men, 3 women) underwent 10 minutes of progressive whole body cooling (water‐perfused suit) while breathing from a scuba mouthpiece during both normoxia and isocapnic hypoxia. Skin blood flow was assessed using laser Doppler flowmetry and cutaneous vascular conductance (CVC) was calculated as red cell flux/mean arterial pressure and normalized to normoxic baseline. During normoxia (SaO2 = 98.4 ± 0.3%), cold stress decreased CVC from 99.9 ± 0.6 to 71.4 ± 4.3 %baseline (P<0.05). During isocapnic hypoxia (SaO2 = 79.9 ± 0.4%), baseline CVC was elevated (P<0.05 vs. normoxia) and cold stress decreased CVC from 124.3 ± 4.5 to 86.9 ± 6.5 %baseline (P<0.05). Since the change in CVC was not different between normoxic and hypoxic conditions (−28.6 ± 4.4 vs. −37.4 ± 4.2 %baseline; P=0.11), the resulting CVC was lower during cold exposure in normoxic vs. hypoxic conditions (P<0.05). Thus, although reflex cutaneous vasoconstriction is functional during isocapnic hypoxia, skin blood flow during cold exposure remains elevated by isocapnic hypoxia relative to values observed in normoxic conditions.
Hyperthermia‐induced mitochondrial damage is exacerbated with aging
Authors: Haak Jodie L , Spitz Douglas R , Kregel Kevin C
Published: June 2021

Heat stress produces exaggerated oxidative damage with aging, and recent studies implicate mitochondria, a primary source of prooxidant production, as a key contributor to this response. In the current study, liver mitochondria isolated from either control or heat‐stressed young (6 mo) and old (24 mo) Fischer 344 rats (n=6/group) were assessed for structural and functional damage as well as endpoints indicative of oxidative stress. Electron microscopy revealed structural damage to mitochondria from both young and, to a greater extent, old heat stressed rats. Consistent with increased lipid peroxidation, 4‐hydroxynonenal‐modified protein was increased above control levels in mitochondria from both young and, again, to a greater extent in old rats exposed to hyperthermia. ATP and immunoreactive cytochrome c protein levels were significantly decreased in mitochondria from old but not young rats exposed to heat stress, suggesting that hyperthermia notably compromised mitochondrial function in aged animals. Taken together, the data indicate that, relative to young animals, mitochondria from aged rats are more vulnerable to oxidative damage caused by heat stress. These results support the speculation that mitochondrial damage and dysfunction could cause amplified prooxidant production that contributes to the increased susceptibility of aged organisms to physiological stressors. (NIH AG12350)
Redox Regulation of Circulating NO Bioavailability & Post‐Exercise Hemodynamics in Hypertension
Authors: New Karl James , Bailey Damian , McEneny Jane , Templeton Catherine , Ellis Gethin , James Philip , Davies Bruce
Published: June 2021

9 pre‐hypertensive males, MAP = 106 ± 5 mmHg were studied for 2‐hours following 30‐minutes of cycle exercise at 70% maximal oxygen consumption. Echocardiography determined cardiac output (CO) and systemic vascular resistance (SVR) by MABP/CO. Venous blood was sampled from an antecubital vein pre‐, immediately post‐, 1‐hour post‐ (P1) and 2‐hours post‐ (P2) exercise and corrected for plasma volume shifts. Plasma nitrate (NO−3) and nitrite (NO−2) was determined fluorometrically, S‐Nitrosothiol (RSNO) concentrations were assayed by the Saville reaction. Lipid hydroperoxides (LOOH) were determined spectrophotometrically whilst blood was also assayed for selective antioxidants. Upper (UL) and lower limb (LL) arterial pulse wave velocity (APWV) was determined pre and post‐exercise via oscillometry.

Ascorbic acid, α‐tocopherol, RSNO and LL APWV were unchanged. UL APWV was 18% lower than baseline at post recovering by P2 being 9% elevated above baseline.

Elevated oxidative stress in the recovery period coincides with a blunting of circulating NO, UL arterial compliance and a return of SVR towards baseline.
Respiratory muscle blood flow steal during exercise in heart failure
Authors: Olson Thomas Patrick , Joyner Michael J , Dietz Niki M , Eisenach John H , Curry Timothy B , Johnson Bruce D
Published: June 2021

Heart failure (HF) patients have reduced cardiac reserve and increased work of breathing (WOB)., Increased demand for locomotor muscle blood flow (i.e. activities of daily living) may result in competition between respiratory and locomotor vascular beds. We hypothesized that HF patients would demonstrate improved locomotor blood flow with respiratory muscle unloading during moderate activity. We studied 10 HF patients (EF=30±8%) and 10 controls during two 10 min. cycling sessions at 60% VO2max. Session 1 (S1): 5 min of normal breathing (NB) and 5 min respiratory muscle unloading with a ventilator. Session 2 (S2): 5 min NB and 5 min of respiratory muscle loading with inspiratory resistance. Measurements included: leg blood flow (LBF), cardiac output (CO), and inspiratory pleural pressure time integral as an index of the WOB. During S1, WOB was reduced similarly in both groups (HF: 50±60%; CTL: 50±56%, p<0.05 for both). In HF, CO was reduced (9.6±1.4 vs 8.2±2.1 L/min, p<0.05) and LBF increased (4.9±2.6 vs 6.2±2.9 L/min, p<0.01). In CTL, there was no change in CO (14.7±3.1 vs 14.8±5.0 L/min) or LBF (10.9±5.6 vs 10.3±5.2 L/min). During S2, WOB was increased similarly in both groups (HF: 111±75%, CTL: 110±85%, p<0.01 for both). In HF, there was no change in CO (10.0±1.3 vs 10.3±2.5 L/min) or LBF (5.0±1.8 vs 4.9±1.4 L/min). In CTL, CO increased (15.4±4.3 vs 16.9±4.7 L/min, p<0.01) with no change in LBF (10.7±5.3 vs 10.3±5.6 L/min). These data suggest that the respiratory muscles of HF patients preferentially recruit blood flow away from the locomotor muscles during moderate intensity, whole body exercise.
Excess dietary fat depletes glutathione, increases mitochondrial oxidant emitting potential and shifts redox balance to a more oxidized state in human skeletal muscle
Authors: Anderson Ethan J. , Boyle Kristen E. , Houmard Joseph A. , Neufer P. Darrell
Published: June 2021

