bluuebunny/crossref_metadata_2025 huggingface dataset:
Antenatal Protein Deprivation in the Mouse: Epigenetic Changes and Developmental Origin of Hypertension in Adult
Authors: Goyal Ravi , Leitzke Arthur , Goyal Dipali , Longo Lawrence D.
Published: June 2021

As a consequence of antenatal maternal low protein diet (AMLPD), studies report changes in the renin‐angiotensin system (RAS), which may be involved in fetal programming of hypertension. We tested the hypothesis that AMLPD may cause epigenetic modifications and altered miRNA regulation of the RAS genes leading to developmental programming of hypertension in the adult mice offspring. We administered isocaloric, normal, 50% (moderate AMLPD) and 33% (severe AMLPD) diet, starting 1 week before mating and continued until delivery of the pups. We measured blood pressure (BP), promoter DNA methylation, mRNA, miRNA, proteins from the mice brain at fetal, 3 weeks and 33 weeks of age. Birth weight was significantly reduced, but there was a rapid catch‐up growth with AMLPD. Both moderate and severe AMLPD lead to significant increase in BP in male and female mice. In both fetal and adult AMLPD brain, we observed an increase in ACE mRNA levels. However, in the fetus increased ACE mRNA was not associated with increased protein levels. miRNA mmu‐mir‐27a increased significantly in fetus from moderate AMLPD group, which can lead to decrease in ACE protein translation. We also observed hypomethylation of DNA promoter of ACE, which can increase mRNA transcription. Overall, our results indicate that AMLPD can lead to epigenetic changes in brain ACE with associated hypertension in adult offspring.
Elevated sympathetic activity precedes the arterial hypertension in the Goldblatt model
Authors: Oliveira‐Sales Elizabeth B , Abdala Ana PL , Colombari Eduardo , Campos Ruy R , Paton Julian FR
Published: June 2021

Despite the extensive use of the Goldblatt model of hypertension (two kidney one clip, 2K1C) sympathetic mechanisms responsible for both the development and maintenance of the elevated blood pressure remain unclear. We tested the hypothesis that sympathetic nerve activity (SNA) is elevated by the third week after clipping and before development of hypertension. Using the in situ working heart brainstem preparation, male Wistar rats were divided into three groups: Sham (n=7), and 2K1C (3 weeks) of which some were hypertensive (2K1C‐H, n=6) or and others normotensive (2K1C‐N, n=9) as determined a priori. At comparable perfusion rates, pressures were higher in both 2K1C groups (2K1C‐H: 76 ± 1; 2K1C‐N: 74 ± 3 vs Sham: 60 ± 2 mmHg, p< 0.05). The respiratory modulation of SNA was significantly elevated in both 2K‐1C groups (e.g. 2K1C‐H: 47.7 ± 6.1; 2K1C‐N: 32.8 ± 2.8 vs Sham: 20.5 ± 2.5 μV, p< 0.05). Pre‐collicular transection reduced SNA in all rat groups (2K1C‐H: 32.5 ± 7.5, 2K1C‐NH: 48 ± 6.9 vs Sham: 13.2 ± 1%, p< 0.05). Subsequent transection at the medullary‐spinal cord abolished SNA in Sham and 2K1C‐N but decreased it by 57± 5.5% in 2K1C‐H. We conclude, SNA can be raised prior to onset of hypertension by the 3rd week after clipping and this, in part, originates from enhanced respiratory modulation. Supported by CAPES and British Heart Foundation.
THE CORONARY ENDOTHELIUM AS A FUNCTIONAL AND PHYSICAL BARRIER FOR ENDOCRINE ANGIOTENSIN II
Authors: Tirado David Torres , Hernandez Jesus Castillo , Ahumada Erika Chi , Diaz Juan Ramiro , Rubio Rafael
Published: June 2021

Circulating/endocrine Angiotensin II (AII) may remain intravascularily, does not diffuse across the vessel wall and acts only in the luminal endothelial membrane (LEM). We synthesized 15,000 kDa polymers of AII (AII‐Pol) and Saralasin (Sar‐Pol) that due to their size, remain intravascularily and act solely in the LEM's AII receptors (L‐AT1).

In isolated perfused rat hearts AII and AII‐Pol both induces equally a positive inotropism and vasoconstriction. However, their ways of action differ; AII causes desensitization but AII‐Pol does not. Simultaneous infusion of both agonists show that as AII‐Pol/Ang II ratio raises, AII's desenzitization dwindles and is blocked. Desensitization is attributed to L‐AT1 internalization and is expected AII to induce L‐AT1 internalization but not so AII‐Pol. In LEM sampled during sustained stimulation with AII or AII‐Pol we show AII decreases L‐AT1 expression but no so AII‐Pol. The effects of both agonists were blocked by the non‐permeant Sar‐Pol. These results indicate that Ang II and Ang II‐Pol act and compete for the same L‐AT1.

To demonstrate the action of AII is restricted to L‐AT1, we intracoronary infused biotin labeled AII (AII‐B). Micrographs reveal AII‐B retained luminally and co localized with endothelial markers.

We conclude that the coronary endothelium is a functional and physical barrier for circulating AII. Funded by CONACYT SEP‐42567, 2004 C01 156.
CHRONIC BLOCKADE OF ENDOGENOUS FORMATION OF HYDROGEN SULFIDE CAUSED INCREASE IN SYSTEMIC BLOOD PRESSURE IN RATS
Authors: Khan Md. Abdul Hye , Roy Alexander P , Islam M Toriqul , Majid Dewan SA
Published: June 2021

