bluuebunny/crossref_metadata_2025 huggingface dataset:
Methyl group partitioning during insufficient and excess availability via guanidinoacetate and creatine supplementation in young pigs
Authors: McBreairty Laura Emily , Robinson Jason L , Brunton Janet A , Bertolo Robert F
Published: June 2021

Methionine is an essential amino acid that provides methyl groups for transmethylation (TM) reactions via S‐adenosylmethionine (SAM), resulting in S‐adenosylhomocysteine (SAH), an inhibitor of TM reactions. Methylation of guanidinoacetate (GAA) to produce creatine (Cr) is proportional to GAA availability while Cr supplementation reduces GAA production and spares methyl groups. Our objective was to determine the partitioning of methyl groups among TM reactions under conditions of excess and insufficient methyl group availability. Three mo‐old pigs (n=18) were fed control, GAA‐ or Cr‐supplemented diets twice daily. On day 18, anesthesia was induced 1–3 h post feeding and a bolus of [methyl‐3H]methionine was intravenously infused. After 30 min, the liver was analyzed for methyl‐3H incorporation into protein, Cr, phosphatidylcholine (PC) and DNA. Compared to control (P<0.05), Cr and GAA led to a 200% and 700% increase in hepatic Cr concentration, respectively; GAA caused a 400% increase in total hepatic Cr methyl‐3H incorporation. Unlike Cr pigs, GAA supplementation decreased methyl‐3H incorporation into PC and decreased hepatic SAM concentration with no change in SAH, suggesting that creatine synthesis resulted in a limited methyl supply for PC synthesis (P<0.05). Creatine supplementation did not spare methyl groups, but GAA did lead to a limited methyl supply (CIHR).

Grant Funding Source: CIHR
The roles of mitochondrial Src tyrosine kinase and zinc in nitric oxide‐induced cardioprotection against ischemia/reperfusion injury
Authors: Xu Zhelong , Liu Xin , Zhang Lijun , Kang Yi , Lou Jianshi , Zhu Yi
Published: June 2021

While nitric oxide (NO) induces cardioprotection by targeting the mitochondrial permeability transition pore (mPTP), the precise mitochondrial signaling events that mediate the action of NO remain unclear. This study tested if NO induces cardioprotection against ischemia/reperfusion by inhibiting oxidative stress through mitochondrial zinc and Src tyrosine kinase. The NO donor SNAP reduced cell death in rat cardiomyocytes subjected to ischemia/reperfusion and this was abolished by the zinc chelator TPEN and the Src tyrosine kinase inhibitor PP2. SANP also prevented loss of mitochondrial membrane potential (¦¤¦·m) at reperfusion, an effect that was blocked by TPEN and PP2. SNAP increased mitochondrial zinc and enhanced mitochondrial Src phosphorylation in a zinc‐dependent manner. SNAP inhibited both mitochondrial complex I activity and ROS generation at reperfusion through zinc and Src tyrosine kinase. Finally, the antiinfarct effect of SNAP was abrogated by TPEN and PP2 applied at reperfusion in isolated rat hearts. In conclusion, NO induces cardioprotection at reperfusion by targeting mitochondria through attenuation of oxidative stress resulted from inhibition of complex I at reperfusion. Activation of mitochondrial Src tyrosine kinase by zinc may account for inhibition of complex I.
Mechanical Tissue Resuscitation (MTR) activates the RISK pathway and widens the therapeutic window for treating acute myocardial infarction
Authors: Jordan James E. , Pereira Beatriz D. , Morykwas Michael J. , Lane Magan R. , Mays Jennifer J. , Colmer Gerard , Hollingsworth Charlotte , Argenta Louis C.
Published: June 2021

Background

Reperfusion injury results in the continued loss of myocardial cells following revascularization of ischemic myocardium. Therapeutics which decrease myocardial cell death may improve the morbidity and mortality associated with AMI. MTR has been demonstrated to reduce infarct size when initiated at the time of reperfusion. Here we hypothesize that this therapeutic approach remains efficacious even when the initiated further into reperfusion.

Methods

A left, anterior free‐wall ischemic lesion (85 min.) was created using reversible tourniquets in swine. Animals were randomized to receive either no treatment (Control) or MTR (−125 mmHg) beginning 90 min. after reperfusion. Following reperfusion, infarct size (IS), PMN accumulation and activation of the RISK salvage pathway were assessed.

Results

MTR reduced IS (24.2±4.8% vs. 50.3±2.7%, AN/AAR, p< 0.001) with no difference in the AAR (p=0.88). PMN accumulation within the AAR was also reduced by MTR (19.7±2.5 vs. 28.9±4.9 units). MTR increased the phosphorylation of ERK1/2 and AKT in epicardial tissue 2.1‐fold (p<0.01) and 2.0‐ fold (p=0.06), respectively.

Conclusions

MTR provides robust protection from MI/R injury by activating the RISK pathway even when initiated 90 min. into reperfusion. This timing defines a clinically relevant window of opportunity for the treatment of MI/R injury and potentially expands the patient population who could derive benefit from such therapeutic intervention.
Reactive oxygen species in saliva related acute myocardial infarction
Authors: Rubio Maria C , Puntarulo Susana , Gonzalez Paula , Ramos Cecilia , Lewin Pablo , Lifshitz Fima , Friedman Silvia , Nicolosi Liliana
Published: June 2021