The worldwide epidemic of Type 2 Diabetes is linked to modern lifestyle, of which high dietary fat intake is a substantial component. To examine the molecular impact of a high fat diet on human skeletal muscle mitochondria, male (18–34 yrs) subjects were recruited and muscle biopsies were obtained following a 12‐hour fast (A), 4 hours after a single high‐fat meal (65–70% fat content, B), and after 5 days of high‐fat diet/12‐hour fast (C). Using a method to measure in situ mitochondrial respiration and H2O2 emission (mH2O2, an index of reactive oxygen species) in permeabilized muscle fibers supported by glutamate/malate (G/M), succinate (S), and palmitoyl‐carnitine (PC), we observed from A to both B and C a striking 2.5‐ and 3‐fold (P<0.05) increase in Vmax of G/M/S and PC‐supported mH2O2 emission, respectively. Surprisingly, from A to C the total glutathione content (GSHt) was decreased by 40% (P<0.05), and the GSH/GSSG ratio decreased by 2.5‐fold (P<0.05). Respiration with all substrates remained unchanged from A to B, but basal and maximal G/M/S‐supported respiration was decreased by 30% (P<0.05) at C. These data demonstrate that an excess dietary fat intake 1) increases skeletal muscle mH2O2 emission and shifts redox balance to a more oxidized state, 2) decreases capacity for scavenging mH2O2 by depleting GSHt, 3) decreases mitochondrial respiration, all potentially contributing to the etiology of Type 2 diabetes.
FOXO Proteins, Downstream of Akt Signaling, Partially Mediate both Loading Induced Hypertrophy and Subsequent Unloading Induced Atrophy In the Fast Twitch Patagialis Muscles of Middle‐Aged Japanese Quail
Authors: Jackson Janna Renee , Alway Stephen E.
Published: June 2021

The aim of this study was to determine the effects of loading and subsequent unloading on AKT and FOXO protein signaling in the Patagialis (PAT) muscle of middle‐aged Japanese Quail (16–24mo). Muscle hypertrophy was induced by attaching a weight representing 10% of the animals’ body weight to one wing for 14 days. Animals were then randomly assigned to either a 0, 7, or 14 day unloading period. The contralateral limb served as the intra‐animal control. Total muscle homogenates were analyzed for the protein content of FOXO1, FOXO3a, AKT, pAKt and pGSK. Fourteen days of loading significantly increased PAT muscle mass by 22%. Muscle mass remained elevated at 7 days of unloading, however, the difference was not significant 14 days after weight removal. FOXO3a protein content was elevated after 14 days of loading (Day 0 unloading), however, there were no significant changes in the protein content of FOXO1, AKT, pAKT or pGSK. FOXO3a remained elevated at both day 7 and day 14 of unloading, although, no other measured proteins showed significant changes after 7 days of unloading. Interestingly, both pAkt and pGSK were significantly elevated after 14 days of unloading, although there were no significant changes in total Akt protein content. These results suggest that FOXO proteins, downstream of AKT, may partially mediate both skeletal muscle hypertrophy and atrophy in middle‐aged Japanese Quail. Funded by NIH: NIHROIAG021530
Skeletal muscle anabolic response to resistance exercise and essential amino acids is delayed with aging
Authors: Drummond Micah J , Dreyer Hans C , Pennings Bart , Fry Christopher S , Dhanani Shaheen , Dillon Edgar L , Sheffield‐Moore Melinda , Volpi Elena , Rasmussen Blake B
Published: June 2021

The loss of skeletal muscle during aging (sarcopenia) leads to an increased risk of falls, fractures and eventually loss of independence. Resistance exercise (RE) is a useful intervention to prevent sarcopenia, however, the response to RE is less when compared to young subjects. Essential amino acids (EAA) increase muscle protein synthesis (MPS) equally in both young and old subjects. We hypothesized that EAA ingestion following RE would restore anabolic signaling and MPS in aging muscle. Young and old men ingested 20g of EAA 1hr following a bout of leg‐RE. Muscle biopsies were obtained before, 1, 3, and 6hr post‐exercise to measure MPS and signaling pathways which regulate translation initiation. MPS increased early in young (1–3hr post‐exercise) and later in old (3–6hr post‐exercise). RE activated ERK1/2 and eIF2α signaling only in the young 1hr post‐exercise. mTOR/S6K1 signaling was similar between groups at all time points and AMPKα phosphorylation was higher in old 1–3hr post‐exercise. We conclude that MPS after RE and EAA ingestion is similar between young and old but delayed with aging. Unresponsive ERK1/2 signaling and AMPK activation in aging may explain the delayed activation of MPS. Notwithstanding, the combination of RE and EAA should be a useful strategy to combat sarcopenia.

Supported by NIH/NIAMS grant # R01 AR049877 and NIH/NIA P30 AG024832
PPAR‐γ activation inhibits TNF‐α induced NF‐κB activity in skeletal muscle
Authors: Remels Alexander , Langen Ramon , Schrauwen Patrick , Gosker Harry , Schols Annemie
Published: June 2021

Peroxisome proliferator‐activated receptor γ (PPARγ) has anti‐inflammatory properties which, in part, rely on its ability to modulate signaling by the cytokine‐inducible transcription factor NF‐κB. NF‐κB activation has been causally related to skeletal muscle atrophy, which is a characteristic of several chronic inflammatory diseases. The goal of this study was to evaluate if PPARγ activation affects TNF‐α induced NF‐κB activity in skeletal muscle cells. C2C12 Myoblasts with a genomically integrated NF‐κB luciferase reporter construct were differentiated during 6 days into myotubes. TNFα (1ng/ml, 4h) induced an 8‐fold increase in NF‐κB transcriptional activity. Pre‐treatment of the myotubes with the specific PPARγ agonist rosiglitazone significantly lowered TNFα induced NF‐κB transcriptional activity in a dose and time dependent fashion, with a maximal inhibitory effect of 80% (150 μM, 1h pre‐incubation). Similar effects were observed when a structurally unrelated PPARγ agonist was applied, or in response to IL‐1β‐induced NF‐κB activation. In addition, a significant decrease in mRNA levels of the NF‐κB target genes ICAM‐1 and KC was observed. These results demonstrate that NF‐κB activation by pro‐inflammatory cytokines in skeletal muscle can be suppressed by PPARγ agonists, which may be a new venue for therapeutic targeting of skeletal muscle atrophy.