Hydrogen sulfide (H2S), an endogenous vasodilator, is produced by two enzymes; cystathionine γ‐lyase (CSE) and cystathionine β‐synthase (CBS). In this study, the effects of chronic inhibition of CSE by DL‐propargylglycine (37.5 mg/kg/day; ip) and CBS by aminoxyacetic acid (8.8 mg/kg/day; ip) given either alone or in combination for 4 weeks on systemic blood pressure (SBP) and renal function were examined in rats (n=6 in each group). Prior to the start and then every 7th day during treatment period, SBP was measured by tail‐cuff method and 24 hr urine collections were made using metabolic cages. Urinary excretion rate of sulfate (USV; marker for endogenous H2S level) was determined by a colorimetric assay. At the end of treatment period, rats were subjected to acute experiments under anesthesia for determination of renal blood flow (RBF; Transonic flow‐probe) and glomerular filtration rate (GFR; inulin clearance). Compared to vehicle treated group, only the combined therapy group showed a decrease in USV (553±71 vs 248±50 nmol/24hr) and an increase in mean SBP (107±3 vs 130±2 mmHg) with its consequent increase in sodium excretion (4±1 vs 13±3 mmol/24hr). This group with combined therapy also showed a decrease in RBF (7±0.4 vs 4±0.3 mL/min/g) but not appreciably in GFR (1.0±0.1 vs 0.9±0.03 mL/min/g). These data suggest that a decrease in endogenous H2S production exerts direct vascular effect leading to an increase in SBP.
The inhibition of NOS down regulates adenosine production in the interstitial space of rat skeletal muscle
Authors: Zhao Yi , Smith Ashley A , Fabris Sergio , MacLean David A
Published: June 2021

It is generally accepted that vasodilatation in skeletal muscle is regulated in part by both adenosine (Ado) and nitric oxide (NO). It has previously been shown by our laboratory that perfusion of 60 μM of ATP into the interstitial space (via microdialysis) more than doubled the interstitial Ado concentration (0.15±0.02 to 0.33±0.04 μM), mediated predominantly by the 5′‐ectonucleotidase. However, a thorough examination of the relationship between NO and Ado production in the interstitial space has yet to be examined. Anesthetized Sprague Dawley rats had microdialysis fibers inserted into the gastrocnemius muscle of each leg and perfused with saline (baseline control) and then with either 10 mM L‐NAME (NOS inhibitor, n = 8 rats, n = 31 probes) or 10 mM L‐NAME + 60 μM ATP (n = 6 rats, n = 14 probes) for 20 minutes at a rate of 5 μl/min. Interestingly, during L‐NAME perfusion interstitial Ado levels were decreased (P<0.05) from baseline levels (0.20±0.01 μM) to below HPLC detection limits. The addition of ATP to the perfusate in an effort to promote Ado production via the 5′‐ectonucleotidase did not restore interstitial Ado concentrations. These data clearly show that the inhibition of NOS dramatically attenuates Ado production, which cannot be reversed by the addition of ATP. These finding strongly suggest that skeletal muscle interstitial Ado production is regulated by NO and/or NOS activity.

Supported by NSERC
Molecular, biochemical characterization and localization of neuronal nitric oxide synthase in human neutrophil
Authors: Jyoti Anupam , Saluja Rohit , Kumar Sachin , Chatterjee Madhumita , Verma Anupam , Barthwal Manoj K , Bajpai Virendra K , Dikshit Madhu
Published: June 2021

Nitric oxide (NO) is a potent free radical with diverse roles in regulating several important functions of neutrophils (PMNs), including chemotaxis, adhesion, aggregation, apoptosis, bacterial killing, however the expression status, biochemical characterization and localization study remains the least defined in human PMNs. The present study was attempted to characterize expression of NOS isoforms and NO generation potential in healthy human PMNs. Real time PCR using NOS primers have identified neuronal NOS (nNOS) as higher copy number (45 fold, *p<0.001) than inducible NOS (iNOS). Identification of nNOS PDZ domain (Exon‐2, N‐terminal) was done by RT‐PCR using exon‐2 specific primer and Western blotting using N‐terminal specific antibody. Localization of nNOS was explored by electron and confocal microscopy. Biochemical study involving NOS activity (L‐Arginine to L‐Citruline conversion) exhibited higher calcium dependent activity (0.21±0.05 pmol/30 min./107cells) than calcium independent (0.039±.007 pmol/30 min/107) which positively correlated with higher expression of nNOS. Immunocytochemical studies exhibited distribution of nNOS isoform primarily in the cytosol, granules, mitochondria and nucleus. The results thus suggest constitutive expression of PDZ domain containing nNOS localized mainly to cytoplasm contributes to calcium dependent activity and NO generation in human PMNs.
Nitroxyl anion mediates vasorelaxation in salt‐loaded AngII hypertensive mesenteric arteries
Authors: Wynne Brandi Michele , Labazi Hicham , Tostes Rita C. , Webb R. Clinton
Published: June 2021

The endothelium regulates vascular tone, releasing various vasoactive substances, including nitric oxide (NO), a vasodilating agent. NO exists in 3 redox states; the uncharged NO, the oxidized state, nitrosonium cation (NO+), and the reduced state, nitroxyl anion (NO−). NO−, has recently become an emerging candidate in vascular regulation. We hypothesized that relaxation to the NO− donor, Angeli's Salt (AS) in resistance arteries from salt‐loaded (4%) Ang II mice (SL) will be decreased. First order mesenteric arteries from Ang II (90ng/min) or sham mice were isolated for functional studies. Concentration‐response curves to AS and ACh were performed in Phe contracted vessels in the presence of the NO and NO− scavengers (carboxy‐PTIO, L‐cystiene, resp.) or the voltage‐gated K+channel blocker (4‐AP). Vessels from SL mice exhibited an endothelium‐independent decrease in NO− mediated relaxation vs. sham (EC50−5.47± 0.17, intact and −5.71± 0.21, denuded vs. −6.16± 0.17*). Sham vessels exhibited a decrease in NO dependent relaxation (Rmax 77.8 vs. 97.2%*), in contrast to SL vessels. In addition, in SL vessels incubated with L‐cystiene or 4‐AP, ACh mediated relaxation was attenuated (EC50 −4.97± 0.24 vs. −5.81± 0.18*) or abolished (−3.32± 17.37 vs. −5.81± 0.18**) resp. These data suggest that SL mesenteric arteries have an increased dependence upon NO−, mainly via K+V channels. (*p<0.05, **p<0.001) HL074167 RCW
K2P‐like current in freshly dissociated mouse thoracic aorta: the impact on thoracic aorta diameter
Authors: Bychkov Rostislav , Hayoz Sandébastien , Soliman Youssef , Cubano Luis , Maldonado Hector
Published: June 2021

Background

It was suggested that K+ channels family identified as "two‐pore potassium channels (K2P)" function as leak K+ efflux that regulates membrane potential complementary to Ca2+, ATP and voltage dependent K+ channels. Does opening of K2P channels dilate vascular beds, or do they tune membrane potential only?