Acute myocardial infarction (AMI) is a highly dynamic event, associated with increasing production of reactive oxygen species (ROS). The imbalance between ROS production and antioxidative defenses, leads to a condition known as oxidative stress that may increase cellular damage. We investigated the presence of an oxidative stress biomarker in saliva of patients after AMI. A prospective study was undertaken. AMI was characterized by laboratory (increased serum creatine phosphokinase (CPK) and troponin T), clinical (chest pain >; 20 minutes at rest) and electrocardiographic (changes in ST/T at least in two contiguous leads) criteria. At 24 and 48 hours, 2′7′ dichlorohydrofluorescein diacetate (DCFH‐DA) levels (spectrofluorimetry), a ROS index generation capacity, were assessed in saliva. Fifteen adult patients were admitted to our hospital with AMI and 12 healthy subjects, as controls. AMI patients showed serum CPK24>;CPK48 (t=3.29 p<0.006). In saliva, AMI group showed >; DCFH‐DA levels (t =3.295; p<0.003) that remained unchanged at 48 hours. In this preliminary study, AMI causes an increase of ROS which was detected in saliva by DCFH‐DA assay. Awarded by UBACyT 2011–2014, C001 and 20020100100613.
Novel mitochondrial mechanisms mediate enhanced vasodilation of rat middle cerebral arteries to mitochondrial depolarization following ischemia‐reperfusion injury
Authors: Rutkai Ibolya , Wappler Edina A , Katakam Prasad V.G. , Liu Dan , Busija David W
Published: June 2021

Mitochondrial depolarization leads to vasodilation via activation of SR/ER Ca2+ release. Our objective was to determine the role of mitochondria in vasodilation of middle cerebral arteries (MCA) following ischemia‐reperfusion injury (I‐RI). Male Sprague Dawley rats were subjected to 90 minutes MCA occlusion followed by 48 hours reperfusion. Vasodilation in response to bradykinin (BK), nitroprusside (NTP) and mitochondrial depolarization by diazoxide (DZ, a mitochondrial KATP channel activator) was determined in isolated MCAs by videomicroscopy. Mitochondrial membrane potential (Ψm)was determined by fluorescence microscopy with specific fluoroprobe, TMRE. Western blots determined the expression of markers of mitochondrial biogenesis [Fusion: mitofusins 1 & 2 (MFNs) and Fission: dynamin‐related protein 1 (DRP1)], voltage‐dependent anion channels (VDAC), ER stress (phosphorylated eukaryotic initiation factor 2 α subunit (p‐eIF2α) and neuronal nitric oxide synthase (nNOS). DZ induced vasodilation was enhanced on ipsilateral MCA (iMCA) compared to contralateral (cMCA) whereas BK and NTP responses were similar. Mitochondrial in iMCA displayed increased baseline Ψm and enhanced depolarization to DZ compared to cMCA. Expression of MFNs and VDAC were unchanged, DRP1 was decreased and nNOS and p‐eIF2α were increased in iMCA. Thus, MCAs respond to I‐RI by reduced mitochondrial fission and increasing Ψm leading to increased ROS response to depolarization, ER Ca2+ release and nNOS activation.
Activation of formyl peptide receptors induces relaxation and reduces contraction in resistance arteries
Authors: Wenceslau Camilla Ferreira , McCarthy Cameron G. , Goulopoulou Styliani , Webb R. Clinton
Published: June 2021

Mitochondrial damage‐associated molecular patterns (DAMPs) are endogenous molecules that are released from damaged and dying cells. The innate immune system responds to cellular damage by recognizing DAMPs. One mitochondrial DAMP is formyl peptides (fMLP) that act on formyl peptide receptors (FPR). FPR 1 and 2 are expressed on neutrophils as well as in vascular cells, and promote inflammatory responses. We hypothesized that activation of both receptors increases vascular contractile response. We used mesenteric resistance arteries from 12 weeks old male Wistar rats to assess vascular function. A single dose of fMLP, FPR‐1 agonist (10 or 30μM) and a concentration response curve of lipoxin A4, FPR‐2 agonist (LXA4, 1–300nM) induced relaxation (~30%). Cyclosporine H (FRP‐1 antagonist: CsH, 1μM) inhibited the fMLP response. Also, concentration response curves to acetylcholine (ACh, 0.1nM‐30μM) and phenylephrine (PE, 1nM‐30μM) were performed in the absence or presence of CsH (1 μM), fMLP (10μM) or LXA4 (1nM). Results indicated that fMLP, but not CsH or LXA4, decreased PE contraction [Emax(%KCl), vehicle: 119±6 vs. fMLP: 96±1, p<0.05], however ACh‐induced relaxation was not altered. These data suggest that FPRs have a functional role in resistance arteries and may contribute to vascular dysfunction in pathologies associated with tissue damage such as sepsis or hypertension.

Support: NIH; CNPq; SWHR
Stathmin modulates microtubule dynamics and controls Rho signaling and vascular permeability
Authors: Birukova Anna A , Sarich Nicolene , Wu Tinghuai , Tian Xinyong
Published: June 2021

Crosstalk between microtubules (MT) and actin cytoskeleton is involved in Rho GTPase‐dependent regulation on endothelial (EC) permeability, however precise mechanisms of Rho regulation by MT remain poorly understood. We studied a role of stathmin, the MT‐associated protein controlling MT stability, in the mediation of agonist‐induced permeability and vascular leak. Thrombin treatment of human pulmonary EC induced rapid dephosphorylation of stathmin leading to MT disassembly and activation of feedback signaling by another MT‐bound protein, tau, which further promoted MT disassembly. Inhibition of stathmin activity abrogated thrombin‐induced cytoskeletal remodeling and Rho‐dependent EC hyperpermeability, while expression of a phosphorylation deficient stathmin mutant exacerbated thrombin‐induced EC barrier disruption. Stathmin inhibition preserved the MT network and prevented thrombin‐induced tau‐dependent MT disassembly, and suppressed release of Rho‐specific guanine nucleotide exchange factor, GEF‐H1. Protective effects of stathmin knockdown were observed in vivo in the two‐hit model of ventilator induced lung injury. These results demonstrate the mechanism of stathmin‐dependent control of MT dynamics and suggest novel potential pharmacological interventions in the prevention of increased vascular leak via modulation of stathmin activity. Funding: NHLBI HL089257, HL107920
Novel Rap1‐dependent regulator of endothelial cell junctions afadin mediates protective effects by oxidized phospholipids in the models of acute lung injury
Authors: Birukov Konstantin G , Sarich Nicolene , Dubrovskyi Oleksii , Zebda Noureddine
Published: June 2021