This abstract was sponsored by Numico Research
Chromatin modification and remodeling during repair of a double‐strand break
Authors: Haber James , Hicks Wade , Kim Jung‐Ae , Dotiwala Farokh , Coïc Eric , Li Jin
Published: June 2021

A single double‐strand break (DSB) can be induced synchronously in a population of budding yeast, allowing us to monitor the kinetics of repair and the dynamics of DNA damage‐induced chromatin modification. When a DSB is induced at the MAT locus, it can be repaired by recombining with the heterochromatic donor loci, HML or HMR. The two donors are in competition with each other, but surprisingly the donor choice is not changed when one of the donors is unsilenced. Nevertheless, there are substantial chromatin changes when the Rad51‐coated single‐stranded DNA at the resected end of MAT invades the heterochromatic donor. These changes can be seen by analysis of the protection of MNase‐resistance of nucleosome‐bound sequences. Although strand invasion and synapsis occur in cells lacking the Swi2/Snf2 homologue, Rad54, we find important differences in the alterations of nucleosome protection relative to wild type cells.

A single DSB provokes modification of about 50 kb of chromatin around the DSB by phosphorylation of histone H2AX (γ‐H2AX) by ATM or ATR. Heterochromatic regions are refractory to modification but do not prevent spreading of γ‐H2AX beyond such regions. γ‐H2AX can be removed by 5′ to 3′ resection of the DSB ends (moving about 4 kb/hr) but also in a more rapid and concerted fashion when the DSB is repaired. Once liberated from chromatin γ‐H2AX is dephosphorylated by Pph3. The roles of various chromatin remodeling complexes in the removal of γ‐H2AX will be discussed. A second modification, independent of ATM/ATR kinases, is the casein kinase II‐dependent phosphorylation of histone H4‐S1. Unlike γ‐H2AX, γ‐H4 is not constitutively found near telomeres. The kinetics and extent of spreading of “γ‐H4” are markedly different from γ‐H2AX. Strikingly, γ‐H4 is found very close to the DSB site, even when these regions appear to be resected and single‐stranded. The loss of γ‐H4 when the DSB is repaired also exhibits different kinetics. The role of phosphatases and chromatin remodelers in γ‐H4 dynamics are under investigation.
Chronic intermittent hypoxia increases chromaffin cell secretory capacity through a reactive oxygen species mediated, protein kinase C dependent pathway
Authors: Kuri Barbara A , Khan Shakil A. , Chan Shyue‐an , Prabhakar Nanduri R. , Smith Corey B
Published: June 2021

Chronic intermittent hypoxia (CIH) augments sympathetic‐evoked catecholamine release from the adrenal medulla. In the present study we examine the effects of CIH on adrenal chromaffin cell secretion. Experiments were preformed on adult male mice (C57/BL6) exposed to 1‐4 days of CIH or normoxia. Perforated patch electrical capacitance recordings were preformed on freshly prepared adrenal medullary slices. Cells were directly stimulated by voltage clamp depolarization. Cells from CIH‐exposed mice exhibited a significant increase in the readily releasable pool (RRP) of secretory granules. Continuous hypoxia (CH) either for 2.5 hours (equivalent to hypoxic duration accumulated for 4 days of CIH) or for 4 days was ineffective in evoking changes in the RRP. Biochemical analysis of adrenal medullae showed both increased PKC expression and activity by CIH. PKC inhibitors prevented the CIH induced increase in RRP. CIH resulted in elevated reactive oxygen species (ROS) formation in chromaffin cells; anti‐oxidant treatment prevented ROS production, blocking the elevated RRP. These results demonstrate that CIH increases the secretory capacity in adrenal chromaffin cells at the cellular level, independent of cholinergic stimulation. Supported by NIH‐HL‐25830 and 076537 to NRP, NS‐053134 and IBN‐0344768 to CS, and BK is supported by NIH–TS HL07653.
Chronic intermittent hypoxia inhibits proteolysis in juvenile rat skeletal muscle
Authors: Fabio Thiago L. , Zanon Neusa M. , Kettelhut Isis C. , Bonagamba Leni G. , Machado Benedito H. , Navegantes Luiz Carlos C.
Published: June 2021

Catecholamines exert anti‐proteolytic effects on rat skeletal muscle in basal conditions. Considering that hypoxia is a powerful stimulus of the sympathetic nervous system, the present study was undertaken to examine in juvenile rat isolated muscles the possible changes in proteolysis that occur after chronic intermittent hypoxia (CIH). For this, 3‐week‐old rats were exposed to 9 days of CIH (6% O2 for 40s at 9 min intervals; 8 h day−1). On the 10th day the weight of CIH was smaller than control rat (83 ± 2 vs 91 ± 2 g). In both soleus and extensor digitorum longus (EDL) muscles, rates of overall proteolysis (nmol Tyr·mg wt−1·2h−1) decreased after CIH. The fall in the rate of protein degradation in soleus and EDL was accompanied by a 50% and 25% decreased activity of Ca2+‐dependent (0,047 ± 0,016 vs 0,100 ± 0,013) and Ubiquitin‐proteasome proteolysis (0,035 ± 0,005 vs 0,047 ± 0,003), respectively. In vitro rates of protein synthesis (nmol Tyr·mg wt−1·2h−1) in soleus from hypoxic rats decreased by 35% (0,371 ± 0,009 vs 0,273 ± 0,016). CIH increased by 75% the content of glycogen (%) in liver (8,9 ± 0,5 vs 5,1 ± 0,3) and soleus (0,71 ± 0,06 vs 0,41 ± 0,02). These data suggest an inhibitory control of intracellular proteolytic systems by adrenergic system and/or insulin in rat muscles exposed to CIH, which may prevent additional loss of body weight during hypoxia on this critical period of the development.