Methods and results

Patch‐clamp technique was used to record an ATP‐γs elicited delayed K+‐current in freshly dissociated myocytes. This current was not inhibited by a "cocktail" of K+ channels blockers (TEA, 4AP, charibdotoxin, apamin, glibenclamide). Tested purinergic agonists (ATP, UTP) – except for ADP ‐ activated delayed K+‐current indicating that at least P2Y11 receptor stimulates delayed K+‐current. 12,13‐didecanoate also stimulated this current. ATPγs‐elicited K+‐current amplitude decreased in staurosporine and G6850 pretreated cells. Acid pH (7 and 6) inhibited when basic pH (8) stimulated amplitude of ATPγs‐elicited K+‐current. ATP‐γs produced a vasodilation in endothelium‐denuded thoracic aorta rings contracted by the K+‐channels blockers and pre‐treated by carbenoxolone. Vasodilation was inhibited by acid pH (6).

Conclusions

Our results demonstrate that ATP‐γs dilates pre‐stretched thoracic aorta rings via stimulation of pH sensitive K+‐current through P2Y receptors. Taken together our data provide new cellular mechanism of vasoregulation via K2P‐like channels.
Atomic force microscope studies demonstrate increased vascular smooth muscle cell stiffness associated with aging
Authors: Zhu Yi , Sun Zhe , Qiu Hongyu , Li Zhaohui , Trzeciakowski Jerome , Hunter William , Vatner Dorothy , Vatner Stephen , Meininger Gerald
Published: June 2021

Vascular stiffness has been shown to increase with aging due largely to changes in the properties of the extracellular matrix (ECM). We hypothesized that increased stiffness of vascular smooth muscle cells (VSMC) could also play a role in vascular stiffness changes associated with aging. Studies were conducted on VSMCs from the thoracic aorta of old (30 mo) and young (6 mo) F344xBN rats and from old (24–25 yr) and young (5–8 yr) male monkeys. Stiffness of single VSMCs was measured using a nanoindentation technique with atomic force microscopy. The estimated elastic modulus was significantly increased (p<0.001) in VSMCs from old rats (30.1±0.4 kPa; 57 cells) compared to young rats (14.±0.2 kPa; 140 cells) and similarly, was significantly higher (p<0.001) in old monkeys (41.7±0.5 kPa; 76 cells) than in young monkeys (12.8±0.3 kPa; 40 cells). Measurements of stiffness for 30 min indicated that stiffness oscillates in VSMCs and that these oscillations are enhanced in older (period ~8 min) vs younger (period ~18 min) animals. These data support the concept that the increased vascular stiffness that occurs during aging includes mechanical contributions from an increased stiffness at the level of the single VSMC.
Phosphatidylinositol 3,5‐bisphosphate increases intracellular free Ca2+ in VSMC and elicits vascular contraction
Authors: Silswal Neerupma , Parelkar Nikhil K , Andresen Jon
Published: June 2021

Phosphatidylinositol 3,5‐bisphosphate (PI(3,5)P2) is a newly identified phosphoinositide that is an agonist for the ryanodine receptor (RyR). Immunofluorescence demonstrated that PI(3,5)P2 was present throughout the wall of the mouse aorta. Application of exogenous PI(3,5)P2 (10 nM to 3 μM) contracted aortic rings whereas vehicle had no effect. Depletion of intracellular Ca2+ stores with thapsigargin (10 μM) and ryanodine (10 μM) abrogated contraction to PI(3,5,)P2. Verapamil (10 μM), however, blocked the sustained, but notthe initial phase of the contraction to PI(3,5)P2. In Fura‐2 Ca2+ imaging studies of dissociated aortic smooth muscle cells PI(3,5)P2 concentration‐dependently elevated intracellular Ca2+ ([Ca2+]in). Removal of extracellular Ca2+, or addition of 10 μM verapamil, substantially decreased the rise in [Ca2+]in caused by PI(3,5)P2. In Ca2+‐free buffer, preincubation with either ryanodine or caffeine prevented PI(3,5)P2 from generating a Ca2+ response. Addition of La3+ (100 μM) to block transient receptor potential channels also reduced, but did not abolish the rise of [Ca2+]in caused by PI(3,5)P2. Likewise, in aortic rings, La3+ impaired the contractile response to PI(3,5)P2. These data suggest that PI(3,5)P2 modulates Ca2+ signaling in vascular smooth muscle cells and contraction in intact arteries. This research was supported by an AHA SDG (0735053N) and UMKC start‐up funds.
The small GTPase RhoA Stimulates the Contraction of Airway Smooth Muscle (ASM) by Regulating Actin Polymerization rather than Myosin Light Chain (MLC) Phosphorylation
Authors: Zhang Wenwu , Gunst Susan J
Published: June 2021

Activation of RhoA has been shown to inhibit MLC phosphatase activity, thus increasing MLC phosphorylation and force in some smooth muscle. In ASM, actin polymerization is required for active contraction in addition to MLC phosphorylation. RhoA can regulate cytoskeletal processes and actin polymerization. We evaluated whether RhoA regulated the contraction of ASM primarily through its effects on MLC phosphorylation or actin polymerization. RhoA activity was inhibited in canine tracheal smooth muscle tissues by expressing the inactive RhoA mutant, RhoA T19N, or by treating tissues with the cell permeant RhoA inhibitor, C3 exoenzyme. RhoA inactivation inhibited ACh‐induced contractile force by 60–70%, inhibited ACh‐induced actin polymerization, and also caused a small (10–20%) decrease in MLC phosphorylation. Tissues were treated with the phosphatase inhibitor, calyculin A, by inactivate MLC phosphatase. Calyculin A induced MLC phosphorylation and contraction in the absence of ACh and potentiated MLC phosphorylation induced by ACh, but it did not affect ACh stimulated contraction or actin polymerization. However, inhibition of MLC phosphatase by calyculin A did not prevent the depression of force caused by the RhoA inactivation. This suggests RhoA activation regulates ASM contraction primarily through its effects on actin dynamics.