Afadin has been described as a regulator of epithelial cell junctions assembly. However, its role in control of vascular permeability remains obscure. We have previously described protective effects of oxidized phospohlipids (OxPL). This study examined a role of afadin and its upstream regulator Rap1 GTPase in the OxPL‐induced cell junction assembly as a novel mechanism of endothelial cell barrier preservation. OxPL induced Rap1‐ dependent afadin translocation to the cell periphery and promoted accumulation of adherens junction and tight junction proteins at the cell membrane. OxPL also stimulated Rap1‐dependent afadin interactions with adherens junction and tight junction proteins p120‐catenin and ZO‐1, respectively. SiRNA‐induced knockdown of afadin or Rap1 suppressed endothelial barrier enhancement caused by OxPL and abolished barrier protective effects of OxPL against thrombin‐induced endothelial permeability. Similar effects were obtained in experiments with ectopic expression of afadin mutant lacking Rap1 GTPase binding domain or inhibitor of Rap1 activity, Rap1‐GAP. Afadin or Rap1 siRNA‐mediated knockdown also abolished protective effects of OxPL against acute lung injury in vivo. These results demonstrate for the first time a critical role of afadin in the regulation of vascular barrier function in vitro and in vivo.

Funding: NHLBI HL76259, HL089257, HL107920
Basal expression of VEGF does not correlate with loss of skeletal muscle capillarity with detraining
Authors: Olenich Sara , Roberts Kathleen , Audet Gerald , Olfert I. Mark
Published: June 2021

Temporal expression of positive and negative angiogenic factors in response to training and detraining is still poorly described. Protein expression of anti‐angiogenic thrombospondin‐1 (TSP‐1) and endostatin as well as pro‐angiogenic vascular endothelial growth factor (VEGF), matrix metalloproteinases ‐2, ‐9 (MMP‐2, MMP‐ 9), and nucleolin (a nuclear protein involved with synthesis and maturation of ribosomes) were examined after training and detraining in mouse gastrocnemius muscle. Mice were placed in one of 5 groups (n=12/group): control (did not run), trained (voluntary wheel ran 21 days), or detrained (21 day trained, then detrained) for 7, 14, or 28 days. When compared to control mice, training increased capillary‐to‐fiber ratio (C:F) and resulted in an increase of basal VEGF and MMP‐9 (p<0.05), all other factors remained unchanged. After 7 days of detraining, C:F returned to control levels, basal VEGF remained elevated and TSP‐1 increased (p<0.05). Following 14 days of detraining, basal VEGF levels were still elevated, TSP‐1 fell to baseline, and nucleolin declined compared to controls (p<0.05). After 28 days of detraining, basal VEGF returned to baseline, TSP‐1 remained unchanged while endostatin and MMP‐2 levels decreased compared to controls (p<0.05). These data suggest that an increase in TSP‐1 and a decrease in MMP's and nucleolin, despite elevated VEGF, correlate with detraining losses of skeletal muscle capillarity. Supported by NIH 5P20RR016477; NIH 5T32HL090610; AHA 10BGIA3630002
Effects of combined aerobic and resistance exercise training on apoptosis and autophagy of CD4+ lymphocytes in sedentary men
Authors: Lin Yi‐Huei , Wang Jong‐Shyan
Published: June 2021

Physical exercise may influence CD4+ lymphocyte activity and potentially modulate adaptive immune function. This study investigated how combined aerobic and resistance exercise training influenced apoptosis and autophagy of CD4+ lymphocytes. Twelve healthy sedentary males were trained on a bicycle ergometer (60%VO2max for 30 minutes) for 3 days/week and an isokinetic dynamometer (alternating 60°–180°/s for 6 to 10 sessions) for 2 days/week for 6 weeks. Apoptotic and autophagic responses in CD4+ lymphocyte were determined by monitoring corresponding biological markers. The experimental results demonstrated that strenuous, acute exercise simultaneously lost mitochondrial membrane potential, activated caspase‐3, and up‐regulated LAMP‐2, which were accompanied by the increases in exposure of phosphatidylserine (PS) and staining of acridin orange (AO) in CD4+ lymphocytes. However, combined aerobic and resistance exercise training decreased CD4+ lymphocyte LAMP‐2 content, AO staining, and PS exposure at rest status. Moreover, this training regimen also suppressed the extent of increased PS exposure by intense exercise. Therefore, we conclude that strenuous, acute exercise facilitates CD4+ lymphocyte autophagy and apoptosis, whereas combined aerobic and resistance exercise training attenuates apoptotic activation of CD4+ lymphocyte induced by vigorous exercise.
Hospital‐based supervised aerobic training effectively improves ventilation efficiency and cardiac output power in patients with systolic heart failure
Authors: Ho Ching‐Wen , Huang Shu‐Chun , Cheng Shu‐Chuan , Chan Chih‐Hsiang , Wang Jong‐Shyan
Published: June 2021

This study investigated the effects of hospital‐based supervised aerobic training on ventilation efficiency and cardiac output power in patients with systolic heart failure (SHF). Thirty‐three patients with SHF who completed hospital‐based supervised aerobic training for 36 sessions were retrospectively collected. The former 18 sessions and the latter 18 sessions were performed at moderate continuous training (constant 60%VO2peak) and aerobic interval ntraining (3‐minute intervals at 40% and 80%VO2peak) for 45 minutes, respectively. Thirty‐three SHF patients who only received community‐based exercise program education were gathered as control group. The results showed that hospital‐based supervised aerobic training 1) increased work‐rate and VO2 at ventilation threshold and peak exercise, 2) reduced the value of VE/VO2 during submaximal exercise, 3) enhanced cardiac output power at peak exercise, and 4) lowered the score of Minnesota Living with Heart Failure Questionnaire (MLHFQ). Despite modestly lowered MLHFQ score, no significant changes in ventilatory/hemodynamic responses to exercise were observed in control group. Hence, we conclude that hospital‐based supervised aerobic training in patients with SHF effectively improves ventilation efficiency and cardiac output power at submaximal and peak workloads, respectively.
The Changes of Lower Limb Muscle Function and Gait with Application of Aquatic Rehabilitation Exercise in Korean Stroke Patients
Authors: Jin Haeng Mi , Jeon In Gon , Kim Hee Eun
Published: June 2021