Financial support: FAPESP and CNPQ
Intracellular Oxygenation During Ischemic Muscle Contractions
Authors: Tevald Michael A , Lanza Ian R , Befroy Douglas E , Kent‐Braun Jane A
Published: June 2021

Proton magnetic resonance spectroscopy was used to investigate the effect of ischemic contractions on human skeletal muscle oxygenation. PURPOSE: To determine if ischemia+contraction produces greater muscle deoxygenation than ischemia alone. METHODS: 5 men performed the following protocols in a 4 tesla magnet: P1) 10 min of ischemic rest, then 4 min of intermittent maximal isometric voluntary contractions (MVCs), and P2) 1 min of ischemic rest, then 3.4 min of intermittent MVCs and 5.6 min of ischemic recovery. Deoxymyoglobin (dMb) was normalized to the peak obtained during 10 min of ischemic rest. RESULTS: In P1, dMb reached a plateau during 10 min of ischemic rest and was not affected by contractions (102 ± 4.1%). In P2, dMb increased rapidly, followed by a plateau that tended to be lower than that obtained during ischemic rest (86.7 ± 2.32%, p = 0.063). During ischemic recovery, dMb gradually increased to the same level observed during resting ischemia (102 ± 4.2%). CONCLUSIONS: The addition of contractions after 10 min of ischemia does not result in further desaturation, suggesting that the muscle is already fully desaturated. Overall, these results validate the use of 10 min of resting ischemia to calibrate the dMb by MRS, and suggest that the observed plateaus in the dMb signal reflect inadequate oxygenation rather than mitochondrial inhibition by metabolite accumulation. Support NIH AG21094, K02 AG023582
Role of Advanced Glycation Products with Oxidative Stress in Resistance Artery Dysfunction in Type 2 Diabetic mice
Authors: Su Jun , Lucchesi Pamela , Suzuki Yasuhiro , Rezk Bashir M , Palen Desiree I , Boulares Hamid A , Matrougui khalid
Published: June 2021

Diabetes is associated with increased advanced glycation end products (AGEs) formation and vasculopathy. We hypothesized that AGEs contribute to resistance artery dysfunction. Type 2 diabetic db−/db− (diabetic) and non‐diabetic db−/db+ (control) mice were treated with the AGEs inhibitor (aminoguanidine: 50 mg/Kg/day) for 3 months. Pressure‐induced myogenic tone (MT) was increased in mesenteric resistance arteries (MRAs) from diabetic mice but was unaffected by aminoguanidine treatment. Nitric oxide donor‐induced endothelium‐independent relaxation were similar in all groups. In diabetic mice, endothelium dependent relaxation in response to shear‐stress or acetylcholine was altered and was associated with reduced eNOS. Aminoguanidine treatment improved endothelial function and restored eNOS expression. Primary cultured endothelial cells (EC) subjected to high glucose for 48 hrs showed decreased eNOS expression and phosphorylation in response to calcium ionophore. High glucose decreased anti‐oxidant protein (MnSOD) and increased pro‐oxidant proteins (gp91phox) expression leading to increased oxidative stress generation, as assessed by DHE staining and endothelial NADH/NADPH oxidase activity. The pre‐incubation of endothelial cells with aminoguanidine restored eNOS‐phosphorylation and expression as well as the balance between pro‐ and anti‐oxidant factors induced by high glucose.
Altered vascular function in endothelial‐specific peroxisome proliferator‐activated receptor gamma (PPARγ) null mice
Authors: Bland Jennifer M. , Kleinhenz Dean J. , Hansen Jason M. , Ritzenthaler Jeff D. , Sutliff Roy L. , Hart C. Michael
Published: June 2021

Objective: To explore the role of PPARγ in vascular regulation, endothelial PPARγ‐deficient mice (ePPARγ−/−) were subjected to studies of systemic vascular function. Methods: Mice with a floxed PPARγ gene were bred with mice expressing Cre recombinase driven by the endothelial promoter, Tie‐2. Subsequent ePPARγ−/− mice were selected by genotyping. Blood pressure of littermate control (Control) and ePPARγ−/− mice was monitored with telemetry. Aortic ring relaxation responses were determined in a muscle bath. Aortic nitric oxide (NO) production was measured with electron spin resonance spectroscopy and Fe(DETC)2. Aortic NF‐kB nuclear binding was detected by EMSA. Plasma levels of cysteine and cystine (Cys:CySS) were determined by HPLC, and derivatives of reactive oxygen metabolites (dROMs) by ELISA. Results: Compared to Controls, ePPARγ−/− animals did not demonstrate significant increases in mean arterial pressure. However, ePPARγ−/− mice displayed: 1) significant reductions in endothelium‐dependent vasorelaxation and aortic NO production, 2) increased aortic NF‐kB nuclear binding, and 3) increased vascular oxidative stress manifested as oxidation of the Cys:CySS redox couple and increased dROMs. Conclusion: Vascular endothelial PPARγ plays an important role in maintenance of redox balance in the vasculature. Supported by grants from the NIH, VA Research Service, and Takeda Pharmaceuticals.
TRPV4 channels contribute to ATP‐stimulated calcium influx in endothelial cells
Authors: Marrelli Sean P
Published: June 2021

Objective: We examined the role of TRPV4 channels in ATP‐stimulated Ca2+ influx in endothelial cells (ECs) of the carotid artery (CA).

Methods: Rat CAs were harvested, cut into segments, and organ cultured for three days with TRPV4 siRNA to selectively knockdown TRPV4 protein. Mock transfection and scrambled siRNA controls were performed in parallel. Knockdown was confirmed by real‐time PCR, Western, and immunofluorescence. Intracellular free Ca2+ concentration was measured (fura‐2) in response to ATP in ECs isolated from organ cultured arteries.

Results: TRPV4 mRNA and protein were significantly knocked down in whole CA in the TRPV4 siRNA group. EC‐specific expression of TRPV4 from organ cultured arteries was also significantly reduced in the siRNA group compared with control (immunofluorescence). Next, endothelial Ca2+ responses to 4α‐PDD (a TRPV4 channel opener) and ATP were evaluated in mock and TRPV4 siRNA transfected arteries. The EC Ca2+ increase to 4α‐PDD was significantly reduced (~50%) following TRPV4 knockdown, demonstrating significant functional loss of TRPV4 channels. Finally, stimulation with ATP revealed a significant reduction in the sustained Ca2+ influx component whereas peak Ca2+ responses were unchanged.