Supported by HL29289, HL074099, ALA, and NIH T32 Grant GM12453‐03
Interaction of fluoxetine and paroxetine with muscle nicotinic acetylcholine receptors
Authors: Andersen John David , Feuerbach Dominik , Ortells Marcelo O , Arias Hugo
Published: June 2021

The interaction of serotonin selective reuptake inhibitors (SSRIs) (i.e., fluoxetine and paroxetine) with muscle nicotinic acetylcholine receptors (AChRs) in different conformational states was compared to that for dizocilpine by using functional and structural methods. The results established that: (a) SSRIs inhibit (±)‐epibatidine‐induced Ca2+ influx with higher potencies than dizocilpine, (b) SSRIs inhibit [3H]TCP binding to the desensitized Torpedo AChR with higher affinities compared to the resting AChR, and (c) fluoxetine inhibits [3H]dizocilpine binding to the desensitized AChR, suggesting a mutually exclusive interaction. This is supported by our molecular docking results where fluoxetine and dizocilpine in the neutral state interact with the valine (position 13′) and leucine (position 9′) rings. Protonated fluoxetine has two potential binding sites, one located at or near the outer ring (position 20′), and another that overlaps the domain for the neutral molecules and for protonated dizocilpine. The high proportion of protonated fluoxetine and dizocilpine at pH 7.4 (>95%) suggests that the protonated drugs can be attracted to the channel mouth before binding deeper within the AChR ion channel, in a site shared with PCP. Support: Science Foundation Arizona.
Pharmacological block of KCa1.1 channel increases the amplitude of nerve‐evoked contractions induced by electrical field stimulation in human detrusor smooth muscle
Authors: Petkov Georgi V. , Kellett Whitney F. , Rovner Eric S.
Published: June 2021

We have previously identified the KCa1.1 channel as a major player in human detrusor smooth muscle (HDSM) contractility. Here, we examined the contribution of KCa1.1 channels in the nerve‐evoked contractions of isolated HDSM strips. HDSM tissues were obtained from patient‐donors during open surgeries (n–number of strips; N–number of patients). Nerve‐evoked contractions were generated by increasing electrical field stimulation (EFS) frequencies (3.5–50 Hz). Blocking the KCa1.1 channel with its specific inhibitor, iberiotoxin (200 nM), caused a statistically significant increase in the nerve‐evoked contraction amplitude at all stimulation frequencies (n=12, N=7). In the presence of 10 μM suramin and 10 μM α,β‐meth‐ATP, iberiotoxin (200 nM) also caused a statistically significant increase in the cholinergic component of the nerve‐evoked contractions (n=10, N=8). In the presence of 1 μM atropine, iberiotoxin (200 nM) still caused a statistically significant increase in the purinergic component of the nerve‐evoked contractions (n=6, N=5). Under block of the KCa1.1 channel (200 nM iberiotoxin), the maximum response of the purinergic component was almost doubled. Time‐controls for each experimental series confirmed the stability of the preparations (n=5–7, N=4). The data indicate that the KCa1.1 channels oppose nerve‐evoked contractions in HDSM. Supported by NIH DK084284 and DK070909 to G. V. Petkov.
Endurance Exercise and Systemic Mitochondrial Rejuvenescence: Run for Your Life!
Authors: Safdar Adeel , Little Jonathan P , Hettinga Bart P , Kujoth Gregory C , Prolla Tomas A , Tarnopolsky Mark A
Published: June 2021

A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the polymerase gamma (PolG) mutator mouse, harbouring a proofreading‐deficient copy of PolG, exhibits an accelerated aging phenotype, systemic mitochondrial dysfunction, multisystem failure, and reduced lifespan. Studies in primary cells from mitochondrial myopathy patients indicate that inducing mitochondrial biogenesis via over‐expression of PGC‐1α has therapeutic potential. We aimed to delineate if endurance exercise‐mediated induction of PGC‐1α metabolic network can prevent premature aging and systemic decline in PolG mice. At 3‐mo, 36 PolG mice (♀ = ♂) were randomly assigned to a sedentary (SED) or forced‐endurance training (END; 15m/min for 45 min, 3x/week for 5 months) group. In skeletal muscle, END increased nuclear abundance of PGC‐1α (76%) while concomitantly decreasing RIP140 (a negative regulator of PGC‐1α; 33%) nuclear content (P<0.05). END also increased mtDNA copy number (2–3 fold), protein content of respiratory chain subunits (CI‐NUFA9, CII‐subunit 70 kDa, C‐III core 2, and COX‐IV; 2–3 fold), and COX activity (22–120%) in skeletal muscle, lungs, heart, brain, and gonads (P<0.05). These molecular adaptations conferred complete phenotypic protection, reduced multi‐organ pathology, and increased lifespan of PolG mice. We conclude that END training promotes PGC‐1α‐mediated systemic mitochondrial oxidative capacity, contributing to the complete rejuvenation of PolG mice. We propose that END training offers a valuable therapeutic intervention for attenuating and/or reversing mitochondrial abnormalities associated with aging. (Funded by CIHR, and Mr. Warren Lammert and family)
Postnatal PGC‐1α gene transfer attenuates acute injury in mdx mice
Authors: Selsby Joshua T , Gardan‐Salmon Delphine
Published: June 2021