The purpose of this study was to investigate the effect of aquatic lower limb muscle strengthening program on lower limb muscle function and gait in Korean hemiplegia stroke patient. Subjects were 20 hemiplegia stroke patients aged from 40 to 60 and they participated in the aquatic lower limb muscle strengthening program during 12 weeks. The exercise intensity was 12–16 (50–85% VO2max) RPE. Gait analysis, lower limb function mobility, balance ability, lower limb muscle were measured before and after the exercise. Collected data were analyzed by paired t‐test and independent t‐test with using SPSS window 18.0 version. After aquatic lower limb muscle strengthening program, gait distance was increased (P<0.001) and gait course angle was decreased (P<0.05). Lower limb function mobility (P<0.05), balance ability (P<0.001), lower limb muscle (P<0.001) were also increased after the program. Interestingly, after aquatic lower limb muscle strengthening program, left hemiplegia stroke patients presented higher increase in lower limb muscle strength than right hemiplegia stroke patients (P<0.05). In conclusion, aquatic lower limb muscle strengthening program improves gait and lower limb function in Korean hemiplegia stroke patients. Therefore, aquatic lower limb muscle strengthening program may be considered as proper rehabilitation exercise program for stroke patients.
Endurance training promotes upregulation in microRNA‐206 on blood and in human skeletal muscle
Authors: Alves Cleber Rene , Junior Jose Ribeiro Lemos , Alves Guilherme Barreto , Negrão Carlos Eduardo , Oliveira Edilamar Menezes
Published: June 2021

Skeletal muscle is a highly plastic organ, capable of altering phenotype in response endurance training (ET). MicroRNAs (miR) are a class of short, non‐coding RNA molecules that reportedly play a central role in regulating post‐transcriptional. Therefore, we evaluated the effects of ET on the blood and in human skeletal muscle in miRNAs‐206 levels.

Twelve healthy volunteers were biopsied in vastus lateralis muscle and collected blood sample. Heart rate (HR), mean blood pressure (MBP), maximal exercise capacity (VO2peak), forearm blood flow (FBF) and forearm vascular conductance (FVC) were evaluated. MiR 206 levels were analyzed by real‐time PCR. Endurance training was performed for 16 weeks. After ET, the individuals showed an increase of 97% in skeletal muscle and 128% on blood in miR 206 levels. These alterations were accompanied by decreased in resting bradycardia (12,5%), increased of VO2peak (18%), FBF (68%) and FVC (63%).

These results show that in healthy subjects the miR‐206 expression is up regulated after endurance training paralleled with improved of vascular function and endurance capacity, suggesting miR‐206 may have a role on metabolism related with high performance.
Dietary nitrate supplementation augments cutaneous reflex vasodilation in healthy humans
Authors: Levitt Erica Lynne , Keen Jeremy Thomas , Wong Brett James
Published: June 2021

Beetroot juice (BRJ) has been shown to increase NO‐dependent dilation through both NOS‐dependent and independent pathways. We hypothesized BRJ supplementation would augment reflex cutaneous active vasodilation. Subjects were equipped with two microdialysis fibers on the forearm and randomly assigned as control (Ringer's) or NOS inhibition (20mM L‐NAME). Whole‐body heating was achieved via water‐perfused suits to raise core temperature (Tc; ingestible telemetric pill) 0.8°C. Maximal cutaneous vasodilation was reached by administering 54mM SNP and local heating to 43°C. Skin blood flow was measured via laser‐Doppler flowmetry and mean arterial pressure was measured; cutaneous vascular conductance (CVC) was calculated and expressed as %CVCmax. Subjects underwent heat stress pre‐ and post‐nitrate supplementation (3 days of BRJ: 5mM, .45g nitrates per day). BRJ increased the plateau CVC at control (pre: 55±3 vs. post: 77±5 %CVCmax) and L‐NAME (pre: 27±3 vs. post: 41±6 %CVCmax; p<0.05 for all conditions) sites. The %NO contribution increased from pre‐ (47±2 %CVCmax) to post‐BRJ (71±4 %CVCmax; p<0.05). These data suggest BRJ augments NO‐dependent component of reflex cutaneous vasodilation.
Microvascular function is reduced in normotensive salt‐sensitive individuals independent of dietary sodium intake
Authors: DuPont Jennifer J , Greaney Jody L , Matthews Evan M , Lennon‐Edwards Shannon L , Farquhar William B , Edwards David G
Published: June 2021

Impairments in microvascular (MV) function are thought to occur early in cardiovascular disease development. We sought to determine if MV function is altered in salt‐sensitive (SS) adults compared to salt‐resistant (SR) adults during low (LS) and high (HS) sodium diets. Seven healthy SS adults and 7 healthy SR adults were studied (SS: 2M, 5F; 48±4 yrs; SR: 2M, 5F; 45±3 yrs). After a run‐in diet, subjects were randomized to a 7 day LS (20 mmol/d) and 7 day HS (300–350 mmol/d) diet (controlled feeding study). Salt sensitivity was defined as a >; 5 mmHg change in 24‐hour mean arterial BP (MAP) from the LS to HS diet. MV function was assessed using laser Doppler flowmetry during local heating (42°C). Cutaneous vascular conductance (CVC) was calculated as red blood cell flux/MAP and all data were expressed as a percentage of the maximum CVC (28 mM SNP, 43°C). 24 hour MAP (mmHg) increased in the SS group between diets, but was unchanged in the SR group (SS: LS: 85±2, HS: 92±3, p<0.05; SR: LS:83±3, HS: 83±2, p>;0.05). Plateau %CVCmax was impaired in the SR group on the HS diet compared to the LS diet (80±2 vs. 90±2%; p<0.05). There were no differences in plateau %CVCmax in the SS group between LS and HS diets (80±4 vs. 82±3%; p>;0.05) but both were impaired (p<0.05) compared to the SR group on the LS diet. These data suggest that MV function is impaired in SS adults independent of dietary sodium intake. Supported by 2 P20 RR016472‐11 & R01 HL 104106.
Endurance training changes the DNA methylation pattern in human skeletal muscle
Authors: Lindholm Malene Elin , Marabita Francesco , Gomez‐Cabrero David , Fischer Heléne , Tegnér Jesper , Sundberg Carl Johan
Published: June 2021

Long‐term endurance training induces many beneficial effects in human skeletal muscle. However, some people respond strongly while others barely improve. Training responsiveness is related to differential gene expression through incompletely understood mechanisms. We hypothesized that DNA methylation pattern changes with endurance training and that these changes could be related to training response.