Conclusion: These results demonstrate that ATP‐stimulated Ca2+ influx in CA endothelial cells is partially dependent on TRPV4 channels. (AHA 0665100Y and 0230353N)
Limb‐specific vascular reactivity and ischemic reperfusion: sex differences?
Authors: Nishiyama Steven Keita , Wray David Walter , Berkstresser Kimberley A , Harris Ryan A , Richardson Russell S
Published: June 2021

We sought to investigate vascular reactivity and ischemic reperfusion (IR) characteristics in peripheral conduit arteries of the upper and lower extremities in men and women. In thirty young, healthy volunteers (n = 15 men, 28 ± 2 yrs; n = 15 women, 23 ± 1 yrs) we utilized ultrasound Doppler to evaluate IR and flow‐mediated dilation (FMD) following 5‐min supra‐systolic cuff occlusion in both the arm (brachial artery, BA) and leg (popliteal artery, PA). Cumulative IR (area under the curve, AUC) normalized for muscle mass was greater in the arm compared to the leg (P < 0.05), but revealed no sex‐related differences in either arm (men: 40 ± 3 ml/100g; women: 42 ± 4 ml/100g) or lower leg (men: 12 ± 3 ml/100g; women: 15 ± 2 ml/100g). When FMD (% change diameter) was normalized for the shear rate AUC, the leg (PA) of both men and women revealed a significantly greater FMD for a given shear rate than the arm (BA) (PA: 9.7 ×10−4 ± 0.1 ×10−4 and BA: 3.6 ×10−4 ± 0.3 ×10−4 % Δ / s−1•s, P < 0.05; female group: PA: 8.1 ×10−4 ± 1.7 ×104and BA: 3.4 ×10−4 ± 0.5 ×10−4 % Δ/s−1•s, P < 0.05). However, there were no sex‐related differences in FMD per unit of shear rate in both limbs. These findings confirm vascular limb‐differences in a female group, while also revealing no sex‐related difference in functional vascular characteristics (IR and FMD) in either limb.
Vasoregulatory mechanism response speed to step increases or decreases in exercise from steady state
Authors: Poitras Veronica , Matusiak Kristine , Pickett Amy , Tschakovsky Michael E.
Published: June 2021

We tested the hypothesis that muscle vasoregulatory mechanisms respond rapidly to step increases and decreases in exercise intensity from steady state. 10 men and 9 women (21.8 ±3.6 yrs) performed repeated step‐transitions between 4 min bouts of low (L) and moderate (M) forearm handgrip exercise intensities. Forearm blood flow (FBF; Doppler ultrasound), mean arterial pressure (MAP, finometer), and heart rate (HR; ECG) were measured beat by beat. A cardiac cycle during each relaxation of the duty cycle (1s contract/2s relax) was used to quantify forearm hemodynamics (FBFrelax ml/min; forearm vascular conductance, FVCrelax ml/min/100 mmHg). Data mean ± SD. FBFrelax increased rapidly following a step increase in exercise intensity (L steady state 243.1 ±103.6 vs. 1st M relaxation 262.0 ±78.1, P = 0.075 vs. 2nd M relaxation 289.8 ±82.2, P<0.001). This was due to a rapid vasodilation (FVCrelax L steady state 258.8 ±93.0 vs. 1st M relaxation 279.6 ± 71.8, P = 0.058; vs. 2nd M relaxation 304.7 ±74.0, P<0.001). The same rapid responses were observed with decreases in exercise intensity (FVCrelax M steady state 462.3 ± 120.5 vs. 1st L relaxation 435.8 ±103.3, P<0.001; vs. 2nd L relaxation 420.4 ±99.0, P<0.001). We conclude that vasoregulatory mechanisms are able to alter muscle blood flow rapidly and with little delay in response to sudden increases and decreases in exercise intensity from steady state. Supported by NSERC Canada.
Effects of angiotensin II on resistance vessels in hamster hypertension
Authors: Kurjiaka David T. , Burt Janis M.
Published: June 2021

Earlier work suggested endothelin‐1 elevated blood pressure in the spontaneously hypertensive hamster (SHH). However, while no differences were observed in vascular responses to endothelin‐1, maximal angiotensin II (Ang II) constriction was greater in the SHH (75% vs 50% normotensive). Interestingly, the potent constrictor was <100% effective in both strains. These experiments investigated this incomplete Ang II constriction in the SHH. In isolated skeletal muscle feed arteries, Ang II constriction was unaltered by the AT‐2 antagonist PD‐123319 (4 μM). Rather, maximal constriction to Ang II were recovered by angiotensin converting enzyme 2 (ACE2) inhibition with DX‐600 (0.1 μM) such that no differences were observed between strains. This suggests that earlier differences in Ang II constriction reflected a reduced ACE2 activity in resistance vessels of the SHH. Since the Ang 1–7 generated by ACE2's action on Ang II did not relax these vessels, the reduced constriction would appear to involve a decrease in Ang II rather than an increased Ang 1–7 action. Interestingly, ACE2 expression was greater in the SHH suggesting that ACE2 activity is dramatically reduced in the SHH. As the elevated blood pressure was completely recovered by acute application of losartan (60 mg/kg), the elevated blood pressure in the SHH appears to result from reductions in ACE2 activity that would increase local concentrations of Ang II.
Angiotensin system is mediating cardiac oxidative stress and p38 expression in SHR
Authors: Bertagnolli Mariane , Viegas Vinicius Urbano , Janner Daiane , Casali Karina Rabelo , Ribeiro Maria Flávia , Belló‐Klein Adriane , Rigatto Katya , Irigoyen Maria Cláudia
Published: June 2021

The objective was to determine the effect of treatment with an ACE inhibitor, enalapril (EN), on cardiac oxidative stress. Male Kyoto and SHR (15 wks) were treated for 10 weeks with EN or control (gavage, 10 mg/kg, n = 7/group). After treatment, blood pressure (BP) and heart rate were recorded from an arterial catheter (Windaq, 2KHz). Rats were euthanized with collection of heart with measurement of cardiac index (ventricle weight/body weight; mg/g), chemiluminescence (CL) and carbonyl assay. Akt and MAPK (Erk and p38) expression were assessed by Western blot. Results are presented as mean ± SD (table). In SHR, enalapril reduced BP, cardiac hypertrophy and p38 expression, probably due to a decreased cardiac oxidative stress caused by renin‐angiotensin system.