A promising strategy to treat Duchenne muscular dystrophy aims to replace the aberrant dystrophin protein with utrophin, a dystrophin related‐protein. Induction of PGC‐1α increases utrophin and may also support damaged mitochondria. Previously, constitutive PGC‐1α over‐expression reduced acute injury in dystrophic skeletal muscle. As dystrophin deficiency results in developmental abnormalities potentially improved by constitutive PGC‐1α over‐expression, it is imperative to determine the extent to which post‐natal PGC‐1α over‐expression prevents acute injury in dystrophic muscle. Neonatal mdx mice were injected in the right limb with 1×1011 gc of AAV 2/6 causing over‐expression of PGC‐1α while the left was injected with null virus. At 6 weeks of age, mice performed downhill running (−17°, 10m/min for one hour) to cause an acute eccentric injury, and were sacrificed 72 hours later. Following injury, PGC‐1α over‐expression in the soleus caused a 50% reduction in necrotic area and Evan's blue dye penetration (p<0.05), and tended to reduce central nucleation when compared to contralateral control muscle. These data indicate that PGC‐1α gene transfer following in utero development protects dystrophic muscle from acute injury.
The cellular force‐frequency response in ventricular myocytes from the varanid lizard, Varanus exanthematicus
Authors: Warren Daniel E. , Galli Gina L.J. , Patrick Simon M. , Shiels Holly A.
Published: June 2021

To investigate the cellular mechanisms underlying the negative force‐frequency relationship in the ventricle of the varanid lizard, we measured sarcomere and cell shortening, intracellular Ca2+ ([Ca2+]i), action potentials (APs) and K+ currents in isolated ventricular myocytes. Experiments were conducted between 0.2–1.0 Hz, which spans the physiological range of heart rates at 20–22°C for this species. As frequency increased, diastolic length, percent change in sarcomere length and relaxation time decreased significantly. Shortening velocity was unchanged. The rate of rise of [Ca2+]i increased, [Ca2+]i transient amplitude decreased, and diastolic [Ca2+]i increased. The first order time constant of the [Ca2+]i transient decay decreased at higher frequencies, indicating frequency dependent acceleration of relaxation, then leveled out and did not change above 0.5 Hz. The AP rate of rise was unaffected, but the AP duration (APD) decreased with increasing frequency. Peak depolarization was only significantly decreased at 1.0 Hz. The decrease in APD was not due to changes in IKr or Ito. Our results suggest that a negative FFR in varanid lizard ventricle is caused by decreased amplitude of the [Ca2+]i transient with an increase in diastolic [Ca2+]i, leading to incomplete relaxation between beats at high frequencies. This coincides with shortened action potential duration (APD) at higher frequencies.
Regulation of heart rate in zebrafish embryos
Authors: Rieger Sandra , Contreras Jorge E.
Published: June 2021

Although zebrafish has emerged as model for studying cardiovascular function, still very little is known about heart rate control in these animals. Here we report that known drugs that modify heart rate in mammals have similar effects in 2 days post fertilization zebrafish embryos. Injection of 0.5mM carbachol or propranolol into the blood stream reduced the heart rate by >25%, while norepinephrine resulted in a ~10% increase. We further discovered that brief exposure of embryos (5 sec) to UV light similarly increased the heart rate, up to 50%. Surprisingly, UV light exposure restored the charbacol‐induced drop in heart rate to normal levels. To determine whether UV light sensitive photoreceptors in the eyes or pineal gland (PG) mediated this effect, we ablated either one or both organs in GFP‐expressing transgenic embryos using a two‐photon laser. Ablation of both eyes and PG reduced the UV light‐mediated heart rate increase by ~30%, whereas ablation of either one led to a partial reduction. Nevertheless, we still observed a small but persistent increase, corresponding to a yet unknown source. Our observations suggest a novel mechanism for the regulation of heart rate by UV light, independent of autonomic function.
Deficiency of Bax promotes compensatory muscle hypertrophy as induced by denervation of agonists
Authors: Siu Parco M , Alway Stephen E
Published: June 2021

The purpose of the present study was to determine the effect of interference of apoptosis on muscle hypertrophy. We tested the hypothesis that the extent of muscle hypertrophy is enhanced in muscle of mice that are deficient of the pro‐apoptotic Bax gene. Experiments were conducted on adult wild‐type mice (n=8) and Bax gene‐deficient mice (n=6). The medial and lateral branches of tibial nerve that innervate the plantar flexor gastrocnemius and soleus were transected close to their neuromuscular junction. This experimental procedure left the contractile activities of plantar flexor plantaris intact which was induced for compensatory muscle hypertrophy. The denervation procedure was performed in right hind limb and sham procedure was performed in left hind limb, which served as the intra‐animal contralateral control. After 14 days of soleus and gastrocnemius denervation, no significant increase in plantaris muscle mass was found in wild‐type mice whereas significant compensatory hypertrophy was evident in plantaris of Bax−/− mice in which the plantaris muscle mass was significantly increased by 24% (P<.05) in Bax−/− mice. The cytoplasmic content of cytochrome c was significantly reduced by 32% (P<.05) in plantaris of Bax−/− mice with hypertrophy whereas no change was observed in that of wild‐type mice. Hydrogen peroxide content in muscle tissue was measured by fluorimetric assay but no changes were found in plantaris of both Bax−/− and wild‐type mice. These data demonstrated the enhancement of compensatory muscle hypertrophy as induced by denervation of agonists is accompanied by suppressed pro‐apoptotic signaling in skeletal muscle that is deficient of Bax. The present findings suggest that apoptotic signaling is involved in the adaptation of muscle hypertrophy.
Force deficits of human type I, IIa, and IIa/IIx muscle fibers following a single in vitro eccentric contraction
Authors: Choi SeungJun , Widrick Jeffrey J
Published: June 2021