17 subjects (males and females) performed three months of supervised one‐legged knee extension endurance training. DNA from vastus lateralis muscle biopsies was used for an Illumina 450K chip. Training response was evaluated using one‐legged performance tests and enzyme activity assays.

The Illumina data showed around 5000 sites that significantly changed in methylation level with training. Using methylation estimates for these sites, two responder groups were identified with different magnitude of change in citrate synthase activity. It was also possible to separate the males and females based on methylation level of these sites (excluding the sex chromosomes), a finding of great interest for the difference between male and female skeletal muscle.

In conclusion, our results indicate that DNA methylation is affected by endurance training and could be one mechanism involved in the variability in response.
Maternal Exercise During Pregnancy Improves Glucose Metabolism of Offspring
Authors: Stanford Kristin I. , Lee Min‐Young , Getchell Kristen M. , Hirshman Michael F. , Goodyear Laurie J.
Published: June 2021

The intrauterine environment during pregnancy is a critical factor in the development of diabetes and obesity in offspring. To determine the effects of maternal exercise during pregnancy on the metabolic health of offspring, 6wk old C57BL/6 virgin female mice were fed a chow or high‐fat (HF) (60%) diet and divided into 4 sub‐groups: Trained (housed with running wheels for 2wks preconception and during gestation); Pre‐Pregnancy Trained (housed with running wheels for 2wks pre‐conception); Gestation Trained (housed with running wheels during gestation); or Sedentary (static cages). Breeders were sedentary and chow‐fed. Offspring were sedentary, chow‐fed and studied for 12 months. From 6 months on, male offspring from Trained dams had decreased serum insulin concentrations, improved glucose tolerance, and decreased % body fat vs. all other groups. This improved metabolic profile occurred in offspring of both chow‐ and HF‐fed dams. Female offspring had similar responses. 12 month old offspring from Trained dams had significant changes in hepatic expression of key genes regulating Krebs cycle activity and fatty acid oxidation, suggesting liver adaptations are central for this effect. In conclusion, maternal exercise before and during pregnancy in mice has profound effects to improve the metabolic health of offspring. Thus, maternal exercise may combat the increasing rates of diabetes and obesity.
Sympathetic nerve activity response to acute intermittent hypoxia (AIH) and development of sympathetic long‐term facilitation (LTF)
Authors: Fong Angelina Y , Hammond Andrew M , Xing Tao , Pilowsky Paul M
Published: June 2021

Recent studies found that AIH produces LTF of splanchnic sympathetic nerve activity (sSNA), as well as the well‐described respiratory LTF. This study aimed to further characterize sympathetic LTF in urethane‐anesthetized, vagotomised, ventilated male Sprague‐Dawley rats. Hypoxia (10% O2, 45s) elicited increase in phrenic burst frequency and amplitude, sympathoexcitation (235.1 ± 3%) with a corresponding pressor effect (+31.4 ± 4mmHg), and a depressor response on return to the hyperoxia (−16.5 ± 4mmHg). Repeated hypoxia (10x, 5 mins apart) produced consistent respiratory stimulation, depressor and sympathoexcitation. Sympathetic‐respiratory coupling is also unchanged between the 10 stimuli. Total SNA power (0–20Hz) increased during the hypoxic stimulation, but the ratio of cardiac and respiratory frequency components was unchanged. The correlation between the sympathoexcitation and the pressor response declined with repeated hypoxia (Stim 1: +31.4 ± 4mmHg, vs Stim 10: +10.0 ± 2mmHg), indicating adaptation of the vasculature or the neuroeffector junction. At 60 mins after AIH, sSNA was elevated by 60±12.7% (n=4) above baseline. The pattern of respiratory‐sympathetic coupling, total power and the frequencies correlating to the cardiac and respiratory components in the sSNA was unchanged. These data support the idea that sympathetic LTF is due to increase in sympathetic tone. Funding: ARC, NHMRC, MQ.
Ventilatory instability persists 1 week after lipopolysaccharide (LPS) exposure in newborn rat pups.
Authors: Mason Ariel Vanessa , Kostuk Eric , Shirahata Machiko , Gauda Estelle B
Published: June 2021

Premature infants are frequently exposed to prenatal and postnatal infections, and have persistent and intractable apnea. Since pro‐inflammatory cytokines affect neural mechanisms that control breathing, we hypothesized that LPS given to rat pups at postnatal day 2 (P2 ) would induce unstable breathing that would persist beyond the acute inflammatory episode. Sprague Dawley littermates (n=8, each group) were given either LPS (50–100mcg/kg) or saline, intraperitoneally at P2, and at P9 ventilation was measured with plethysmography and the inter breath interval (IBI) was calculated at baseline and in response to hypoxia and hyperoxia. At baseline, LPS animals had a 3 fold greater number of breaths with an IBI >;0.6 secs in comparison to control animals (6±2 vs 2%, p<0. 01), which persisted during hyperoxia (16±3%vs6±1%, p<0.02). Hypoxia shortened the IBI in both groups with <2% of breaths with a IBI >;0.6 secs. From hypoxia to hyperoxia there was an 14±3% increase in breaths with IBI >;0.6 secs in LPS animals vs a 5±2% increase in control animals (p<0.03). 70±6% and 53±11% of the breaths had IBI <0.35 secs in control and LPS animals, respectively. These data suggest that LPS exposure in newborn rat pups reconfigures the respiratory network leading to breathing instability at baseline and in response to hyperoxia that persist for at least 1 week after the acute inflammatory event. Support: HL81345, R25DA021630
Hypoglycaemia‐induced hyperpnoea: a role for epinephrine and the carotid body?
Authors: Thompson Emma L , Ray Clare J , Coney Andrew , Kumar Prem
Published: June 2021