Supported by: CNPq and FAPERGS.
Persistent PAR2 vasodilation in hypertensive mice
Authors: McGuire John J , Vliet Bruce N Van , Halfyard Sarah J
Published: June 2021

Proteinase‐Activated Receptor 2 (PAR2) caused vasodilations that were preserved in genetic hypertensive mice. In the current study we examined the effects of infusion of angiotensin II (AII) on PAR2‐mediated vascular smooth muscle relaxations and PAR2 expression on high salt diet‐induced hypertension in mice. Wire myographs were used to measure PAR2 and acetylcholine‐induced relaxations of mesenteric arteries from saline‐treated and AII‐treated mice. PAR2 relaxations persisted in the AII‐treated mice despite the reduction of acetylcholine‐induced relaxation. Blood pressures measured by radiotelemetry determined the severity of hypertension in background and PAR2 knockout mice fed high salt diets over a two week period. Baseline blood pressures and high salt diet‐induced increases in blood pressure in controls were similar to PAR2 knockout mice. We conclude that PAR2 expression did not affect blood pressure response to a high salt diet and therefore, PAR2 expression did not play a role in the blood pressure‐natriuresis relationship. Further investigation is warranted to investigate the interactions of PAR2 with endothelial dysfunction‐resistant mechanisms of vascular smooth muscle relaxation in cardiovascular diseases such as hypertension.
Collagen metabolism and renal damage in 2k1c rats
Authors: Skogstrand Trude , Hultström Michael , Iversen Bjarne M.
Published: June 2021

We have found a clear difference in gene expression for the enzymes of collagen metabolism between the inner and outer renal cortex in old spontaneously hyptertensive rats with renal damage. The progress of fibrosis in 2k1c rats is less well studied. In the present study we have investigated 2k1c rats six weeks after clipping using western blot and real time PCR to compare the gene and protein expressions in the inner and outer cortex. Using real‐time PCR we studied collagen type‐1‐A1, procollagen‐n‐ and ‐c‐peptidase, MMP‐2, MMP‐9, TIMP‐1 and ‐2. This was complemented with protein quantification using western blot for MMP‐9 and TIMP‐2 as well as the superoxide dismutases EC‐SOD and MN‐SOD.

No differences were found between the inner and outer cortex of the kidney for any of the genes and no difference was found between the expressions in 2k1c and controls. This while the systolic pressure after six weeks was 126±2 mmHg in controls and 150±4 mmHg in the 2k1c rats.

Our conclusion is that: While 6 weeks is pleny of time for hypertension to develop, it is not enough for the collagen metabolism to be significantly altered at the gene expression level.

The study was founded by Helsevest, Norway.
Pressure‐induced renal injury is attenuated in norepinephrine‐induced hypertensive rats
Authors: Polichnowski Aaron James , Kaldunski Mary , Cowley Allen W
Published: June 2021

We investigated the role of elevated renal perfusion pressure (RPP) versus circulating norepinephrine (NE) on renal outer medullary (OM) injury in Sprague Dawley rats. Rats were prepared with carotid and femoral arterial catheters, a femoral venous catheter, and a servo‐controlled aortic balloon occluder positioned between the renal arteries. Rats were infused with NE (0.5, 1, and 2 ug/kg/min on Days 1, 2, and 3–14, respectively) for 14 days while sham rats received saline. On day 14 of infusion, kidneys were removed and prepared for histological analysis. Sections were stained with Gomori trichrome to identify tubular necrosis, and immunostained with antibodies for α‐SMA for fibrotic tissue and for ED‐1 positive cells for macrophage infiltration. Over the period of NE or saline infusion, RPP of the uncontrolled kidney (149.1 ± 4.2 mmHg) was significantly higher (p < .05) versus the controlled (117.1 ± 0.4 mmHg) and sham (110.7 ± 0.4 mmHg) kidneys. The uncontrolled, controlled, and sham kidneys had similar levels of tubular necrosis (0.4 ± .17, .04 ± .02, and .01 ± .01 %, respectively), OM fibrosis (1 ± .19, 0.9 ± .19, and 1.4 ± .28 %), and macrophage infiltration (35.2 ± 7.9, 27.2 ± 7.3, and 21.0 ± 1.6). In conclusion, neither elevated RPP nor NE results in significant OM renal injury in NE‐induced hypertension. This is in contrast to AngII‐induced hypertension, in which OM renal injury is largely pressure dependent.
Contribution of T‐type Ca2+ channels to blood pressure regulation in genetically hypertensive rats
Authors: Alonso‐Galicia Magdalena , Regan Hillary K. , Barrow James C. , Selnick Harold G. , Stauffer Shaun R. , Friesen Richard W. , Ellis Joan D. , Uebele Victor N. , Renger John J. , Connolly Thomas , Feuerstein Giora Z.
Published: June 2021

L‐type Ca2+ channels (LCC) play a major role in Ca2+ influx into smooth muscle cells and cardiac myocytes. However, other Ca2+ channels have been recognized to operate in the cardiovascular system of which the T‐type Ca2+ channels (TCC) have been the focus of attention. Using a novel, highly selective TCC blocker we aimed to determine the contribution of TCC vs. LCC to blood pressure regulation in conscious SHR. Vehicle and equipotent doses of various calcium channels blockers were given IV for 24h. Mibefradil (0.25 mg/kg.h), Amlodipine (0.1 mg/kg.h), Verapamil (0.66 mg/kg.h), and Diltiazem (0.66 mg/kg.h) significantly lowered mean blood pressure (MBP) over 24h by −24±3, −20±4, −22±3, and −22±2 mmHg, respectively and caused tachycardia (5–15%), except Mibefradil. The TCC antagonist (T‐and L‐type IC50: 14 and 823 nM), given at 2.95 mg/kg.h IV lowered MBP transiently by −18 mmHg for 1–2h only without changing heart rate. Unbound plasma levels of the novel TCC blocker in vivo were almost 2 times the T‐type IC50 and 0.03 times the L‐type IC50 determined in vitro suggesting that the majority of TCC were blocked over 24h. Our studies suggest that the pharmacological effects of the calcium channel blockers tested, including Mibefradil, are due mostly to inhibition of L‐type Ca2+ channels and that inhibition of T‐type Ca2+ channels causes acute, transient decreases in blood pressure in conscious genetically hypertensive rats.
Soluble fms‐like tyrosine‐1 (sFlt‐1) production in response to placental ischemia and hypoxia in placenta of pregnant rats
Authors: Murphy Sydney Roberts , LaMarca Babbette , LaMarca Ellen , Cockrell Kathy , Gilbert Jeffery , Granger Joey P.
Published: June 2021