Fast muscles from small laboratory animals show greater force deficits after eccentric contractions than slow muscles. How this applies to humans is unclear as humans do not express the same fast myosin heavy chain (MHC) isoforms as these animal models. To investigate relationships between eccentric damage and fiber type in human muscle, we measured peak Ca2+ activated force (15°C) of chemically skinned fiber segments (vastus lateralis, 6 subjects, age = 24 ± 2) before and after a single eccentric contraction (25% strain, 50% of pre‐eccentric unloaded shortening velocity). Fiber type was determined by gel electrophoresis. Fibers showing visible post‐eccentric myofibrillar tearing (N = 8) were excluded from analysis. At baseline, type IIa (125 ± 6 kN/m2; n = 28) and IIa/IIx (133 ± 7 kN/m2; n = 12) fibers produced greater Ca2+ activated force than type I fibers (111 ± 3 kN/m2; n = 38). A significant fiber type × time interaction revealed similar force deficits for type I (11 ± 1 kN/m2 or 10 ± 1% of pre force) and IIa (14 ± 2 kN/m2, 11 ± 1%) fibers but a 3‐fold greater deficit for IIa/IIx fibers (40 ± 5 kN/m2, 30 ± 3%). Control fibers subjected to a single isometric contraction showed force deficits of only 2 ± 1 kN/m2 (1 ± 1 %) with no difference between fiber types. These results indicate that human fast hybrid fibers (IIa/IIx) are more sensitive to a standardized in vitro eccentric contraction then either type I or type IIa fibers.
Post‐exercise feeding attenuates proteolytic gene expression in human skeletal muscle
Authors: Harber Matthew , Konopka Adam , Jemiolo Bozena , Trappe Todd , Trappe Scott , Reidy Paul
Published: June 2021

The purpose of this investigation was to assess the influence of feeding after a single session of aerobic exercise on the expression of genes involved in skeletal muscle remodeling. Eight recreationally active males (25±1y; VO2max: 52±2 ml·kg−1·min−1) performed 60‐min of cycle ergometry at 72±1% intensity on two occasions in a counter‐balanced design. Trials differed only in the post‐exercise nutritional intervention. Subjects ingested a non‐caloric placebo (FAST) beverage or a beverage containing (per kg body weight): 5kcal, 0.83g carbohydrate, 0.37g protein and 0.28g fat (FED) immediately and 1h after exercise. Muscle biopsies were obtained at 2 and 6h post‐exercise for the assessment of mRNA expression. Feeding attenuated (P<0.05) the mRNA expression of proteolytic factors MuRF‐1 (6h) and calpain‐2 (2 and 6h) post‐exercise but did not alter FOXO3A, calpain‐1, CASPASE3, or myostatin mRNA expression compared to FAST. Myogenic regulatory factor (MRF4) mRNA was also attenuated (P<0.05) at 2 and 6h post‐exercise in FED compared to FAST. The mRNA expression of mitochondrial factors PGC‐1α and TFAM were not influenced by feeding in the post‐exercise state. These data indicate that post‐exercise nutrient intake attenuates the expression of factors involved in the ubiquitin‐proteosome and Ca++ dependent protein degradation pathways.

Supported by a grant from the Gatorade Sports Science Institute
Scleraxis and Fibroblast Specific Protein‐1 Identify Fibroblast Cells in Skeletal Muscle Tissue
Authors: Shallal‐Ayzin Mark V , Gumucio Jonathan P , Bakhurin Konstantin I , Mendias Christopher L , Brooks Susan V
Published: June 2021

Fibroblasts are thought to play an important role in the production and maintenance of the extracellular matrix (ECM) of skeletal muscle, but very few markers are known to identify fibroblasts in the cellular milieu of skeletal muscle ECM. Scleraxis (Scx) is a bHLH transcription factor that is critical for limb tendon development, and is a marker for fibroblasts from tendon. Fibroblast specific protein (FSP‐1), also known as S100A4, plays a role in cell motility and proliferation and has been used to identify fibroblasts in tissues from the liver, kidney, lung and heart. To identify markers for fibroblasts in skeletal muscle ECM, we tested the hypotheses that (i) Scx and FSP‐1 are expressed in fibroblast cells in mouse EDL muscles, and (ii) that the number of Scx and FSP‐1 positive cells would increase following muscle injury. Using immunohistochemistry, we identified a population of cells in skeletal muscle ECM that co‐express Scx and FSP‐1. Following injury, there was a dramatic increase in the number of Scx/FSP‐1 positive cells in the muscle ECM. The Scx/FSP‐1 positive population of cells was distinct from the satellite cell (c‐met), macrophage (F4/80) and neutrophil (Ly6) populations of cells. The results from these studies indicate that Scx and FSP‐1 are useful markers for fibroblasts in skeletal muscle ECM and may serve as potential therapeutic targets for the prevention of fibrosis following skeletal muscle injury.
Enhanced transforming growth factor β (TGF‐β) signaling and fibrogenesis in fetal skeletal muscle of obese sheep at late gestation
Authors: Huang Yan , Yan Xu , Zhu Mei J , Ford Stephen P , Nathanielsz Peter W , Du Min
Published: June 2021

INTRODUCTION

Maternal obesity is increasing at an alarming rate. The objective of this studywas to evaluate the effect of maternal obesity on fibrogenesis in fetal skeletal muscle in late gestation when ovine fetal muscle matures.

METHODS

Non‐pregnantewes were assigned to a control (Con, fed 100% of NRCnutrient recommendations, n = 6) or obesogenic (OB, fed150% of NRC recommendations, n = 6) diet from 60 days before to 135 days after conception (gestation =148 days) when the fetal semitendenosus (St) muscle wassampled for trichrome staining, immunoblotting and real‐time quantitative PCR analyses.

RESULTS

The total area of collagen in the cross‐sections of St muscle increased by 105.1 ± 5.9% (P = 0.05) in fetuses of OB compared with Con mothers. The expression of precursor TGF‐β in OB fetal muscle was 177.3 ± 47.6% higher (P < 0.05) than Con fetal muscle, indicating activation of TGF‐β signaling. p38 promotes TGF‐β signaling; the concentration of phospho‐p38 was 74.7 ± 23.6% higher (P < 0.05) in OB fetal muscle, as well as the ratio of phospho‐p38/total p38 (83.2 ± 20.4%, P < 0.05). The mRNA expression of Smad7 and fibronectin is regulated by TGF‐β signaling. An increase of 327.9 ± 168.0% (P < 0.05) and 188.9 ± 82.1% (P < 0.05) respectively was observed for the mRNA expression of Smad7 and fibronectin in OB fetal muscle compared with Con.