Hypoglycaemia in vivo induces a carotid body‐dependent increase in ventilation suggested to reflect a direct action of reduced blood glucose on the CB, but an indirect effect via an action of counter‐regulatory hormones, including epinephrine, has not been discounted. Ventilation (VE) was recorded during insulin‐induced hypoglycaemia (2.9±0.08mM) and during normoglycaemic epinephrine infusion (1μg.kg‐1.min‐1), in Alfaxan‐anaesthetized, male Wistar rats. All procedures were carried out in accordance with current UK legislation. Epinephrine mimicked the increased VE (ca.15–20%) observed during hypoglycaemia, without significant change in PaCO2 in either condition. Hyperoxia reduced baseline VE and decreased the VE response to both stimuli. Propranolol reduced baseline VE, reduced the VE response to epinephrine and abolished the VE response to hypoglycaemia, with concomitant increase in PaCO2 in both conditions. Hyperoxia and propranolol in combination produced additive effects. Both hypoglycaemia and epinephrine increased the hypercapnic ventilatory response, which was blocked by propranolol. These data show that the increased VE observed in hypoglycaemia is, in large part, a hyperpnoea associated with a β‐mediated, but O2‐independent effect of epinephrine. We propose that hypoglycaemia acts to increase VE via an epinephrine‐mediated change in CO2 chemosensitivity in the carotid body.
Two potassium‐channel blockers differently affect afferent signal of avian intrapulmonary chemoreceptors
Authors: Lonjaret Jean‐Guillaume , Lee Sheridan Krystal , Hemplema Steven C
Published: June 2021

We investigated the roles of IA and IDR potassium channels in action potential generation of avian intrapulmonary chemoreceptors (IPC). IPC are CO2‐sensing vagal afferents in avian lungs, with fast and inverse response to airway CO2 partial pressure (PCO2). Discharge frequency of a single cell IPC was recorded in 11 anesthesized, ventilated ducks. FICO2 was electronically controlled. Intravenous injection of voltage‐gated potassium‐channel blockers 4‐aminopyridine (4AP) or tetraethylammonium (TEA) disrupted the response to CO2. 4AP blocks the transient K current (IA), and TEA blocks the delayed rectifier current (IDR). A dose of 7 mg/kg of 4AP eliminated both phasic and tonic IPC sensitivities to FICO2, resulting in constant high‐frequency AP firing (p < 0.05). Low dosages of TEA had no effect on that sensitivity, although at high TEA doses (>; 20 mg/kg) the phasic (p < 0.05) but not tonic response to a step change in FICO2 disappeared. Our results suggest that transient voltage gated K channels (affected at low dosages of 4AP and high dosages of TEA) are involved in setting resting membrane potential in IPC. If IA in IPC is increased by CO2/H+ as it is in slowly adapting mammalian pulmonary stretch receptors, IA channel modulation by CO2/H+ may also underlie CO2 signal transduction in avian IPC. Support: NIH 2R15HL087269
Brain‐derived erythropoietin modulates the hypercapnic ventilatory response during early postnatal life
Authors: Soliz Jorge
Published: June 2021

Apart from neuroprotective roles against stroke, erythropoietin (Epo) in brain (i.e. Epo synthesized in neurons and astrocytes) modulates the ventilatory response to hypoxia of mice and human. This effect was attributed to the presence of Epo receptors (EpoR) in neurons involved in respiratory rhythmogenesis (e.g., the pre‐Bötzinger complex), sensory integration (nucleus tractus solitarius) and in catecholaminergic neural populations (A6 and A5 in the pons; A2/C2 and A1/C1 in the medulla oblongata). Apart from hypoxia however, central and peripheral chemoreceptors regulate the ventilatory response to carbon dioxide. In this study we used C57Bl6 mice at postnatal ages 10, 15 and 21 days, to test the hypothesis that brain‐derived Epo impacts the hypercapnic ventilatory response during the postnatal development. Animals were injected (i.p.) with Epo (500 U/mouse) 24h before experimentation. Subsequently, baseline ventilation (normocapnia; 5 min), hypercapnic ventilatory response (5% CO2, 21% O2 in N2; 5 min), and recovery (normocapnia; 5 min) were evaluated by whole body plethysmography. Our preliminary results show that Epo increases the hypercapnic ventilatory response at P10, has no effect at P15, and decreases the hypercapnic ventilatory response at P21. Furthermore, by using antibodies to paired‐like homeobox 2b (Phox2b), Epo and Epo receptor were localized to the retrotrapezoid nucleus (RTN) at postnatal day 10. These novel findings imply that Epo plays a key‐regulating role in the neural response to carbon dioxide in a period in which the central respiratory network undergoes full development and maturation.
Pulmonary neuroendocrine H146 cells as hypercapnic sensors
Authors: Ludlow Katherine , Buttigieg Josef
Published: June 2021