Reduction in uteroplacental perfusion (RUPP) during pregnancy is associated with abnormalities consistent with features of human preeclampsia (PE). Recent studies have shown that an imbalance of pro‐angiogenic factors and the anti‐angiogenic factor, sFlt‐1, exists in PE. While abnormalities in sFlt‐1 synthesis are thought to play a role in the pathogenesis of PE, the mechanisms that lead to its production are unclear. The purpose of this study was to determine the role of placental ischemia and hypoxia in mediating the increase in sFlt‐1. Plasma concentrations of sFlt‐1 in response to RUPP in pregnant rats are elevated (660±270 vs 82±26 pg/mL). In addition, we found sFlt‐1 to be significantly elevated in placentas from RUPP rats. Moreover, we report that sFlt‐1 from placental explants is higher in RUPP rats (2603±187 vs 2025±183 pg/ml) under normoxic conditions (6%O2). Finally, we found that placental explants from pregnant rats grown in hypoxic conditions (1%O2) enhanced the production of sFlt‐1 (4066±561 vs 2025±183 pg/ml). In summary, we found that plasma and placental sFlt‐1 production are significantly elevated in response to placental ischemia in pregnant rats, in placental explants from RUPP rats, and in placental explants from normal pregnant rats exposed to hypoxia. These data suggest that placental ischemia/hypoxia may be an important mechanism that leads to production of sFlt‐1 in preeclampsia.
Angiotensin II‐induced hypertension stimulates thick ascending limb NaCl absorption by increasing O2−
Authors: Silva Guillermo B , Garvin Jeffrey L
Published: June 2021

Renal medullary O2− increases in angiotensin‐induced hypertensive (Ang‐IH) rats. We have shown that O2− increases thick ascending limb (TAL) Na transport, but the effect of Ang‐IH on the TAL is unknown. We hypothesized that TAL Na absorption in Ang‐IH rats is enhanced due to elevated O2− levels and protein kinase C (PKC) α activity. We measured O2− levels, furosemide‐sensitive oxygen consumption (FSQO2) as a measure of Na transport and PKCα activity in TAL suspensions from control and Ang‐IH rats. In TALs from Ang‐IH rats O2− levels were 2.5±0.5 compared to 0.8±0.3 arbitrary units (AU)/min/mg protein for controls (p<0.05). FSQO2 also increased (25±3 to 36±2 % of total QO2; p<0.05). Tempol, a O2− scavenger, infused into 1 kidney of Ang‐IH rats decreased O2− levels from 2.3±0.4 to 1.3±0.2 AU/min/mg protein (p<0.05) and FSQO2 from 33±2 to 24±2 % of total QO2 (p<0.05). Tempol had no effect on O2− or FSQO2 in controls. Tempol did not alter blood pressure of Ang‐IH rats. In Ang‐IH rats PKCα activity was 3.7±0.9 compared to 1.1±0.2 AU/mg protein (p<0.05) in controls. Tempol infusion into 1 kidney of Ang‐IH rats reduced PKCα activity from 3.0±0.6 to 0.6±0.1 AU/mg protein (p<0.05). Infusion of the PKCα inhibitor Gö6976 into one kidney of Ang‐IH rats reduced FSQO2 from 38±3 to 26±3 % of total QO2 (p<0.05). We conclude that in Ang‐IH rats TAL NaCl absorption is enhanced due to elevated O2− and PKCα activity rather than increased blood pressure.
Salt sensitive modulation of the interaction between efferent and afferent renal nerve activity mediated by PGE2 and endothelin (ET)
Authors: Kopp Ulla , Cicha Michael , Smith Lori
Published: June 2021

Increases in efferent renal sympathetic nerve activity (ERSNA) increase afferent renal nerve activity (ARNA) which in turn decreases ERSNA via activation of the renorenal reflexes in the overall goal of maintaining low ERSNA to limit sodium retention. We examined if dietary sodium modulates the interaction between ERSNA and ARNA and the norepinephrine (NE)‐induced activation of renal sensory nerves. Thermal cutaneous stimulation (HOT) to increase ERSNA was produced by placing the rat's tail in 47 and 49°C water in rats fed high sodium (HNa) and low sodium (LNa) diet (lower heat activation threshold in HNa vs LNa rats). HOT increased ERSNA and ARNA 100120 ± 1830 and 7660±1380 % sec (AUC) in HNa rats and 4370 ± 1290 and 970 ± 420% sec in LNa rats. In isolated renal pelvises from HNa rats, NE 10 pM increased the release of substance P from 7 ± 1 to 12 ± 2 pg/min and PGE2 from 67 ± 1 to 150 ± 13 pg/min. In pelvises from LNa rats, NE 10‐6250 pM had no effect. Renal pelvic administration of the ET B receptor (ETB‐R) antagonist BQ788 blocked the ARNA response to HOT by 76 ± 6% in vivo and the NE‐induced increases in substance P and PGE2 by 65 ± 16 and 34 ± 15% in vitro. Conversely in LNa rats, the ETA‐R antagonist BQ123 enhanced the ARNA response to HOT from 970 to 7360 ± 2124 % sec and the renal pelvic release of substance P produced by NE 6250 pM. NE increased substance P release from 5 ± 0 to 7 ± 1 pg/min (NS) before and from 5 ± 1 to 9 ± 1 pg/min (P<0.01) during BQ123. BQ123 had no effect on the NE‐induced release of PGE2. BQ788 and BQ123 have no effect in normal sodium diet rats.