CONCLUSIONS

These data demonstrate that maternal obesity enhanced fibrogenesis in fetal muscle in late gestation associated with up‐regulation of the TGF‐β/p38 signaling pathway. These changes are expected to negatively affect the properties of offspring skeletal muscle. (Supported by NIH 1R03HD057506 and Wyoming INBRE P20RR016474)
Soleus muscle of Down syndrome (DS) mice exhibits elevated markers of oxidative and nitrosative stress
Authors: Cowley Patrick M , DeRuisseau Keith C
Published: June 2021

Individuals with DS have a reduced capacity for voluntary force generation compared to individuals with and without other forms of intellectual disability. Oxidative stress (OS) is thought to be a key factor in DS pathologies that may be the result of triplication and over‐expression of copper‐zinc superoxide dismutase (CuZnSod). Our aim was to assess markers of OS and nitrosative stress (NS) in soleus muscle of a well established mouse model of DS, the Ts65Dn mouse, which is segmental trisomic for CuZnSod. We hypothesized that soleus muscle of Ts65Dn mice would exhibit elevated markers of OS and NS compared to soleus from wild‐type (WT) controls. Eighteen (n=9 per group) 4–7 month old male Ts65Dn and WT mice were used in these experiments. Western blot analysis was performed to quantify the relative expression of markers of OS (4‐hydroxy‐nonenal (4‐HNE)) and NS (3‐nitrotryosine (3‐NT)). Levels of soleus muscle 4‐HNE and 3‐NT were 476% and 276% higher in Ts65Dn mice (p<0.05), respectively. These findings suggest that soleus muscle from Ts65Dn mice exhibits elevated markers of OS and NS, which is consistent with the human phenotype of DS. Additionally, preliminary microarray analysis identified 43 up‐regulated and 66 down‐regulated genes in soleus of Ts65Dn compared to WT mice (p<0.001, ± 1.5‐fold). We are currently categorizing these differentially expressed genes.

Supported by Syracuse University SOE
Hydration effects on vascular fluid dynamics during acute exposure to 3,048 m
Authors: Castellani John William , Muza Stephen R , Kenefick Robert W , Sawka Michael N
Published: June 2021

Vascular fluid dynamics [plasma volume (PV) and total circulating protein (TCP)] during acute (8‐h) high‐altitude (ALT) exposure are poorly described and the impact of hypohydration (HYPO) on these responses has not been reported. Seven men (25 ± 7 yr; 44.1 ± 4.9 ml/kg/min V02peak completed 4 experimental trials in a counterbalanced design. Subjects were sea level (SL) residents who had completed 4 days of heat acclimation prior to the trials. Trials were: a) SL‐Euhydrated (SL‐EUH); b) SL‐HYPO; c) ALT‐EUH; and d) ALT‐HYPO. The day before each trial, subjects completed a standardized exercise‐heat stress bout either with (EUH) or without (HYPO, −4% body mass) fluid replacement. Subjects maintained hydration status overnight and the following morning entered a hypobaric chamber (27°C) and rapidly ascended to either 150 m or 3,048 m (O2 saturation = 89 ± 3 %). During each 8 h trial, subjects were sedentary except for completing a standardized submaximal exercise bout (60 min) between 1–2.5 h of exposure. Plasma osmolality was not altered by ALT but was increased (291 ± 5 to 300 ± 6 mosmol/kg) by HYPO. PV was not altered by ALT (3.37 ± 320 L) but was reduced (320 ± 120 ml, 9.5%, p<0.01) by HYPO. TCP (268 ± 30 g) was not altered by ALT or HYPO. These data indicate that vascular fluid dynamics are not altered during the initial 8h of exposure to 3,048 m regardless of hydration status.

Not reflecting official views of the Army or DoD.
Increasing Intensity of Hypoxia Augments Fos Expression in Hypothalamic Paraventricular Nucleus (PVN)‐Projecting Neurons in the Nucleus of the Tractus Solitarius (nTS)
Authors: King Titia Luise , Heesch Cheryl M , Kline David D , Hasser Eileen M
Published: June 2021

Acute hypoxia (AH) activates peripheral chemoafferents. Sensory information is sent to the nTS, integrated and relayed to other brain regions to increase breathing, sympathetic nerve activity and blood pressure. The nTS sends direct projections to the PVN which may be excitatory or inhibitory, but the role of the projections in the chemoreflex is unknown. We used Fos‐immunoreactivity (IR) to determine activation of PVN‐projecting nTS neurons to AH. We hypothesized that increasing intensity of AH augments activation of nTS neurons, particularly PVN‐projecting cells. Fluorogold (FG) was microinjected bilaterally into the PVN to label nTS PVN‐projecting neurons (n=8 rats). After recovery, rats underwent normoxia (N, 21% O2) or AH (12, 10, or 8% O2; 3hr). Fos and GAD67 (marker for GABAergic cells) immunohistochemistry was performed. Positively labeled nTS neurons were counted. AH increased the number of Fos‐IR nTS cells (N, 15±0; 12%, 43±2; 10%, 157±49; 8%, 164±37). The percent of PVN‐projecting nTS cells co‐expressing Fos was greater at higher AH intensity (N, 3±0%; 12%, 11±2%; 10%, 32±12%; 8%, 30±4%). Fos‐IR was not altered by AH in GAD67‐IR cells. FG and GAD67 colabeling was low. Data suggest increasing AH intensity activates more nTS cells, especially non‐GAD67 PVN‐projecting neurons. The PVN may play an important role in cardiorespiratory responses as chemoreflex stimulation intensifies. HL55306, HL085108
Variability in pulmonary function changes in United States Antarctic Program participants following rapid transport to the South Pole
Authors: Lalande S , Ceridon M L , Anderson P J , Miller A D , Beck K C , O'Malley K A , Johnson J B , Johnson B D
Published: June 2021