Singular pulmonary neuroendocrine cells (pNEC) or clusters called pulmonary neuroepithelial bodies (NEBs) are widely distributed within the airway mucosa of human and mammalian lungs. NEB cells produce amines (e.g. 5‐HT), and are thought to modulate the control of breathing in response to low O2 conditions (hypoxia). The mechanism by which these cells detect hypoxia is thought to occur via activity of NADPH oxidase in conjunction with K+ channels. Exposure of NEBs to hypoxia causes K+ channel closure, membrane depolarization and 5‐HT release. In other chemosensory organs (e.g. the well studied carotid body) the sensory cells are often polymodal, capable of responding to high CO2 levels (hypercapnia) and/or hypoxia (usually via different mechanisms). However it is currently unknown whether NEBs can sense hypercapnia in addition to hypoxia. Using molecular biology, immunocytochemistry, and electrophysiology we explore whether H146 cells (a model cell line of pNEC/NEB) are capable of responding hypercapnia. In other hypercapnic sensing organs (e.g. adrenal chromaffin cells), carbonic anhydrase 2 (CA II) is thought to play a role in mediating the hypercapnic response. Although native NEB do express this isoform (among several others), CA II is not expressed in H146 cells. Nevertheless they do respond to hypercapnia via membrane depolarization and 5‐HT release, as determined using electrophysiology. This finding suggests that another isoform of CA or alternate mechanism may be involved in mediating hypercapnic sensitivity in H146 cells. Funded by: NSERC Discovery Grant
Medullary raphé transcriptome comparisons among inbred rat strains differing in ventilatory sensitivity to CO2
Authors: Hodges Matthew Robert , Yang Chun , Mouradian Gary Charles , Liu Pengyuan , Liang Mingyu
Published: June 2021

Brown Norway (BN) rats exhibit reduced ventilatory CO2 sensitivity relative to the Dahl salt‐Sensitive (SS) rats. The medullary raphé nuclei contain serotonergic (5‐HT) neurons, which are putative CO2 chemoreceptors and provide neuromodulatory input to respiratory control nuclei. Here we tested the hypothesis that the reduced CO2 sensitivity in the BN rat is due in part to altered medullary raphé gene expression using mRNA sequencing and qPCR validation. cDNA libraries (n=3/strain, 3–4 rats/library) were prepared from caudal and rostral raphe punches from adult BN (n=11) and SS (n=11) rats and sequenced. We found 74 differentially expressed (p=0.005, q<0.25) genes between caudal and rostral raphe in both strains, which pointed to the caudal raphe having a greater capacity for excitatory neuromodulation. Strain differences were also detected in multiple 5‐HT‐specific genes in the rostral, but not caudal medullary raphé, predictive of a reduced quantity of monoamines in the BN rat by bioinformatics pathway analyses. Brain tissue HPLC verified this as 5‐HT and norepinephrine levels in multiple brain regions including the medulla were lower in the BN despite equal numbers of 5‐HT‐producing neurons. The expression data suggest altered gene regulation and perhaps function of rostral raphe 5‐HT neurons in BN rats relative to SS rats, which may contribute to the reduced ventilatory CO2 sensitivity in BN rats.
Transcriptional mechanisms that mediate upregulation of thrombospondin‐1 expression by leptin in vascular smooth muscle cells
Authors: Sahu Soumyadip , Chavez Ronaldo , Ganguly Rituparna , Raman Priya
Published: June 2021

Hyperleptinemia is an important pathophysiological trigger for development of atherosclerotic complications. However, molecular mechanisms activated by leptin that modulate vascular gene expression are poorly understood. We recently reported a novel regulatory effect of leptin via JAK2/ERK/JNK signaling on thrombospondin‐1 (TSP‐1), a potent proatherogenic and antiangiogenic matricellular protein, in human aortic smooth muscle cells (HASMC). The goal of this study was to explore transcriptional mechanism(s) that mediate TSP‐1 upregulation by leptin. Promoter deletion analysis revealed a long fragment (−1270/+750) that is required for activation of TSP‐1 promoter by leptin in HASMC. Using protein/DNA array (Panomics), we identified several potential transcription factors (TFs: GAS/ISRE, IRF1, STAT1/3, CREB) activated by leptin, with predicted binding sites in distal fragment of TSP‐1 promoter determined by MatInspector (Genomatix). These results were further confirmed in EMSA where leptin increased IRF1 and CREB binding activity to TSP‐1 promoter. Additionally, JAK2‐, ERK‐, and JNK‐specific inhibitors significantly attenuated activation of IRF1 and CREB‐binding protein complexes in leptin treated cells. These data suggest that specific TFs such as IRF1 and CREB mediate leptin induced TSP‐1 transcription which may contribute to vascular complications associated with hyperleptinemia.
TNFα receptor 1 causes endocytosis‐dependent NF‐κB and ‐ independent JNK activation in vascular smooth muscle cells
Authors: Choi Hyehun , Lamb Fred S
Published: June 2021

Tumor necrosis factor‐α (TNFα), a pro‐inflammatory cytokine can cause either vascular smooth muscle cell (VSMC) apoptosis (JNK‐dependent) or proliferation (NF‐κB‐dependent). We hypothesized that TNFα receptor 1 (TNFR1) endocytosis balances these two pathways. We quantified NF‐κB activation and MAPK (JNK, p38, ERK1/2) phosphorylation by TNFα in the presence of siRNA targeting TNFR1 or 2 in cultured murine mesenteric VSMC. Both NF‐κB activation (Fig. 1, n=3–4) and JNK phosphorylation are TNFR1‐dependent, and NF‐κB activation is potentiated by JNK inhibition (SP600125, 3μM). Impairing endocytosis (Dynasore, 15μM) reduced NF‐κB activation by TNFα (1.5‐fold, n=5) and prevented this effect of SP600125 (2.2‐fold, n=4). TNFα also caused phosphorylation of p38 and ERK1/2. JNK and ERK1/2 phosphorylation were TNFR1‐dependent while p38 activation was altered by both TNFR1 and 2 siRNA. Thus, TNFR1 activates both NF‐κB and JNK activation and JNK modulates endocytosis‐dependent NF‐κB activation in VSMC.
Implication of lysophosphatidic acid in hypoxic pulmonary vascular remodeling: effects on cell adhesion and migration
Authors: Shlyonsky Vadim , Naeije Robert , Mies Frédérique
Published: June 2021