Conclusion: Activation of ETB‐R contributes to the increased PGE2 synthesis and increased interaction between ERSNA and ARNA in HNa rats. Activation of ETA‐R contributes to the decreased interaction between ERSNA and ARNA in LNa rats by a mechanism distal to PGE2 synthesis.
Effect of heart failure on cardiac and respiratory variability and cardioventilatory coupling
Authors: Kruper Gregory John , Fishman Mikkel , Hoit Brian D , Chandler Margaret , Loparo Kenneth A , Dick Thomas E
Published: June 2021

Changes in heart rate (HRV) and respiratory variabilities are associated with worsening heart failure (HF). We hypothesized that changes in non‐linear dynamic properties of respiratory pattern track the development of early HF. In Sprague Dawley rats (n=6, 3 with sham surgery) were implanted with wireless blood pressure (BP) transducers (DSI transmitters) and the left main cardiac artery was ligated. Heart rate (HR), BP and respiration was monitored simultaneously by placing a plethysmograph on the DSI‐receiver. Animals were exposed to: 21%, 100% and 8% O2 each wk following surgery. Animals were echoed at 6 wk to assess cardiac function. Breath‐to‐breath variability (Ti and Te) was assessed by systematically applying non‐linear analytical tools to epochs of stationary data, and compared the results to 19 surrogate data sets, in which the auto‐correlation function was preserve but the time dependent properties of the data set were destroyed. Mutual information increased and sample entropy of the interval decreased from 3 to 8 wks post‐surgery in HF animals compared to animals those received sham surgery. These changes in variability were especially apparent when animals were breathing a hypoxic mixture. These preliminary data suggest that nonlinear sources of breathing variability are sensitive to cardiac performance decrease even during the early stages of HF (HL‐080318).
Chronic volume overload alters cardiac fibroblast secretion of collagen and matrix regulatory proteins
Authors: Hutchinson Kirk R , Stewart James A , Lord Kevin C , Varner Kurt J , Lucchesi Pamela A
Published: June 2021

Hearts with volume overload undergo progressive left ventricular dilatation with decreased extracellular matrix (ECM), associated with dynamic changes in bradykinin, angiotensin II, oxidative stress and the balance between pro‐ and anti‐fibrotic proteins. This study determined phenotypic changes in primary cultures of cardiac fibroblast (CF) isolated from rats subjected to aortocaval fistula (ACF) for 15 wk vs. aged‐matched shams. Western blot analysis and zymography measured basal and agonist induced expression of Ang II type 1 (AT1R) receptors, iNOS, and secretion of type I collagen, matrix metalloproteinases (MMPs) and plasminogen activator inhibitor‐1 (PAI‐1). ACF CFs demonstrated a 50% decrease in basal collagen type I and PAI‐1 expression and a 2‐3 fold increase in MMP‐9 and iNOS. Treatment with bradykinin (100 nM, 24hr) induced collagen secretion to a greater extent in CF from 15 wk sham vs. ACF. On the other hand, the NO‐donor DETA‐NONOate (100 μM 24 hr) increased MMP‐2/9 secretion by CF from 15 wk ACF to greater extent than CF from age‐matched shams. These data suggest that changes in CF function may underlie the persistent ECM degradation observed in vivo in chronic volume overload and may be associated with the transition to decompensated heart failure. Supported by R01HL063318 and P20RR18766 (PAL).
Cardiovascular autonomic control in healthy adolescents
Authors: da Silva Prado Juliana Martuscelli , da Costa Priscila Standke , Marmo Denise Barbieri , Martins Luiz Eduardo Barreto , Gallo‐Junior Lourenço , Golfetti Roseli
Published: June 2021

The purpose of this study was to identify differences in cardiovascular autonomic control in a sample of healthy adolescents. We studied 58 adolescents (21 females, 37 males) between the ages of 11–18. Heart rate variability (HRV) measures were adopted to assess autonomic heart rate function. The short‐term HRV in the time and frequency domains was obtained using 24‐h dynamic ECG. Analysis of the HRV in the time domain showed a longer R‐R mean (medians; 871 ± 191 ms vs 758,5 ± 109,5 ms; P<0,05) in boys in the early adolescence group than similarly aged girls. Girls in the late adolescence exhibited significantly higher normalized low frequency (LFun; 84 ± 11% vs 70 ± 11%; P<0,05) and lower normalized high frequency (HFun; 16 ± 11% vs 30 ± 9%; P<0,05) versus boys in the same aged group. Girls in the early adolescence had significantly lower values for LFun (medians; 67 ± 19,6% vs 84 ± 11%; P<0,05) and higher values for HFun (medians; 33 ± 19,5% vs 16 ± 11%; P<0,05) as compared to girls in the late adolescence. No significant difference in LF/HF ratio was detected between both groups. There is an increase in LF power, an indicator of predominantly sympathetic activity, during pubertal development. These results indicate the need to account for gender‐ and age‐related differences in cardiac autonomic function measures.

Supported by CAPES and FAEP/UNICAMP
SOS Induction by Stabilized Topoisomerase IA Cleavage Complex occurs via the RecBCD pathway
Authors: Sutherland Jeanette Helen , Tse‐Dinh Yuk‐Ching
Published: June 2021

Enzymes known as topoisomerases regulate the topology of DNA. Antibacterial drugs such as quinolones target type II topoisomerase by trapping the cleavage complex formed between the enzyme and cleaved DNA. This subsequently leads to accumulated double stranded breaks that result in bacterial cell death. Due to increasing resistance to these antibacterial drugs, it is essential to identify new drug targets. One such target is topoisomerase IA, which cleaves a single strand of DNA. The overall purpose of this study is to investigate the cellular response to stabilized type IA DNA topoisomerase cleavage complex in Escherichia coli. A mutant of the Yersinia pestis topoisomerase IA has been shown to form a stabilized cleavage complex resulting in SOS induction and extensive bacterial cell death. This topoisomerase mutant was used to determine whether the RecBCD or RecFOR enzyme complexes were involved in SOS induction and the repair of this stabilized cleavage complex. The study concluded that RecBCD was required to induce the SOS response in E. coli cells expressing the recombinant mutant topoisomerase and for repair of the topoisomerase cleavage complex. This suggests that the single‐stranded break associated with the topoisomerase IA cleavage complex is first converted to a double‐stranded break for repair by the RecBCD enzyme and subsequent induction of the SOS response and homologous recombination.