Maximal expiratory flows (MEF) are dependent on gas density, lung volume, subject effort and airway characteristics. Variable changes in MEF occur after ascent to altitude which may represent an interaction between gas density changes, catecholamine stimulation and mild interstitial edema. Hypoxia induced catecholamine up‐regulation has also been associated with acute mountain sickness (AMS). This study examined the association between MEF variability and AMS symptoms in United States Antarctic Program personnel (n=198, age 37±1 yrs, 3% asthma) rapidly transported to the South Pole (SP, 9302 ft). Spirometric parameters, epinephrine (Epi), norepinepherine (NE) and SaO2 levels were recorded at sea level (SL, McMurdo Station) and after the 2nd night at SP. AMS was assessed using Lake Louise questionnaires (score ≥ 3). FVC, FEV1, FEF25, FEF75 and FEF25–75 increased by 0, 3, 17, 10 and 14% from SL to SP, respectively. On day 2, AMS incidence was 31% and Epi and NE increased by 44 and 71%. Changes in MEF ranged from −64 to +93% in FEF25–75 and −63 to +70% in FEF75. Subjects with the greatest increase in MEF had higher incidence of AMS (24 vs 41%) and higher NE levels, while the subjects with the greatest decrease in MEF had lower SaO2 values (88 vs 90%). Subjects with increased MEF had increased AMS and bronchodilation while subjects with decreased MEF displayed values consistent with mild interstitial edema.
Peripheral chemoreflex sensitivity and sympathetic nerve activity are normal in breath hold divers during training season
Authors: Breskovic Toni , Ivancev Vladimir , Banic Ivana , Jordan Jens , Dujic Zeljko
Published: June 2021

Breath hold divers are exposed to repeated massive arterial oxygen desaturation, which could perturb chemoreflexes. Therefore, we tested the hypothesis that peripheral chemoreflex regulation of ventilation and sympathetic vasomotor tone is present during the training season. We determined ventilation, heart rate, blood pressure, cardiac stroke volume, and muscle sympathetic nerve activity (MSNA) during isocapnic hypoxia in 10 breath hold divers and 11 matched control subjects at the end of the season of intense apnea trainings. Baseline MSNA frequency was 30±4 bursts/min in control subjects and 25±4 bursts/min in breath hold divers (P=0.053). During hypoxia burst frequency and total sympathetic activity increased similarly in both groups. Sympathetic activity normalized during 30 minute recovery period. Hypoxia‐induced stimulation of minute ventilation was similar in both groups. Hypoxia increased heart rate and cardiac output whereas total peripheral resistance decreased. Blood pressure remained unchanged. We conclude that peripheral chemoreflex regulation of ventilation and sympathetic vasomotor tone is paradoxically preserved in apnea divers during the training season.

This study was supported by the Croatian Ministry of Science, Education and Sports.
Monocyte Chemoattractant Protein‐1(MCP‐1) released from hypoxic alveolar macrophages activates systemic mast cells
Authors: Donham Paula , Chao Jie , Viets Zachary , Wood John G , Gonzalez Norberto C
Published: June 2021

Alveolar hypoxia (FIO2 = 0.1) produces a rapid and widespread systemic inflammation in rats. We have shown that the inflammation is not initiated by low tissue PO2 but by a circulating mediator(s) released from alveolar macrophages (AMØ). This substance(s) activates mast cells (MC) which, in turn, initiate the inflammation. The MC secretagogue MCP‐1 is released by primary cultures of hypoxic AMØ, suggesting a role in this process. If MCP‐1 mediates the inflammation: 1) substances that prevent AMØ activation by hypoxia would also prevent MCP‐1 release, and 2) MC immersed in hypoxic AMØ supernatant would show MCP‐1 –dependent degranulation.

Primary cultures of rat AMØ exposed to hypoxia (10, 5 and 0 % O2) showed a respiratory burst evidenced as a release of H2O2; this was accompanied by a release of MCP‐1 into the supernatant; 2) both the respiratory burst and the release of MCP‐1 were blocked by pretreatment of rats with dexamethasone; 3) normoxic MC immersed in supernatant of hypoxic AMØ underwent degranulation; 4) MC degranulation was blocked by pretreatment of MC with MCP‐1 receptor blocker.

Conclusions

The results show that AMØ activated by hypoxia release MCP‐1 which, in turn, produces MC degranulation. These data support a role for MCP‐1 in the initiation of the systemic inflammation of alveolar hypoxia in intact animals.

Supported by NIH 39443‐18, AHA 0815652G and APS Frontiers in Biology
Hypoxic exercise responses in lean and obese humans
Authors: Limberg Jacqueline K , Evans Trent , Blain Gregory , Zillner Caitlin , Proctor Lester , Sebranek Josh , Schrage William
Published: June 2021

No studies have tested whether obese humans exhibit reduced skeletal muscle blood flow during dynamic exercise under conditions of hypoxia. We hypothesized that exercise‐induced blood flow to skeletal muscle would be lower in young healthy obese subjects (BMI 38.5±2.0 kg/m2) compared to lean subjects (BMI 23.3±0.6 kg/m2). We measured forearm blood flow (FBF, using Doppler Ultrasound) and oxygen levels (pulse oximetry) during rest and steady‐state dynamic forearm exercise (20 contractions/min at ~20% maximal voluntary contraction) under two conditions: normoxia (0.21 FiO2, 98% SaO2) or hypoxia (~0.10 FiO2, 80% SaO2). Forearm blood flow relative to muscle mass (rFBF) during steady‐state normoxic exercise was similar between lean (n=18) and obese (n=14) subjects (p>0.05). Forearm exercise under conditions of hypoxia significantly increased rFBF from normoxic levels in lean (p<0.05) but not in obese subjects (p>0.05). Our results suggest that relative forearm blood flow is maintained during dynamic exercise under normoxic conditions in obese subjects. Under hypoxic conditions, lean controls further increase rFBF, whereas obese subjects do not. These results suggest under conditions of hypoxic stress obese, otherwise healthy, adults may not respond appropriately to hypoxia. Research Support: AFAR A08235, AHA 0815622G, NIH HL091397.