Pulmonary hypertension is characterized by cellular and structural changes in vascular wall of pulmonary arteries. Intimal thickening, fibrosis, medial hypertrophy, adventitial fibroblast activation involve progenitors cells and fibrocytes. We hypothesized that lysophosphatidic acid (LPA), a bioactive lipid, is implicated in this vascular remodeling. We investigated the rat model of hypoxic pulmonary hypertension. Chronic hypoxic exposure induced increase of serum levels of circulating LPA (21.6 (11.0 – 42.3) μM vs 40.9 (23.4 – 71.7) μM, p=0.037). In addition, we observed perivascular LPA immunohistochemical staining in lungs of hypoxic rats (antibody against LPA provided by L‐Path Inc.), which co‐localized with lysophospholipase D. Serum from the hypoxic group of animals had significantly higher chemoattractant properties towards rat primary lung fibroblasts, and this increase in cell migration could be prevented by the LPA receptor 1 and 3 antagonist, VPC12249. LPA increased also adhesive properties of human pulmonary artery endothelial cells and of human peripheral blood mononuclear cells via the activation LPA receptor (1 or 3). We suggest that chronic hypoxia increases circulating and tissue level of LPA which might induce fibroblast migration and recruitment of mononuclear cells in pulmonary vasculature that contributes to pulmonary vascular remodeling.
Effects of hypoxia on regulators of G‐protein signaling 2 (RGS2) regulation of pulmonary arterial constriction
Authors: Jain Neha , Tu Yaping , Abel Peter W
Published: June 2021

RGS2 protein inhibits Gq‐coupled G protein coupled receptor (GPCR) signaling. Our goal was to determine whether hypoxia reduces RGS2 in pulmonary arterial smooth muscle causing enhanced GPCR agonist calcium (Ca) signaling and constriction. Human pulmonary arterial smooth muscle cells (SMC) and RGS2 KO and WT mice were used. RGS2 protein was knocked down by RGS2 siRNA. SMC were incubated in hypoxia (1% O2) or normoxia (21% O2) for 1–48 hrs. We measured RGS2 expression by western blotting, Ca mobilization with Ca dyes and constriction of pulmonary arteries and SMC by video morphometry. RGS2 siRNA caused knockdown (KD) of RGS2 by 68%±1 in SMC. Ca mobilization for U46619 and endothelin1 (ET‐1) was 2‐fold higher for RGS2 KD compared to control and constriction by ET‐1 and U46619 was increased by 21%±2 and 14%±2, respectively. Pulmonary arteries of lung slices from RGS2 KO mice had increased constriction to serotonin (26%±4) and U46619 (20%±4) compared to WT. Hypoxia significantly reduced RGS2 at 8 (54%±9), 24 (59%±8) and 48 (71%±6) hours in SMC. 48 hours of hypoxia increased constriction for ET‐1 and U46619 by 19%±3 and 21%±2, respectively while Ca was increased 2‐fold for both agonists. We found that RGS2 plays a role in GPCR constriction of SMC and pulmonary arteries. Hypoxia downregulates RGS2 causing augmented Ca signaling and constriction which may contribute to pulmonary hypertension. Support: Am Heart Asso; Am Asthma Fdn
Characterization of ryanodine receptor‐gated store‐operated calcium entry in rat pulmonary artery smooth muscle cells
Authors: Lin Amanda H. Y. , Sun Hui , Jiang Yong‐liang , Lin Mo‐jun , Sham James S. K.
Published: June 2021

Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs) involves interactions between Ca2+ influx and release pathways. Depletion of sarcoplasmic reticulum (SR) Ca2+ content activates the Ca2+ sensor, stromal interacting molecule (STIM), which induces store‐operated Ca2+ entry (SOCE). Previous studies on SOCE focused on inositol 1,4,5‐trisphosphate receptor (IP3R)‐ related pathways and how ryanodine receptor (RyR)‐gated store regulates SOCE remained largely unknown. Here we characterized the RyR‐gated SOCE in rat PASMCs. Intracellular Ca2+ was measured in the presence of 1 μM nifedipine to prevent voltage‐dependent Ca2+ entry and extracellular Ca2+ was introduced to induce SOCE after 20‐minute store depletion in Ca2+‐free environment. The RyR agonist, 4‐chloro‐m‐cresol (4‐CmC; 1mM), induced SOCE in rat PASMCs. This Ca2+ response was blocked by a low concentration of the SOCE inhibitor, Gd3+ (1 μM). The RyR1 and RyR3 inhibitor, dantrolene (30 μM), did not inhibit 4‐ CmC‐induced SOCE. In addition, maurocalcine (400 nM), a RyR1 agonist, failed to induce RyR‐gated Ca2+ release in rat PASMCs and hence did not activate SOCE. These data shows that RyR2 is the major RyR subtype that mediates SOCE in rat PASMCs. (This project is supported by NIH grant NL‐071835)
Elevated peripheral blood mononuclear cell‐derived superoxide production in healthy young black men
Authors: Deo Shekhar H , Holwerda Seth W , Keller David M , Fadel Paul J
Published: June 2021

Several studies have demonstrated that blacks exhibit elevations in plasma oxidative stress. However, the sources and mechanisms contributing to the elevation in oxidative stress remain unclear. Given recent work indicating that peripheral blood mononuclear cells (PBMCs) can be a major source of angiotensin II type 1 receptor (AT1R)‐NADPH oxidase derived superoxide production, we sought to examine this pathway in mediating the increase in systemic oxidative stress in black men. PBMCs were isolated from whole blood in 8 black and 8 age‐matched white men. Intracellular superoxide production was measured using dihydroethidium fluorescence and protein expression of AT1R and NADPH oxidase subunit, gp91phox were assessed using western blot. Black men showed elevated resting intracellular superoxide production (7.5±1.7 blacks vs. 3.1±1.1 whites, RFU; P<0.05) as well as protein expression for AT1R and gp91phox (P<0.05 vs. whites). Furthermore, for all subjects, AT1R expression was related to gp91phox (R = 0.67; P<0.05). These preliminary findings suggest that young black men exhibit greater resting PBMC derived superoxide production potentially via an up‐regulation of the AT1R‐NADPH oxidase pathway.

Supported by NIH Grant R15HL096103 (DMK) and RO1 HL093167 (PJF)